Preface

Genetics is one of the fastest developing areas of natural sciences, which brings more and more knowledge and important information to our society. This information reaches us through scientists, physicians and other experts in the form of prenatal diagnostics, the latest trends in the field of healthy living, new possibilities for personalised disease therapy, modern methods in criminology etc. The correct understanding of scientific and technical procedures, which include personalised medicine or the use of genetically modified organisms, places increasing demands on every member of modern society. The rational response of all of us to the challenges associated with these technologies, as to the understanding of global pandemics or the climate crisis, thus requires an increasingly strong voice of educated experts and a cultured dialogue between experts and the public. The effort of today's educational institutions to provide quality information is additionally complicated by the spread of fake news, hoaxes and the distortion of reality by non-expert Internet debaters. If our interest is to teach people to critically accept the information that directly affects them and not to be swayed by half-truths and conspiracy theories, a strong foundation in genetics is a necessity today.

This book is intended for teachers of biology students, students of biological and non-biological fields, as well as those interested in genetics from the public. Its aim is to bring the basic genetic principles and discoveries closer together and to discuss them in the context of current events. The book also provides information on the use of knowledge from genetics in other fields, including medicine, criminology, pharmacy and history.

About the authors

This work was originally published in Slovak for the 200^th Anniversary of Johan Gregor Mendel, by Andrea Ševčovičová at the Comenius University in Bratislava, Slovakia. The text has been translated to English in collaboration with all authors, and put together by Samantha Hughes at the Vrije Universiteit Amsterdam, so that everyone can enjoy and understand Genetics.

The full list of contributors are shown below

Chapter	Chapter name	Author(s)
1.	In the beginning there was Mendel	A. Sevcovicova¹
2.	How does a scientist work?	F. Cervenak¹
3.	Meet DNA, the bearer of genetic information	K. Veljacikova¹
4.	How do you work with DNA?	R. Sepsiova¹
5.	Mutations - how they arise and what to do with them	A. Sevcovicova¹
6.	How the environment can affect our genes	A. Sevcovicova¹, V. Vozáriková¹
7.	From epigenetics to human diseases	V. Vozáriková¹
8.	Your cells are stressed too	M. Petková¹
9.	When cells go crazy	V. Vozáriková¹
10.	Gene therapy	F. Brazdovic^{2, 3}
11.	Plants as an inspiration in biomedicine	T. Zajickova⁴, E. Gálová¹
12.	When the environment changes our hormones	I. Kyzekova¹, S. Hughes⁶
13.	Etiquette in our genes	K. Veljacikova¹
14.	The principle of evolution	R. Sepsiova¹
15.	DNA as evidence – forensic genetics	R. Sepsiova¹
16.	Genetics in sport	K. Reichwalderova⁵
17.	Genetically modified organisms	F. Cervenak¹
18.	The Most Common Hoaxes in Genetics - Myths & Facts	S. Kyzek¹
19.	Genetics in science fiction and pop culture	F. Cervenak¹
20.	How to become a model...in biology	L. Tomaska¹, S. Hughes⁶

Affiliations:

¹ Department of Genetics, Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia

² Department of Biochemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slovakia

³ Laboratory of Regulation of Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic

⁴ Department of Medical Genetics, National Oncology Institute, Bratislava, Slovakia

⁵Sensible Biotechnologies, s.r.o., Bratislava, Slovakia

⁶ A-LIFE, Environmental Health &Toxicology, Vrije Universiteit Amsterdam, The Netherlands

As you can read in chapter 2, scientists are more than what is shown in popular culture. Below you can read a bit about what each of the contributors do in their scientific career! If you want to read more about each author, you can click on the ORCID link.

Andrea Ševčovičová (ORCID 0000-0003-2912-5184) is a Professor at the Department of Genetics, Faculty of Natural Sciences, Comenius University in Bratislava. In her scientific work, she mainly focuses on genetic toxicology studying bioactive substances that could find potential application in medicine and the potential genotoxic effect of environmental pollutants. She has published numerous studies in prestigious international journals. During her professional career she stayed at laboratories in Switzerland, Portugal, the Czech Republic and Bulgaria.

Samantha Hughes (ORCID 0000-0001-8447-2563) is an Assistant Professor at the Vrije Universiteit Amsterdam, the Netherlands. Her research focuses on using the model organism Caenorhabditis elegans to explore the phenotypic and genetic impact of environmental pollutants, like PFAS. Samantha has been involved in teaching throughout her career and has a passion for genetics and developmental biology, and you will still find her at the lab bench helping her internship and PhD students.

Filip Červenák (ORCID 0000-0001-6886-8883) is an assistant professor at the Department of Genetics, Faculty of Natural Sciences, Comenius University Bratislava. His scientific work is focused on the evolution of chromosomal ends and nucleo-protein structures (telomeres) that protect them from degradation and fusion. As a teacher, Filip participates in several courses and supervises students at both bachelor’s and master’s levels. He is also involved in various science-popularisation activities organized by the department.

Regina Sepšiová (ORCID 0000-0002-8709-7882) is assistant professor at the Department of Genetics, Faculty of Natural Sciences, Comenius University in Bratislava. She participates in teaching of genetics and molecular and cell biology, and supervises bachelor and master students in the laboratory. Her scientific work is focused on the study of telomeres and telomeric proteins of different yeast species, mechanisms of alternative telomere lengthening and evolution of telomeric sequences. She has several publications in influential scientific journals.

Katarína Veljačiková (ORCID 0000-0003-2977-1354) is a PhD student at the Department of Genetics, Faculty of Natural Sciences, Comenius University in Bratislava. As part of professor Tomáška's team, she researches the molecular mechanisms of communication between the nucleus and mitochondria in the eukaryotic cell. Her primary interest is nuclear-encoded mitochondrial proteins.

Veronika Vozáriková (ORCID 0000-0003-0852-9032) is a researcher at the Department of Genetics, Faculty of Natural Sciences, Comenius University in Bratislava, where she studies the basic mechanisms of human diseases, especially focusing on Parkinson's disease. Her work helps to understand how these diseases develop. Alongside her research, Dr. Vozáriková teaches a variety of courses in areas like biology and genetics. She has published her research in well-known international journals and is recognized for her teaching qualities and graphic expertise.

Maria Petkova (ORCID 0000-0001-9415-4219) is a researcher and teacher in Department of Genetics at the Faculty of Natural Sciences, Comenius University Bratislava. Her research is primarily focused on the molecular mechanism of non-thermal plasma effects on different organisms (yeasts and crops). Recently she is interested in flow cytometry analysis on various cell types. In addition to her research, she also teaches various courses and supervises students.

Filip Brázdovič (ORCID 0000-0002-1566-5967) is a researcher at the Insitute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic. He is interested in the molecular genetics and genomics of microorganisms withe th focus on the expression of genetic information. Dr. Brázdovič does publish, when he must, but he prefers to bake cakes.

Ivana Kyzekova (ORCID 0000-0003-3116-246X) is a researcher and teacher at the Department of Genetics,Faculty of Natural Sciences, Comenius University in Bratislava. In the scientific part of her work, she focuses on the genotoxic effects of endocrine-disrupting chemicals, e. g. bisphenols. She has published several studies in prestigious international journals, and has worked for a few months at the laboratory of Dr. Hughes in the Netherlands.

Stanislav Kyzek (ORCID 0000-0001-8841-4002) is a researcher at the Department of Genetics, Faculty of Natural Sciences, Comenius University in Bratislava. His research primarily investigates the molecular mechanisms in various model organisms following non-thermal plasma treatment. Additionally, he explores the potential of non-thermal plasma to induce adaptive responses across different organisms. Dr. Kyzek has authored numerous studies published in prestigious international journals, which have received significant citation recognition.

Eliska Galova (ORCID 0000-0003-3494-4432) works at the Department of Genetics, Faculty of Natural Sciences, Comenius University in Bratislava. In her scientific work, she deals with various agents, their genotoxic and antigenotoxic effects, focusing on the study of bioactive substances that could find potential application in medicine and pharmacy. She has been working on secondary metabolites of the Hypericum perforatum plant for a long time, focusing on the study of the mechanism of action of these substances on various model organisms. During her professional career, she worked in laboratories in the Czech Republic, Poland and Bulgaria.

Terezia Zajickova (ORCID 0000-0002-5528-3939) works at the Department of Medical Genetics, National Oncology Institute in Bratislava. In her scientific work, she has focused on genotoxic profiling of secondary metabolites and their potential drug-drug interactions, with a particular emphasis on their impact on cancer cell behaviour. Her research explores the molecular mechanisms underlying these interactions, aiming to identify novel combinations and more effective therapeutic strategies.

Katarína Reichwalderová (ORCID 0000-0003-4280-6785) is a Laboratory Manager at Sensible Biotechnologies. In her PhD studies at the Department of Genetics, Faculty of Natural Sciences, Comenius University in Bratislava she focused on polymorphisms of gene and their influence on sport performance.

Ľubomír Tomáška (ORCID 0000-0003-4886-1910) is a professor of genetics with a long-standing interest in the mechanisms of nucleo-mitochondrial communication within eukaryotic cells. Specifically, alongside his younger colleagues, he employs various species of yeast to understand the maintenance of chromosomal termini (telomeres), pinpoint the evolutionary paths resulting in distinct toolkits involved in telomere protection, and uncover the role of post-translational modifications in the regulation of mitochondrial functions.

Chapter 1: In the beginning there was Mendel

Since time immemorial, people have been interested in why some children resemble their parents more than others, or why certain diseases occur more often in some families than in others. Similar questions are asked when growing plants and raising animals. People have subsequently tried to use their observations in order to obtain offspring with the most suitable characteristics. Questions like: "Why do ornamental plants offer so many colors and shapes?" or "Is it possible to somehow increase the yields of commonly grown crops?" were also dealt with by an Augustinian monk working in nearby Brno in the 19th Century, Gregor Johann Mendel (Figure 1.1). His curiosity led him to patiently search for answers to these questions in long-term experimental work. Perhaps his origin also predestined him for this, as he came from a peasant family that was engaged in agriculture and was therefore close to nature. Through experiments with plants, Mendel wanted to understand the principles of inheritance of various characteristics of plants and animals, and we can say today (almost two hundred years later) that he was successful in his endeavor.

Figure 1.1 Johann Gregor Mendel – the founder of genetics 1822-1884

Mendel's work with plants and bees

In 1856, Mendel began experimenting with the crossing of one quite ordinary plant - the pea, Pisum sativum. As it turned out later, this choice was very lucky, because pea plants grow quickly, are quite undemanding, can be propagated in a controlled manner, and what was also very useful, especially in the 19^th century, peas can also be used in the kitchen! However, whoever thought that it was an easy job would be very mistaken. In order to keep everything under control, Mendel removed all the stamens (the male parts of the flower that produces pollen) from the undeveloped bud and using a brush transferred pollen from another plant to the scar (the place on the plant main stem/trunk where a leaf has fallen off). He then covered the flowers with paper cones or gauze bags. In this way, Mendel combined pollen with egg cells found in pistils (the female parts of the flower) with different characteristics and watched how individual characters were transmitted to the next generation. In total, he studied more than 29,000 pea plants. Mendel statistically evaluated the results of his long-term observations and presented them at two meetings of the Brno Natural History Association. However, his appearance had only a lukewarm response, and Mendel's results did not find a response in the scientific community even at the time of their publication in 1866, in a publication entitled "Experiments with plant hybrids" (from the German original “Versuche über Pflanzen-Hybriden“).

On the recommendation of Carl Nägeli, a scientific authority at the time, Mendel wanted to test his rules on another plant species. His choice fell on the hawkmoth (Hieracium), which had one feature that Mendel had no idea about, and which disqualified the hawkmoth as a model organism for studying the foundations of heredity. This genus of plants reproduces apomictically, which means that a new plant can arise from the egg without fusing with the male sex cell. Therefore, Mendel could not obtain the same results for this type of organism as for the pea, which reproduces sexually.

Mendel did not limit himself to plant breeding in his experiments on controlled crossing. As an enthusiastic beekeeper, he tried to breed stingless bees. Unfortunately, even this selection of a model organism did not lead to easy-to-understand results, mainly due to the specific method of determining the sex of bees. Mendel's results were based on mathematical and statistical models, which were not very popular in biology at that time. This fact, and Mendel's underestimation of his own results, meant that his extraordinary research was not understood by the natural scientists. As has often happened in history, Mendel's experiments gradually fell into oblivion, and it was not until two decades after his death that he received the attention he deserved. Thus, Mendel was unlucky in two of his three important experiments. However, the first (and most important) experiment finally made up for it, and when years later he was appreciated by the scientific community, Mendel was found to have laid the foundations for our current understanding of genetics and ensured him an important place in the history of natural sciences.

Experiments with peas

Mendel's exact thinking was significantly manifested in the methodology of his pea crossing experiments, as he was the first to realise that the laws of nature are of a mathematical nature. A very important discovery in his work was that the plant does not have to be examined as a whole, but the focus is on individual characteristics/properties. Mendel chose seven different pea characteristics for his experiments (Table 1.1), but he had no idea that each of these traits is governed by one gene with two variants, which was the basic premise of his success.

Table 1.1 Pea characteristics. Mendel selected several characteristics to study in his peas, which are all governed by a single gene with two variants, dominant or recessive, which give rise to different traits.

	Dominant trait	Recessive trait
Seeds	Yellow colour	Green colour
Seeds	with a smooth surface	with a wrinkled surface
Flowers	Red	White
Flowers	terminally located	axially located
Pods	Inflated	Constricted
Pods	green colour	yellow colour
Plant	Tall	Short

At the time, it was believed that a plant organism develops only from an egg cell without being fertilised by a pollen cell. Mendel guessed that what determines the hereditary characteristics of the offspring must be stored in the germ cells (the egg and pollen cells) of both parents, and this information is combined during fertilisation. He therefore decided to make crosses between different plants and analyse their offspring.

In the first generation, which Mendel designated as the parental generation (P), he crossed a red-flowered plant with a white-flowered one. He made crossings in both directions – that is, the red-flowered plant was the donor of the female reproductive cell (ovum) and the white-flowered plant was the donor of the male (pollen) cell, while in another cross it was the other way around. All individuals of the first generation of offspring (first filial generation – F1) had a red flower, i.e., the parental characteristics did not mix and only one trait was visible. The F1 plants were self-pollinated and the seeds of the F1 generation collected and sown by Mendel. The resulting plants of the new generation, which he called the F2 generation, or the second filial generation, were again crossed with each other. It was surprising that white flowers appeared in the F2 generation, a feature that did not appear in the F1 generation. The ratio between red-flowered and white-flowered plants was approximately 3:1 (Figure 1.2). Such a procedure was repeated for several generations in a row with other traits, with the ratio of characteristics in the P, F1 and F2 generations being key and forming the basis of Mendel's laws.

Figure 1.2 Crossing diagram of field pea. In the parental generation (P), individuals for red (homozygous dominant) or white (homozygous recessive) flowers are crossed with each other. The F1 (first filial generation) resulting from thus cross are uniform and phenotypically identical to the homozygously dominant individual (red flowers). In the F2 generation, which is created by crossing two individuals of the F1 generation, there are 75% individuals with a dominant phenotype (red flowers) and 25% individuals with a recessive phenotype (white flowers). This is a classic 3:1 ratio of red:white flowers.

Having carried out hundreds of such crosses, Mendel came to the following conclusions:

The external appearance of the organism (phenotype) is conditioned by paired elements, which Mendel named hereditary factors. Today we know that these hereditary factors are our genes. In a diploid organism (such as humans) the individual will contain one set of chromosomes from each parent, with each gene represented in two forms – alleles - where the individual receives one from each parent after the fusion of sex cells.
The hereditary factor/allele for white flower colour did not disappear in the F1 generation (Figure 1.2), but its expression was suppressed by the allele for red flower colour. The red colouring is therefore a dominant trait, conditioned by the dominant allele of the gene. The white colour of the flower, on the other hand, is a recessive trait, for which the recessive allele is responsible. If an individual received the same alleles from both parents, they are termed a homozygote. If both alleles are dominant, the individual is a dominant homozygote (i.e., red flowers), while if both alleles are recessive, the offspring is a recessive homozygote (as in the case of white flowers). If an individual received different alleles of a gene (one dominant and the other recessive), the offspring is called heterozygous.
During the formation of gametes (sex cells), the number of chromosomes is reduced to half the original number, so that the two alleles of one gene segregate - they separate into two different sex cells. As a result, the gamete contains only half of the parent's genetic information. During fertilization, during which the gametes of the parents fuse, the original number of chromosomes, which is typical for each species, is restored.

From Mendel to the present

One of the reasons why the public initially did not understand Mendel's laws was perhaps the fact that at the time, it was not possible for scientists to find out which molecule in the cell is the carrier of these hereditary characteristics. Mendel's conclusions were thus forgotten for some time, but fortunately not for very long. At the beginning of the 20^th century, specific formations - chromosomes - were discovered in the sex cells of plants and animals, whose behaviour during cell division and gamete formation corresponded to the behaviour of hereditary factors in the experiments described by Mendel. This discovery, together with the experiments of three important botanists - Hugo de Vries, Carl Correns and Erich Tschermak von Seysenegg - helped to rediscover Mendel's laws. Further important discoveries were not long in coming. Once the DNA molecule was identified as the material carrier of genetic information, genetics was transformed into an independent scientific discipline, and research in this area began to progress by leaps and bounds. The influence of many discoveries related to genetics and molecular biology on our everyday lives can no longer be underestimated. In this book, we will explain the secrets of genetics and point out significant discoveries and applications that each of us may encounter in our everyday life.

Right at the beginning, we will take a closer look at how a scientist works (Chapter 2). The public often imagines scientists as people who wear white coats, thick glasses, are oblivious to their surroundings and hold smoking test tubes of coloured liquid in their hands. However, real scientists, in contrast to this stereotypical image, use a wide range of approaches (many of which do not require test tubes or white coats at all) to solve various professional and practical problems. More than this, scientists are united by a scientific way of thinking, which is crucial in the design of experimental tests, but in many ways also facilitates everyday life.

In the section “Meet DNA: the bearer of genetic information“ (Chapter 3) we will take a closer look at the DNA molecule as the carrier of genetic information and its spatial structure. We will also describe how genetic information is realized - how the information stored in DNA is transcribed into RNA, and then proteins are created according to it, which perform the necessary activities in cells.

Are you interested in how scientists obtain material for genetic analysis, or how they navigate the vast amount of information written in DNA? Would you like to know how it is possible to "read" the DNA sequence and write it in the form of ordinary letters? Turn to Chapter 4 - How do you work with DNA. In this chapter you will find answers to these questions, as well as the principles of the most used methods, which geneticists and molecular biologists rely on in their research. In addition to basic research, these methods are also used in diagnosis of disease, food inspection, genealogical studies, archaeology and many other disciplines.

As mentioned above, Mendel's work was not properly received and appreciated during his lifetime, likely because variability played a significant role in his experiments. Today, it is already clear that DNA is not a static molecule, on the contrary, it is surprisingly dynamic and undergoes daily changes. These changes can cause DNA damage that, if not repaired, leads to mutations. You can read how DNA damage becomes a mutation and what effect it can have on the organism in Chapter 5 - Mutations - how they arise and what to do with them.

The information stored in the DNA (genotype of an individual) is important for the development of the organism. However, the resulting external manifestation of the genotype (phenotype), i.e., what an individual looks like, what characteristics it has and how it behaves, is also dependent, to some extent, on the external environment. Research in this area already indicates that the expression of many genes can be influenced by external factors. The scientific field of epigenetics deals with this kind of research. The basic principles of epigenetics, as well as issues of the influence of the environment on the activity of genes in a multicellular organism, are covered in Chapter 6 - How the environment can affect our genes.

A lot is already known about the fact that hereditary factors are responsible for the emergence of many human diseases. However, much less is known about the role of epigenetics in this context. Knowledge from this scientific field can help us understand many of our health problems. The ambition of Chapter 7 – From epigenetics to human diseases, is to present the influence of the environment and nutrition on our health, with a focus on individual phases of human life, heredity based on the experiences of previous generations, as well as heredity tied to a specific parent. The disease examples described in this section demonstrate why epigenetics as a modern, progressive scientific discipline deserves the attention of the general public.

Almost everyone knows stress as a widespread phenomenon of modern times. Most of us have already experienced a state where, under extreme mental or physical pressure, we feel a rush of adrenaline and for a moment we feel that we can handle everything. But with long-term exposure to stress, things change for the worse. Lack of sleep, irritability and mood swings are the order of the day for stressed people. All this negatively affects the physiological processes taking place in our body, which ultimately affects our health. But did you know that even individual cells can be under stress? And not only our human cells, but also the cells of plants, animals, even microorganisms. In Chapter 8 – Your cells are stressed too, we will therefore focus on cell stress. We will talk a little more about how and why it is produced, why it is necessary for cells, and what effect it can have on them if there is too much of it.

One of the most important topics of modern biomedicine, related to the content of several previous chapters, is cancer. Cancer has been linked to humanity since time immemorial, and along with it, people's efforts to understand its principles and discover a method of treatment. In Chapter 9 - When cells go crazy, we explain how and why a healthy cell becomes a tumour cell, what types of tumours we know and what therapy options are available for them.

Since various genetically determined diseases - among them some types of cancer - are currently among the most widespread health problems in the developed world, we focus on a promising technology that aims to replace or repair faulty genes and thereby eliminate the cause of the disease in Chapter 10 - Gene Therapy. In other words, how can gene therapy allow the treatment of so-called incurable diseases. In addition to cancer, these are mainly cardiovascular and neurodegenerative diseases or diseases of the musculoskeletal system. But why are we learning about gene therapy only now? Why is it not a regular part of our lives? The reason is that this promising technology also hides several potential pitfalls, which you can learn more about in this chapter.

Plants, like all organisms, constantly produce essential substances without which they would not survive (including sugars, fats and proteins). In addition, plants are able to produce compounds known as secondary metabolites, or phytocompounds, which although not essential for their survival, play a number of important roles, including signalling, protection against pathogens or attracting pollinators. However, these phytocompounds may not only be beneficial for plants, but also have meaning for humans. Many currently commercially available drugs originate from these plant components and, thanks to their wide spectrum of biochemical properties, find application in several branches of medicine and pharmacy. In Chapter 11 - Plants as an inspiration in biomedicine, we will imagine why and how such substances are formed in plants and explain with examples how we can use them to our advantage.

All living organisms, including humans, are in an environment in which they are surrounded by a wide range of substances. Some of them, whether natural or man-made, can have an adverse effect on us. In Chapter 12 - When the environment changes our hormones, we will look at substances that can affect the endocrine system and its proper functioning. Since hormones, as products of the endocrine system, regulate a number of processes in our body, their incorrect secretion or altered properties can lead to an undesirable physiological response of the body. How substances with excessive occurrence in the environment can change processes regulated by hormones will be explained using the example of bisphenol A, which is used for the production of plastics and is currently considered a virtually ubiquitous environmental pollutant.

Decent behaviour, ethics, or etiquette are a long-term part of civilized humanity. In fact, the term "behaviour" does not apply only to humans, but also to other living organisms, including the smallest ones - microorganisms. The genetic nature of this phenomenon is investigated by the genetics of behaviour (behavioural genetics). In Chapter 13 - Etiquette in our genes, you'll learn why it's good to study insect behaviour and whether our intelligence, sexuality, and behaviour are rooted in our genes or just a result of our upbringing.

Many years have passed since Charles Darwin published his work "On the Origin of Species by Means of Natural Selection" in 1859, and during that time the theory of evolution has become a relevant scientific theory. However, not everyone knows what principles this theory is based on, what is the real driving force of evolution, or how natural selection works. We will answer these and other questions in Chapter 14 - The principle of evolution.

Knowledge from the field of genetics has found its application not only in science and medicine, but its use covers various areas of life. Identification of persons based on DNA analysis is the essence of forensic genetics, is addressed in Chapter 15 - DNA as evidence. In this chapter, you will be able to take a look at the work of forensic scientists and criminologists who, based on knowledge of the DNA sequence, can recognize unknown persons and obtain incriminating evidence to identify the perpetrators of crimes.

Almost everyone who is involved in sports would like to win a race at least once. But how to achieve this goal? Is it enough to train persistently, or does success also depend on our genes? So far, approximately 200 genetic markers that influence sports performance have been identified. Some represent an advantage for endurance sports, others for speed or power. In Chapter 16 – Genetics in sport, we will talk about the variants of these genes and whether we need genetic testing when choosing a sports discipline.

Genetically modified organisms, which are covered in Chapter 17, are already around us today in the form of food, producers of drugs and hormones or in a scientific research environment. It is likely that in the future human society we will encounter this type of organism more and more often, so it is important for all of us to have at least a basic awareness of what a "genetically modified organism" actually is, what properties it has and what it is used for.

Vaccines cause autism! All people with blond hair will disappear within 200 years! Humans evolve from monkeys! There is a lot of information on the Internet that appears to be fact, but upon closer examination, it turns out to be untrue. Such false information does not bypass genetics either. Chapter 18 - The Most Common Hoaxes in Genetics: Myths and Facts, provides an overview of such false information and attempts to explain why it is incorrect or nonsensical. Knowing some characteristic features of false information can be important not only for processing knowledge from the field of biology, but also in everyday life.

Genetics as a science, but also as the basis of various modern technologies, is often depicted in pop culture - in films, books or computer games. In some cases, science fiction is elaborate and based firmly on scientific knowledge. In others, the term "genetics" is used as a magic word to justify the most incredible characteristics of the main characters. But how are we to distinguish between what makes sense on the TV screen, for example, and what is just a cheap stereotype? In Chapter 19 - Genetics in science-fiction and pop culture, we will use specific examples to show different ways of presenting genetics, thanks to which you too will learn to distinguish interesting fiction from superficial cliches.

To finish, chapter 20 takes a look at some key model organisms used to better understand the basis of genetics and biology more widely. You might be surprised at how diverse the organsims used are!

Want to read more?

Gregor Mendel: His life and legacy by Daniel J Fairbanks

Gregor Johann Mendel: From peasant to priest, pedagogue, and prelate (2022) D.L. Hartl. PNAS 119(30)

The gene: An intimate history by Siddhartha Mukerjee

Principles of genetics: 7th edition, a textbook by D.P. Snustad and M.J. Simmons

Chapter 2: How does a scientist work?

There are many different ideas, myths and assumptions about what the work of scientists looks like. These ideas are often based on a relatively monotonous depiction of science work in literature and pop culture. The consequence of this stereotypical portrayal is the common misconception of general public about what a scientist looks like (they wear a white coat, are dishevelled, distracted...) and what is the purpose of their work.

The social significance of science in its beginnings - solving common problems

Most people associate the term "science" primarily with later historical periods, in which humanity possesses a broad knowledge of basic physical laws, can construct sophisticated measuring devices, and systematically develops individual disciplines of natural and social sciences. Today, scientific research constantly expands the boundaries of knowledge and employs new ideas in various areas of human activity, including the creation of previously unknown objects that improve the quality of our everyday life. However, the beginnings of scientific research lie in a completely different time and occur for a completely different reason. In ancient Egypt and Mesopotamia, astronomical observations began during the fifth millennium BC – at a time when written language already existed, but a reliable and stable numeric system had not yet been established. In those times, other scientific disciplines, especially mathematics and medicine, started to attract public attention. The reason for their emergence was quite simple – people needed them for their daily activities. Astronomy made it possible to effectively track time and create a functional calendar, mathematics and geometry became the basis for construction and architecture, medicine was needed to provide treatment of diseases and injuries. Indian and Chinese science played similar role in oriental cultures. In India, early forms of surgery were developed (e.g., surgery of a torn auricle – the outer ear), measuring devices and construction methods were improved (e.g., the production of bricks with an optimal edge ratio of 4 : 2 : 1), and there were significant advances in mathematics (gradually the numeric system used throughout the world emerged, and around the year 628 the Indian scholar Brahmagupta introduced the use of the number "0"). In the same period, social sciences – linguistics (e.g., the formulation of the rules of Sanskrit – the written language in ancient India) and political science (e.g., Arthaśāstra – a book concerning the relationship between individual parts of the state) were established. In ancient China, astronomers were able to predict solar and lunar eclipses, in the year 132, the first seismometer – a device that detects earthquakes – was built, and Chinese scientists brought many useful inventions to the world, including matches, wheelbarrows and the crossbow.

The social significance of science in its beginnings - understanding the world around us

Ancient Greece was a key culture for the development of science and the scientific thinking. Greek philosophers, representing various schools of thought (e.g., Athenian school, Stoic school, Epicureans, Sceptics, Sophists), brought forth new ideas, setting the ground for all kinds of natural and social sciences. The philosophers also devoted themselves to correct thinking, a critical view of the world and the art of discussion. In the 6^th Century BC, the famous philosopher Thales from Miletus (generally known for the so-called Circle of Thales) claimed that earthquakes are not caused by the anger of the god Poseidon, but they are due to the strong impact of sea waves on the coasts. While we now know that this explanation is obviously wrong, however, it did open the way to investigate natural explanations for various natural phenomena. In addition, Thales calculated that the calendar year lasts 365 days and divided it into 4 periods that we still know today – spring, summer, autumn and winter. Among other scientists and philosophers, Hippocrates was important in the field of medicine (he described many diseases and introduced the Hippocratic Oath for doctors), while Euclid, Archimedes and Pythagoras had prominent positions among mathematicians. At that time, the astronomer Aristarchus was the first to describe the heliocentric model of the solar system (with the Sun in the centre, instead of Earth), while Socrates, Plato and Aristotle were among the most influential thinkers. In addition to practical problems, their focus was mainly on key concepts of mathematics, physics, and other scientific disciplines, which enabled them to explain several natural phenomena that had been previously attributed to gods. In particular, Aristotle's teaching served as the inspiration and source of knowledge even several centuries later, during the Renaissance period.

The social significance of science in its beginnings - the proper way of thinking

One of the most significant contributions of ancient philosophy to the work of modern scientists are the foundations of scientific thinking and properly conducted discussion. In this context, the key figure was Socrates, who used the so-called Socratic method. This is a method of conversation in which the students first try to define a general concept, and then the teacher asks them questions, with which they try to manoeuvre the students into a situation where their answers would contradict each other. If the teacher succeeds, the definition was not chosen correctly. If all the students' answers are consistent, the definition can be considered correct. In later years, other important figures of European science built on the scientific thinking of ancient philosophers – Roger Bacon (13^th Century), William of Occam (14th century), Galileo Galilei (16th and 17th centuries), Pierre Fermat (17^th Century), Gottfried Leibniz (17^th Century) and Isaac Newton (17^th Century). By analysing the thought and experimental procedures of individual scientists, three basic ways of thinking were described:

Inductive reasoning proposes generally valid conclusions based on several empirical observations of specific situations. This means that if we observed swans swimming on a lake for several days in a row, and each of the observed swans was white, we can come to the general conclusion that all swans are white (if we did not know this before). In this way, we can create hypotheses about possible general properties of the things or situations we observe.
Deductive reasoning follows a logical chain of evidence or arguments and identifies the true conclusion by eliminating all incorrect options. In this case, we could say that if there are two white birds swimming on the lake, and at the same time we know for sure that the only white birds that swim on lakes are swans, we can say with certainty that the birds on our lake are swans (if we didn't know that before). This type of reasoning is best illustrated by film detectives, who always combine and evaluate individual evidence, exclude all innocents from the circle of suspects and convict the culprit.
Abductive reasoning is based on a limited amount of information and comes up with a probable conclusion. Therefore, if we know that swans are white birds that swim on lakes, and at the same time we see a white bird on a lake, it is likely that it could be a swan (if we didn't know it before). This type of reasoning can be illustrated by virtually any situation where we say "90% of the time it's true".

Scientific method

A combination of knowledge about the correct procedures in experimental sciences (both natural and social) and scientific reasoning ultimately set ground for the emergence of a generally valid description of scientist's work – the scientific method. It is the employment of this method that unites all scientists, and on its basis, experts in individual fields design their experiments and work procedures. If we look closely at the scientific method, we can notice that it is a cycle of six actions – observing the given phenomenon, obtaining enough reliable information, formulating a hypothesis, experimental testing, analysing, and evaluating the test results, and formulating conclusions. The conclusions can be later subjected to criticism and added to the list of usable information to enrich the available knowledge (Figure 2.1).

Figure 2.1 Green arrows represent actions based mainly on inductive reasoning, red and blue arrows symbolize deductive and abductive reasoning.

Importantly, different types of reasoning are usually employed in different phases of scientific work. Inductive thinking allows scientists to comprehend the large collections of data, identify generally applicable rules and formulate a hypothesis, which is central to any scientific project. Deductive and abductive reasoning then allows researchers to approach their own hypotheses critically, test them and refute them, if necessary. The more rigorous the experimental tests are and the more intensively the scientist tries to disprove their own hypothesis, the more credible the hypothesis becomes if it fails to be disproved.

Experimental design

To test a hypothesis in the right way, scientists must precisely design experiments that will bring them the necessary information. Experimental design is therefore a key part of scientific work, especially if the scientist is studying a complicated issue where it is necessary to approach it carefully. Even the experiment that seems to test a given hypothesis perfectly might still be prone to a certain type of unexpected error or produces misleading results due to an incorrect design. To effectively avoid such faulty experimental design, scientists generally follow these rules when planning experiments:

The experiment should be unambiguous and controlled. As a rule of thumb, if we are investigating the effect of certain factor (e.g., the ability of an antibiotic to kill specific type of bacteria), it is important to compare the experimental setup in which this factor is present with the experiment in which it is not present. We call this second type of experiment, which differs from the main test in only one factor, a control experiment. Since both experiments differ only in this respect, we can attribute the different results to the effect of this particular factor (in case of antibiotics, we would cultivate two groups of bacterial cells in the same environment and at the same temperature, but we would add an antibiotic to only one group). If our experiment wasn’t unambiguous, we would not be able to evaluate its outcome with certainty (if we treated bacterial cells with two antibiotics at the same time, we would not know whether the first antibiotic, the second antibiotic, or the combination of both killed them). If we didn't have a control in the experiment, we wouldn't be able to tell which factor caused the observed result (if we added an antibiotic to the cells but didn't have control cells, we wouldn't know whether the bacteria were not previously infected with a virus that killed them regardless of the presence of antibiotic). Since a properly designed control experiment can be sometimes hard to set up, in complex experimental designs we can encounter a whole series of control experiments that cover all possible uncertainties.
Test subjects should be randomly distributed. The random distribution of tested people, laboratory animals or cells that we use in experiments is important because if we do not select our test groups carefully, we may end up with unwanted significant differences between them (e.g., in one group there is larger proportion of older participants). In such case, the observed results could possibly be due to differences in age, rather than the effect of the tested substance. The more complex the studied phenomenon is, the more properties (often unpredictable) of the test subjects might influence the results of the experiment.
The experiment should be reproducible. Another guiding principle of experimental design is that if we have truly identified a factor that is responsible for a particular experimental outcome, then that factor will be still effective if our experiment is replicated by other scientists in other laboratories. If it is not, that would indicate that not only the studied factor (e.g., antibiotic) but also another unknown circumstance, present only in our laboratory, is responsible for the observed results. For this reason, it is important to successfully repeat every significant experiment at least in one's own laboratory (ideally it should be confirmed by an independent team of scientists elsewhere).

Scientific method in psychology

Even though the working tools of scientists active in the social sciences are different from the tools of natural scientists, the essence of their work – use of the scientific method to learn about the world around us – is identical. As an example of the scientific procedure in the field of psychology, we can look at the so-called "Bobo" doll experiment, which was carried out in 1961 by the team of psychologist Albert Bandura. This experiment tested the hypothesis that children learn how to behave mainly by observing and imitating adults. During the experiment, the tested children were divided into three groups. The first group was seated in a room where the organisers played them a video in which an adult was behaving aggressively towards a "Bobo" doll (a large inflatable doll that is easily hit, kicked, etc.). In the second group, children were shown a video in which an adult behaved neutrally or friendly towards the doll. The third group did not watch any video. The children were then moved to a room with a doll and the scientists recorded their behaviour. It turned out that children who saw aggressive behaviour on video were much more likely to behave aggressively towards the doll in comparison to other children. This difference was even more pronounced if the person in the video was of the same gender as the child (more boys were aggressive if the person on the video was male than if it was a female and vice versa). By comparing the tested children (those who were shown the videos) with the control group (without the video), it was subsequently possible to formulate conclusions and propose further experiments.

A YouTube clip showing this experiment can be found here.

Scientific method in sociology

In sociology, at the end of the 1960s, the so-called "bystander effect" was intensively studied. The central idea of this phenomenon is that the number of participants in a crisis situation affect how quickly (or if at all) any of them will react and contribute to its resolution. To study it experimentally, John M. Darley and Bibb Latané designed an experiment in 1968, placing a volunteer in a room, from which he could communicate with a person located in another room (in fact, this person was a member of the experimenter's team). If necessary, the volunteer could also connect with the head of the experiment. During the interview, which was about an unrelated topic, the experimenter began to pretend that he was having a seizure and needed immediate medical attention. Subsequently, the leaders of the experiment observed the behaviour of the volunteer. The results showed that if a person in need is in contact with a single volunteer, this volunteer reacts almost immediately and tries to help (talks to him, contacts the head of the experiment, and asks him for help). If the circle of volunteers is wider (3-5 people are involved in the conversation), their reaction time increases significantly, as if everyone is hesitating and hope that someone else will react. By comparing the control setting (one volunteer) with the main experiment (multiple volunteers), it is possible to conclude that the presence of several participants in a crisis really complicates their ability to act. Afterwards, the psychological and sociological causes of this phenomenon were investigated in other experiments, but to a certain extent the bystander effect can be observed in everyday life as well (Figure 2.2).

Figure 2.2 The scientific method in sociology. A control experiment with two participants (left) and the main experiment with several participants (right) is depicted.

Scientific method in biology

As an example of the same scientific procedure in a completely different discipline – biology, we can investigate the experiment known as the "Fluctuation test". This experiment was carried out in 1943 by Max Delbrück and Salvador Luria, who were interested in the mechanism by which mutations occur (you can read more about mutations in chapter 5). The basic goal of the experiment was to find out whether mutations arise randomly or are part of a regulated response of the organism to changing environmental conditions. Delbrück and Luria cultured bacteria sensitive to infection with a virus (bacteriophage) and then plated equal numbers of bacteria on Petri dishes (culture vessels) containing the growth medium as well as the virus (Figure 2.3). After a few days, they observed how many bacterial colonies grew on the dishes (cells that grow on the dishes must have undergone a mutation that makes these cells resistant to virus).

Figure 2.3 The Fluctuation test. Purple circles indicate individual colonies of bacteria, cells in which the mutation has occurred (they are resistant to the virus) are marked in red. The result expected if mutations arise in a targeted manner is depicted on the left, the result expected if mutations arise randomly is shown on the right.

If mutations arise as an organism's response to changes in the environment (the presence of a virus), then approximately the same number of colonies should grow on each dish (all dishes are identical, as well as the type and number of cells). However, if the mutations were to occur randomly, it would be possible to observe different numbers of colonies on different plates. Since the results of the experiment showed that different numbers of colonies grew on different dishes, it was possible to conclude that mutations arise randomly. Dishes without virus, on which colonies grew uniformly, served as a control. The results of this simple experiment were crucial not only for geneticists and molecular biologists, but also helped to explain some questions of evolutionary biology. You can read more about the role that the fluctuation test played in the study of evolution in Chapter 14.

Application of the scientific method in everyday life

As we can see from the examples listed above, the scientific method and scientific way of thinking can be adapted and used in all types of scientific work. However, this way of thinking is also applicable in everyday life. It is necessary to choose the right sources of knowledge (e.g., media, politicians, advertisements), critically evaluate them (confront articles with each other and at the same time with your own knowledge) and, if necessary, conduct an experiment (e.g., if we are not sure, whether we will like the food, we can taste a little bit first). Like in the scientific research, a combination of inductive, deductive, and abductive reasoning can lead us to the right life decisions.

Did you know that......

...scientific and especially technological progress in recent years brings scientists not just the new knowledge and information, but also fundamental problems with the organization of their work? Modern devices, capable of analysing tens of thousands of samples of various substances in parallel, create such a large amount of data every day that it is practically impossible to process them manually. Thus, a large part of scientific work has been transferred to the world of mathematics and informatics, which enable machine processing of experiments and statistical evaluation of results. However, there is also a certain danger lurking in this world – relying on the statistical evaluation of a large number of measurements without additional experimental evaluation can increasingly produce incorrect results. Scientists are therefore trying to tighten the experimental and statistical criteria of their work, but finding the correct balance is complicated in many cases. Therefore, the question becoming more and more important in the scientific community today is: "How should we make sense of this sea of data and how do we find the really important pieces of information there?"

Want to read more?

To Explain the World: The Discovery of Modern Science, Weinberg, S., Harper (2015).

Understanding Philosophy of Science, Ladyman, J., Routledge (2001).

Chapter 3: Meet DNA, the bearer of genetic information

The discovery of the structure of DNA marked a major milestone in molecular biology and genetics. In 1953, James Watson and Francis Crick presented the first spatial model of DNA, explaining many of its unique properties. Nine years later, in 1962, together with Maurice Wilkins, they were awarded the Nobel Prize for this discovery. The experiments of Rosalind Franklin, who made X-ray diffraction images of DNA, also made a significant contribution, but her merits in the discovery of the structure of DNA were initially overlooked and received more attention only after her death. Also useful were the observations of Erwin Chargaff, who noticed that the representation of individual nitrogenous bases in DNA follow certain rules, which led to the discovery of the principle of base pairing. A new era of molecular genetics began from this moment.

What is DNA made of?

The basic structural unit of nucleic acids are nucleotides, which consist of a phosphoric acid residue, a specific sugar and nitrogenous bases (Figure 3.1). We distinguish between 2 types of nucleic acids, which differ at the nucleotide level. Deoxyribonucleic acid, called DNA, is usually a double-stranded molecule that contains the sugar deoxyribose, the nitrogenous bases adenine, thymine, cytosine, and guanine. RNA (ribonucleic acid) is usually a single-stranded molecule containing ribose. The arrangement of nitrogenous bases differs from DNA where thymine is replaced by uracil in RNA. Structurally, thymine and uracil are similar, but the production of uracil is less energy consuming. A relatively large number of RNA molecules are involved in various cellular processes, so it is advantageous to use less energy-demanding components. The presence of thymine in DNA ensures the necessary protection of the DNA molecules against damage that can occur in the cell nucleus, where most DNA molecules are located.

Figure 3.1 Structure of DNA. Phosphoric acid residues (orange) together with deoxyribose (gray) form the sugar-phosphate backbone of DNA. Chemical interactions occur between nitrogenous bases, which are based on the principle of base pairing. Two hydrogen bonds form between adenine and thymine (dashed lines), while cytosine and guanine are joined by three such bonds. The figure shows the orientation of individual DNA strands starting from their 5'-end towards the 3'-end. The designation of the ends follows from the chemical structure.

Individual nucleotides, placed one behind the other, are interconnected and thus form a DNA strand. The sequence (order) of nucleotides defines the genetic information, and is especially important. The connection of the two strands occurs through a hydrogen bond, which is formed between the nitrogenous bases of the nucleotides. The principle of base pairing is applied, according to which adenine pairs with thymine, while cytosine pairs with guanine (Figure 3.1). The complete genetic information of an individual is called the genome, which in humans consists of approximately 3.2 billion (3.2x10⁹) base pairs. Only about a quarter of the base pairs contain information for the creation of products (RNA or proteins), and we refer to such sections as genes - genes encoding different types of RNA and genes encoding polypeptides from which proteins are formed. The human genome contains about 20,000 genes whose products are proteins, which represents about 1 to 2 % of the genome, and about 24 % of the genome is made up of genes for RNA.

All genetic information is organized into chromosomes. Human germ cells contain 22 autosomal chromosomes and the X or Y sex chromosome. The chromosomes differ from each other in size, shape and number of genes they contain. When a new individual is created by fertilisation, the germ cells of the parents fuse and a zygote is formed, which contains 23 pairs of chromosomes originating from both parents, with 22 pairs representing autosomes and one pair of sex chromosomes. The set of all chromosomes in the nucleus of a cell is called a karyotype (Figure 3.2). The total length of all the DNA located in the nucleus of one human cell exceeds one meter in length, although the largest of the chromosomes, which is chromosome 1, is only 10 µm long when viewed under a microscope. Thus, it is clear that the long DNA molecule undergoes several levels of packaging. The first level of packaging is represented by nucleosomes, in which DNA wraps around histone protein complexes. Subsequently, there is the formation of a chromatin fibre, which wraps around the protein scaffold of the chromosomes. In this way, DNA molecules shorten their length up to ten thousand times and form relatively small formations (spiralised chromosomes) that can be observed in the cell during division.

Figure 3.2 Human karyotype. Humans have 22 pairs of autosomes and one pair of sex chromosomes, X and Y. This karyotype is of an individual with Klinefelter syndrome, as they have an XXY genotype. Typically a male would be XY and a female XX.

DNA can make exact copies of itself

Cell division is the process by which genetic information is passed between generations. The sequence of the individual phases leading to cell division is referred to as the cell cycle. However, the actual division and creation of daughter cells is preceded by the activity of various preparatory mechanisms, which include DNA duplication, i.e. DNA replication. This process takes place during the S-phase of the cell cycle and is governed by the semi-conservative model of replication, which was introduced by the discoverers of the DNA structure, Watson and Crick, and later experimentally confirmed by Matthew Meselson and Franklin Stahl. The Meselson and Stahl model states that each newly formed DNA molecule contains one strand originating from the parent molecule, which serves as a template for the synthesis of the second strand (Figure 3.3). Synthesis of the new strand takes place on the basis of the principle of base pairing, which means that if the sequence of the template is known this can be used to derive the sequence of the new, just emerging strand.

Figure 3.3 The cell cycle and a semiconservative model of DNA replication. In the S-phase of the interphase of cell division, genetic information is duplicated, which is then evenly redistributed to the daughter cells. DNA replication takes place on the basis of a semiconservative model, according to which each DNA molecule consists of one parent strand, which provides a template for the synthesis of a new DNA strand.

Several different enzymes and proteins are involved in DNA replication. DNA helicase unravels the double stranded molecule into two separate strands, which can then be replicated. When they are unravelled, tension is created, which is reduced by the DNA-topoisomerase enzyme, which also prevents the separated strands from knotting. After successful separation, SSB-proteins (Single Strand Binding proteins) bind to prevent the stands from rejoining and reforming a double strand. DNA polymerases play the most important role in DNA replication. Polymerases are a group of enzymes with similar properties responsible for the synthesis of a new DNA strand (Figure 3.4). Some polymerases also show exonuclease activity, which means that after creating a short section of newly synthesied DNA, the enzyme can check whether the correct nucleotides have been included and possibly remove and repair their mistake. In this way, the cells eliminate a large number of potential mutations that could have harmful consequences. The synthesis of a new strand takes place in the direction from the 5'-end of DNA to its 3'-end (Figure 3.4). In a simplified way, it can be imagined that DNA polymerase always extends the 3'-end of the DNA strand. Cells cannot synthesise a new strand de novo, that is, from nothing. DNA polymerase always requires the presence of a template that serves as a model for the formation of a strand. At the same time, deoxynucleoside triphosphates, or dNTPs for short, are needed, which represent the basic building blocks from which DNA polymerases can assemble a new DNA strand. For their activity, DNA polymerases also need magnesium ions (Mg²⁺) and a short RNA-primer that borders the replicated section and at the same time provides the enzyme with a free 3'-OH group (at the 3'-end of DNA) to which nucleotides are added during DNA synthesis.

Replication always begins at an area known as the replication origin. While prokaryotic cells, e.g., bacteria have one origin of replication present in their circular DNA molecules, large linear chromosomes that are part of eukaryotic cells have many more origins of replication. At the origin of replication, the DNA helix is locally untangled, and a replication bubble is formed. The place where the double strand of DNA has split into two separate strands is referred to as a replication fork. Replication forks move along the DNA molecule and ensure its replication (Figure 3.4).

Figure 3.4 Initiation of replication and synthesis of the leading strand of DNA. DNA replication begins with the unfolding of the double strand at specific sites termed the origin of replication. This creates a replication bubble, which, in addition to moving replication forks, also includes enzymes and proteins that bind to DNA (topoisomerase and SSB proteins). The enzyme helicase unravels the double strand of DNA and the synthesis of a new strand is catalysed by DNA polymerase. The leading strand of DNA is replicated continuously in the presence of RNA primers that are recognized by DNA polymerase. The gradual incorporation of free nucleotides (dNTPs) leads to the synthesis of the leading strand, which is a copy of the original chain.

Replication occurs in three basic phases:

Initiation,
Elongation,
Termination.

After the double strand is unravelled, each strand replicates independently and at different rates. We distinguish the continuous synthesis of the leading strand and the synthesis of the lagging strand, which takes place in smaller sections and is slower.

The leading strand of DNA is synthesized continuously

The first step in the replication of the leading strand is the creation of a replication fork. Subsequently, an RNA-primer is attached to the strand, which provides a free 3'-end in the direction of DNA replication. This allows the DNA polymerase to recognize the site to be replicated. The whole process thus gradually passes from the initiation stage onto the phase called elongation. During elongation, a new strand of DNA is synthesized based on the template. The entire process is ensured by the enzyme DNA polymerase, which faithfully replicates the original mother strand according to the principle of base pairing. The last step is termination of replication. In this phase, the new DNA strand is complete and the DNA polymerase leaves the molecule (Figure 3.4). Since the synthesis of the strand is continuous and occurs relatively quickly, it is referred to as leading strand synthesis.

Discontinuous synthesis of the lagging strand

The synthesis of the second, oppositely oriented strand differs from the replication described so far. While the leading strand replicates continuously, the opposite strand is generated discontinuously through Okazaki fragments and is therefore called the lagging strand. Okazaki fragments were named after their discoverers, husband and wife team Reiji and Tsuneko Okazaki, who described the formation of the fragments in 1968 during their experiments focused on DNA replication .

After the formation of the replication fork, the lagging strand not provide a free 3'-end in the direction of DNA replication (because it is turned in the opposite direction). However, cells can deal with this problem with the presence of RNA primers. Several RNA primers are attached to the lagging strand, creating sections with a free 5'-end which is recognizable by DNA polymerase. After the synthesis of short DNA sequences, a hybrid DNA strand is formed, where the DNA is interrupted by several RNA (from the RNA primers). However, the presence of the RNA primers is not desirable, and so they must be removed. This is done by another enzyme, RNase, which can cleave RNA sequences. In this way, larger gaps will again appear on the synthesised strand, which can be filled by DNA polymerase. The result is that the emerging strand is formed only from DNA. Since the synthesis did not proceed smoothly, but in fragments, it is necessary that the individual sections of DNA are joined into a single, uninterrupted DNA strand. The joining of DNA sections across these small gaps is achived by DNA ligase, which terminates replication and forms a complete strand (Figure 3.5).

Figure 3.5 Lagging strand synthesis. Synthesis of the lagging strand takes place via Okazaki fragments. A. Several RNA primers are attached to the DNA strand. B. Sections between RNA primers are synthesized by DNA polymerase. C. A hybrid molecule formed by RNA and DNA sequences is formed. D. RNA primers are removed from the molecule by RNase. E. Spaces between short sections of DNA are synthesized by DNA polymerase. F. The integrity of the strand is ensured by the DNA ligase enzyme. G. Complete synthesized lagging strand of DNA.

The chromosome will get shorter by 50-250bp each replication cycle, which can be detrimental. To ensure this does not happen, important and specific structures are present at the ends of linear chromosomes, which are called telomeres. Their replication is ensured by the enzyme telomerase, whose activity is influenced by several factors. Telomerase is highly active in healthy human cells during embryonic development, when intensive cell division occurs, and in sex or stem cells. In other cells, telomerase is inactive and so the telomeres are not restored, which is manifested by their gradual shortening and the associated aging of the cells. Cells that have undergone repeated replication and division may harbour accumulated mutations with a potentially deleterious effect. Therefore, it is necessary for such cells to be recognised and safely eliminated. On the contrary, one of the characteristics of tumour cells is the presence of active telomerase, which ensures the continuous replication of telomeres. Thus, they do not shorten and the cells do not age, therefore cancer cells become "immortal" (you can read more about cancer in chapter 9 - When cells go crazy).

How are genes expressed?

In order for the genetic information encoded in DNA molecules to manifest itself even at the level of the phenotype, it is necessary that the genes are expressed. The term "gene" was introduced by Wilhelm Johannsen in 1909, when he defined it as a unit of genetic information that determines a specific phenotypic trait. This definition, although fairly accurate, does not describe the properties of a gene at the molecular level. The acquisition of new knowledge is gradually changing the formal definition of a gene. In terms of the expression of genetic information, the central dogma of molecular biology has long been valid, according to which genetic information is usually copied from 1) DNA to DNA during intergenerational transmission and 2) from DNA to proteins during gene expression. To take the genetic information from DNA to proteins, the transfer requires two steps. The first step is transcription, where the information from DNA is transcribed into an RNA molecule. From this, the information is subsequently translated into the sequence of amino acids forming the polypeptide chain. Such a process is called translation and leads to the formation of proteins (Figure 3.6).

Figure 3.6 The central dogma of molecular biology. The flow of genetic information is from DNA to RNA (transcription) and from RNA to proteins (translation). In specific cases, the transfer of information is also possible in the opposite direction (from RNA to DNA) through the process of reverse transcription.

Transcription of genetic information from DNA to RNA

While bacteria have DNA molecules stored freely in the cell, eukaryotes store genetic information in the cell nucleus. Within the nucleus, DNA is thus separated from the rest of the intracellular space by a nuclear membrane, which also ensures its necessary protection. For gene expression to occur, the genetic information must be transferred from the nucleus to the cytoplasm, where protein synthesis takes place. The first step of transfer is provided by transcription, where the individual genes from DNA are transcribed into messenger RNA, or mRNA for short. The mRNA molecules are small enough to pass through the pores of the nuclear membrane and carry the information into the cytoplasm.

Like replication, transcription takes place in three basic phases, which are initiation, elongation and termination. During initiation, the RNA polymerase enzyme recognizes the promoter region that is located just before the start of the gene. From this site, RNA polymerase moves in both directions until it recognizes specific regions characterizing the presence of the coding region. Then the transcription goes into the second phase; elongation. The enzyme RNA polymerase can unravel the double strand of DNA, which, similar to replication, creates a so-called bubble. In this case, we are talking about a transcription bubble, where the separated template strand serves as a blueprint for the transcription of information into the mRNA molecule. After transcription of the entire section of DNA, transcription is terminated. The RNA polymerase is separated from the DNA molecule and the released mRNA strand is referred to as the primary transcript.

The primary transcript, or otherwise called pre-mRNA, must be edited before leaving the nucleus. A “cap“ is added to the 5'-end of the pre-mRNA, which is most often formed by a 7-methylguanosine molecule. Also, a special polymerase adds a large number of adenines to the 3'-end. Such an end is then referred to as the poly(A)-tail. Both the cap and the tail protect the mRNA from degradation.

In addition, the coding sequences of many eukaryotic genes are interrupted by sequences called introns. These are non-coding regions of DNA, which means that they do not participate in the creation of the resulting protein. Editing of the primary transcript also includes the cutting out of possible introns (splicing) by specific enzymes. Mature mRNA is thus formed only by coding sections, which are called exons (Figure 3.7). When the primary transcript of eukaryotic cells has undergone all three types of editing, including capping, tailing, and excision of introns, the mRNA can leave the nucleus and undergo translation.

Figure 3.7 Editing of the primary transcript of eukaryotic cells. A. A cap and tail are added to the ends of the pre-mRNA (blue). B. Excision of introns occurs within the transcript. The resulting mRNA after editing contains only exons, a cap and a tail.

It is not always the case that one pre-mRNA produces the same protein each time. In eukaryotic cells, a process called alternative splicing takes place. Its essence is the creation of different mRNAs from one primary transcript. If the gene consists of several exons, one of the exons can be cut out at the same time during the splicing of introns, resulting in different combinations of used and unused exons. Such diverse mRNAs are then translated (in some cases) into multiple, mutually different proteins (Figure 3.8). The possibility of such alternative splicing of primary transcripts saves the amount of genetic information stored in the nucleus of eukaryotic cells, since instead of genes or their parts being duplicated, one gene can encode different (albeit partially similar) proteins.

An example of alternative splicing is the sex determination of the fly Drosophila melanogaster. The main regulator of this process is the Sex-lethal (Sxl) gene, which is located on the sex chromosome X. In both sexes, one universal primary transcript is formed, which subsequently undergoes alternative splicing. It is the combination of exons of the resulting mRNA and the length of the resulting protein that determine the sex of D. melanogaster. Specifically, exon 3 contains a termination codon (a stop codon) that signals the end of translation. If exon 3 is included in the resulting mRNA, protein synthesis is terminated prematurely at this location resulting in a short product which has no regulatory function. This signals the cell to activate a set of genes that determine that the embryo will develop as a male. Conversely, if exon 3 is excised together with the introns, the resulting mRNA is translated at full length. A regulatory protein is formed that activates the genes responsible for the development of the female sex of D. melanogaster. In this way, alternative splicing can significantly influence the phenotype of the developing individual.

Figure 3.8 Schematic of alternative splicing of the primary transcript. The removal of introns from the pre-mRNA, which may simultaneously involve the excision of some exons, produces different combinations of coding sequences. These subsequently lead to the formation of different proteins.

It is also worth mentioning the fact that eukaryotic organisms (with the exception of the simplest ones) do not differ so much in the number of genes, but they differ in their complexity, and alternative splicing is one of the mechanisms involved in increasing their complexity.

Translation of genetic information from RNA molecules into the sequence of amino acids

After transcription and the necessary modification, the mRNA molecules pass from the nucleus to the cytoplasm, where the second stage of the expression of genetic information, which is translation, takes place. The result of this process are polypeptide chains forming proteins. Living cells expend more energy on protein synthesis than on anything else.

Besides mRNA, ribosomes and transfer RNA (tRNA) also play an important role in the translation process. tRNA molecules have a specific hairpin structure, which enables them to function as amino acid carriers. Based on the anticodon, i.e. the sequence found in their structure and which is complementary to the codon on the mRNA strand, they act as adapters between the mRNA molecules and the amino acids that make up the resulting proteins. The entire process of translation takes place in ribosomes, which can be stored separately in the cytoplasm of cells, or as part of the rough endoplasmic reticulum. They are composed of a small and a large subunit, and functional ribosomes are created only by joining both subunits together.

An interesting feature of ribosomes is that they have sufficient capacity to synthesize any protein that is encoded in mRNA, even if it comes from cells of another species. This is because the process of translation follows the rules of the genetic code, which is universal. The sequence of nucleotides in the mRNA molecule are read by the translation machinery as codons, i.e. three nucleotides, which determine the inclusion of specific amino acids in the emerging protein. Each amino acid is represented by one or more codons in this code. Out of all 64 possible codons, up to 61 encode different amino acids (20 different in total). Two of them simultaneously function as initiation signals (so-called start codons – AUG, exceptionally also GUG) and three signal the end of translation, which are called termination signals (stop codons – UAA, UAG and UGA) (Figure 3.9). With the help of anticodons present in the tRNA structure, specific amino acids are arranged in the order encoded in the mRNA molecule during the translation process. The result is a chain of amino acids that is released and forms a functional protein.

Figure 3.9 The genetic code. Individual codons code for one of 20 amino acids: phenylalanine (Phe), leucine (Leu), isoleucine (Ile), methionine (Met), valine (Val), serine (Ser), proline (Pro), threonine (Thr), alanine ( Ala), tyrosine (Tyr), histidine (His), glutamine (Glu), asparagine (Asn), lysine (Lys), aspartic acid (Asp), glutamic acid (Glu), cysteine (Cys), tryptophan (Trp), arginine (Arg), glycine (Gly).

Like the previous two mechanisms, the process of translation can also be divided into initiation, elongation and termination. Initiating tRNAs, which have a specific structure and always carry the amino acid methionine (in some cases the methionine is chemically modified), play an important role at the start of the process. At the same time, initating tRNAs participate in the assembly of the ribosome, which is why they follow a slightly different mechanism of interaction with its structurally significant sites (Figure 3.10).

Figure 3.10 Translation progress. A. The picture shows the assembly of the ribosome with the help of the initiator tRNA (blue). B. Subsequently, additional tRNA molecules with bound amino acids enter the ribosome through the A site, between which a peptide bond is formed in the P site. If the tRNA no longer contains a bound amino acid, it leaves the ribosome via the E site. C. Translation is terminated by a termination signal (black), which leads to the release of the polypeptide and disintegration of the ribosome.

Initiating tRNAs bind to the large subunit of the ribosome directly through its ‘P‘ site. Their subsequent binding to the mRNA molecule leads to the joining of both subunits and the formation of a functional ribosome. The translation then enters the elongation phase. Other tRNAs already enter the ribosome by default through the ‘A‘ site. Based on the codons present in the mRNA sequence, they bring with them the corresponding amino acids. A peptide bond is formed between adjacent amino acids in the ‘P‘ site of the ribosome. At the same time, this creates an "empty" tRNA that leaves the ribosome through the ‘E‘ site. There is a shift, which again makes room for the arrival of another tRNA molecule. In this way, translation takes place until the moment when the ribosome encounters a termination signal. Then the translation is terminated and the resulting polypeptide (protein) chain is released. At the same time, the ribosome breaks down into subunits, which can later be used again in the translation of another polypeptide (Figure 3.10).

What regulates gene expression?

Cells contain a large number of genes, not all of which are used all the time. From this point of view, and at the same time to save energy, the process of gene expression regulation is very important. However, not all genes can be safely turned off or on. The products of some genes are so necessary that their absence would be fatal for the cells. They are called housekeeping genes and are expressed under all conditions. Other genes can be regulated depending on whether their products are needed by the cell at a given time. We refer to the process of turning on gene expression as induction, and repression refers to when gene expression is turned off. By turning off the expression of genes, the organism saves energy, which it can use in the synthesis of other products. Various cascades of enzymatic reactions or signalling pathways are part of the regulation of gene expression. For example, prokaryotic cells of bacteria respond to the presence of nutrients by triggering the expression of genes that allow them to efficiently process these nutrients. Their specific feature is that by reacting to one stimulus, they can trigger the expression of all necessary genes at once. This is ensured by the formation of polycistronic transcripts, which are long mRNA transcripts encoding the entire set of necessary proteins.

In eukaryotic cells, gene activity is also influenced by chromatin structure. Regions of packed chromatin, called heterochromatin, generally contain inactive genes. A slight loosening of the structure makes genes accessible to regulatory factors that trigger their transcription (this unfolded form of chromatin is called euchromatin). Gene expression can thus be regulated by chromatin remodelling, which you will read more about in Chapter 7 - From epigenetics to human diseases.

In the human body, there is a large number of different types of cells that contain the same genetic information. It is the regulation of gene expression that ensures the differences between cells with a specialized function. This means, for example, that although the same DNA is found in the nucleus of a neuron and a muscle cell, the specific switching on and off of certain genes can differentiate them sufficiently and adapt them to a specific function and thus be either a neuron or a muscle cell. The regulation of the expression of genetic information also includes various signalling pathways that lead to activation, or suppression of the transcription of certain genes. Similarly to the case of bacteria, a certain impulse triggers the cascade reaction. The trigger can come from the surrounding environment, or it can be a molecule located directly in the cell. It is often various ions, hormones, enzymes or other signalling proteins that are recognized by receptors on the surface or inside the cell. Their capture triggers the transmission of a signal between several molecules, which leads to the activation of transcription factors. These proteins then trigger the expression of a wide variety of genes. Cell signalling plays an important role especially in multicellular organisms, as it allows specialised (and often distant from each other) cells to communicate with each other, which leads to a complex response of the organism to a certain stimulus.

Did you know that...

...DNA chromosomes can be analysed even before a person is born? Such a method is called prenatal diagnostics and is used to examine the proper development of the foetus. One of the common cytogenetic methods is the direct collection of amniotic fluid, which also includes released foetal cells. From them, laboratory diagnosticians can isolate entire sets of chromosomes, which are after staining analysed under a microscope. In addition to the structure of individual chromosomes, diagnosticians also monitor their total number. If they detect a deviation from the standard number of 46 chromosomes, it means the presence of one of the severe diseases. The most well-known type of such a disease is Down's syndrome, which is caused by an extra chromosome 21. The condition where there are three chromosomes in the cells instead of two is called trisomy. Currently, the analysis of freely circulating DNA is relatively widespread as it is a non-invasive method. Subsequently, the presence of the most common trisomy is analysed using molecular biological methods (quantitative PCR). The disadvantage of such analysis is that it does not reveal structural changes of chromosomes. In addition to monitoring the proper development of the foetus, cytogenetic analysis of chromosomes are used to determine the cause of frequent miscarriages and infertility, as well as to determine the prognosis of cancer diseases.

Want to read more?

Okazaki R et al. (1968). Mechanism of DNA chain growth. I. Possible discontinuity and unusual secondary structure of newly synthesized chains. Proc. Natl. Acad. Sci. USA. 59(2): 598-605.

Genetics: A Conceptual Approach, Pierce, B. A., Seventh Edition, W. H. Freeman and Company (2020).

Genetics: From Genes to Genomes, Hartwell, L.H., Goldberg, M.L., Fischer, J.A., Hood, L., Aquadro, C.F., Fifth Edition, McGraw-Hill Education (2018).

Chapter 4: How do you work with DNA?

Genetic information is stored in the molecule of DNA, the properties of which were described in the previous chapter (Chapter 3 - Meet DNA). In this section, we present some of the most used methods of DNA analysis, which are applied not only in basic research, but also in the diagnosis of diseases, genealogical studies, crime investigation, food control, and other fields.

If we want to study the DNA molecule, we must first obtain it from the cells. We call this process DNA isolation or purification. The first isolation of DNA was performed (albeit unintentionally at the time) as early as 1869, long before its spatial structure was discovered. Friedrich Miescher, a Swiss physician, took leukocytes (a type of immune cell) from bandages he used to dress his patients' wounds and wanted to focus on isolating various proteins. In his experiments, he was interested in a particular substance isolated from the cell nucleus that, unlike proteins, did not contain sulphur. This substance precipitated under acidic conditions, while it dissolved under alkaline conditions. Since it came from cell nuclei, he called it nuclein (the name nucleic acid was introduced later by his student Richard Altman).

Since DNA is located inside cells, the first step in DNA isolation is cell disruption or lysis. Cells have a cytoplasmic membrane on their surface, and some also have a stronger cell wall (e.g., plant cells, yeasts, bacteria). We need to break these surface structures to release the cell contents into solution. In principle, there are 3 approaches to breaking cells, and they are usually combined. We can break up the cells mechanically, e.g., by rubbing them in a rubbing bowl or shaking them with special glass or ceramic beads. Mechanical stress on the cell surface is also achieved by ultrasound and sudden temperature changes by freezing and thawing. The so-called digestion of the cell wall is made possible by enzymes isolated from various organisms, e.g., zymolyase isolated from bacteria cleaves the cell wall of some yeasts, lysozyme isolated from saliva or tears cleaves the cell wall of bacteria, and cellulase isolated from some fungi cleaves the cell wall of plant cells. In addition, there are chemical substances that destroy the integrity of the cell surface, and among them the most used are various detergents since lipids are the main component of the membrane. Most laboratories use sodium dodecyl sulphate (SDS) or Triton X-100 as detergent, but any detergent will suffice for DNA isolation outside of laboratory.

Nucleic acid is naturally surrounded by a variety of proteins, lipids, sugars (polysaccharides), and other substances that remain in contact with it even after cell lysis. Separation of DNA from these components is the next important step in DNA isolation and is referred to as DNA extraction. Efficient DNA extraction is possible thanks to the different chemical properties of each molecule surrounding the DNA. Detergents, as mentioned earlier, are used to break down the remnants of membranes, including the nuclear membrane and other lipid structures. Large amounts of proteins in the cell can be precipitated and separated with organic substances - for example, a mixture of phenol, chloroform, isoamylalcohol, or guanidine chloride. After mixing the sample with phenol and chloroform or with chloroform and isoamylalcohol, two layers are formed. The lower layer is organic, in which dissolved impurities remain, i.e., the unwanted components such as lipids and parts of proteins. A protein ring forms between the two layers, and above this is the upper, aqueous layer, which contains solubilized DNA, usually together with RNA. A major obstacle in isolating nucleic acids is the presence of nucleases - enzymes that degrade nucleic acids in cells. Most nucleases are localized in lysosomes, but when the cell is disturbed, they are released (lysosomes also disintegrate) and therefore pose a threat to the isolated DNA. Suppression of nuclease activity is usually achieved by addition of anionic detergents or EDTA (ethylenediaminetetraacetic acid), which absorbs divalent ions (mainly Mg²⁺) that serve as nuclease cofactors.

After removal of the aqueous phase, which also contains DNA, the DNA must be precipitated from the solution to successfully purify and isolate it. Pure ethanol or isopropanol is used for precipitation as the addition of alcohol changes the properties of the nucleic acid making it less soluble in water. Precipitation is also supported by the addition of monovalent ions (usually Na⁺), so sodium or ammonium acetate is added to the mixture. After centrifugation, the precipitated DNA settles, and the remaining alcohol must be thoroughly removed and the DNA dried. Since the precipitated DNA is very often isolated together with RNA, we dissolve the nucleic acids and incubate them with the enzyme RNase, which only cleaves RNA molecules. Then, the DNA must be repeatedly precipitated to get rid of the RNase and the excess solution. Ethanol or isopropanol at low temperature is used for this step. After centrifugation, we need to thoroughly remove the alcohol, because even the residual amount of ethanol could interfere with the subsequent reactions in which we want to use the DNA. Finally, we can dissolve the prepared DNA in water.

Because DNA isolation is often a necessary step for further analysis, many companies have developed commercial kits for their isolation to speed up and simplify the whole procedure and avoid working with chemicals that are hazardous to health, such as phenol and chloroform. However, the principle of such isolation is not much different from the one which has just been described.

In the polymerase chain reaction (PCR), a limited section of DNA is amplified

When analysing DNA, we are often not interested in the sequence of the entire molecule, but only in a specific part of it, for example, a gene under investigation (for an explanation of the term gene, see Chapter 2 - Getting to Know DNA). Within the DNA molecule, this section often occurs in only one copy, so we would need a huge amount of input DNA to obtain the desired section in sufficient quantity for various experiments. This problem was solved by a revolutionary method that is now an almost everyday part of DNA work - the polymerase chain reaction, or PCR for short. Behind its invention is the American scientist Kary Mullis, who began working on this method in 1983, but it was not published until 1985, when he and his colleagues succeeded in amplifying the human gene for beta-globin (the protein subunit of haemoglobin A). He was awarded the Nobel Prize in Chemistry in 1993 for the discovery of PCR.

As input components of the reaction, we need the template DNA to be amplified, free deoxynucleotide triphosphates (dNTPs), primers (short, single-stranded DNA segments, usually 20 nucleotides long that are used to start the synthesis), DNA polymerase, suitable buffer solution, and water. In the first step of the reaction, the DNA strands are separated from each other (denatured) by high temperatures (94-98 °C). In the second step, after lowering the temperature (50-60 °C), the primers are bound to the single strands of the template DNA according to the principle of base complementarity. The binding of primers is called annealing. After increasing the temperature to 68-72 °C, the third step is carried out by the action of DNA polymerase, the synthesis of a new DNA strand – polymerization, or extension. All these steps are repeated 20-40 times, resulting in amplification of desired DNA segment (Figure 4.1).

The optimal amount of DNA added to start the PCR is 1-50 ng. However, the PCR method is very sensitive, and it is possible to begin the reaction with an amount of about 15 pg (15 pg of DNA can be obtained from about 2 cells). Since the PCR method is basically an exponential reaction, if the input amount is one double-stranded DNA molecule, we can obtain 2 molecules after the first cycle, 4 after the next, 8 after the third, or 2ⁿDNA molecules after the n^th cycle under ideal conditions of reaction.

To perform PCR, we use a device called a thermocycler because it cyclically repeats the selected temperature program. The first prototype of such an automatic device was built in 1986 and became possible only when a thermostable DNA polymerase isolated from the high-temperature organism Thermus aquaticus was used for PCR purposes. The first automatic PCR cycler was launched in 1988, and in 1989 the thermostable Taq DNA polymerase was named Molecule of the Year by the journal Science.

Figure 4.1 Steps of polymerase chain reaction (PCR).

Gel electrophoresis enables visualization of DNA.

However, after performing the PCR reaction, we see nothing in the test tube that we take out of the instrument. The DNA is dissolved in the solution, and we need to find out if the desired section has been amplified and if it is a section with the correct length. To visualize and separate DNA molecules according to their size or molecular weight, we mostly use gel electrophoresis. We can use it to detect the presence of any DNA or RNA, it does not have to be just a PCR result. The main component of gel electrophoresis is a gel made from a polymer, usually agarose, which forms a network of pores after boiling and solidification. The PCR sample is mixed with reagents that allow the DNA to be visualized after analysis and then the DNA sample is applied to one end of the solidified gel using a pipette. As DNA is negatively charged it will therefore move towards the positive electrode (anode) in the electric field. Consequently, after filling the electrophoresis apparatus with a current-conducting solution, the movement of the DNA molecules occurs in the direction to the positive pole, with shorter molecules (of lower molecular weight) moving faster through the pores of the agarose gel and longer fragments moving more slowly (Figure 4.2). If we turn off the apparatus after a certain time, we can track where the DNA has gone. We can see this thanks to the fluorescent dye incorporated into the double-stranded DNA, and when the gel is illuminated with light of the appropriate wavelength, we can see bands corresponding to the individual DNA fragments. To determine the actual length of these fragments, we use a DNA molecular weight marker, which is a mixture of fragments of known length. By comparing the position of band in the lane with the bands in the DNA marker (as with a ruler), we can then determine the length of the fragments we analysed.

Figure 4.2 Agarose gel electrophoresis. The samples are loaded into wells of the agarose gel. An electric current is applied so that the DNA which has a negative charge, will move towards the positive electrode. After a period of time, the bands on the gel can be visualised by the addition of UV light.

RT-PCR allows determination of the input amount of nucleic acid in the reaction

In connection with the test for COVID-19, there has been a lot of talk in public media about PCR testing or RT-PCR testing. The abbreviation RT stands for real-time, so RT-PCR means real-time PCR reaction. PCR is an exponential reaction, so its progress is expressed by an exponential function (Figure 4.3). The number of products increases slowly at first, followed by a sharp increase. Since the reaction has its limits, after a certain time the individual components, especially free nucleotides, are consumed until finally the number of copies of the product no longer increases (plateau phase). Depending on the reaction setup, this usually occurs between the 30^th and 40^th PCR cycle. This is usually the phase in which we analyse the resulting product of standard PCR, for example by agarose gel electrophoresis.

Figure 4.3 Exponential curve illustrating the course of the PCR.

In RT-PCR analysis, fluorescent dyes that can incorporate into double-stranded DNA are added to the reaction. In their free state, they do not emit a signal (we cannot detect them), but when they are embedded, their activation occurs, and we can record the signal. The process of RT-PCR is then very similar to classical PCR. At high temperature, the double-stranded DNA is denatured, after lowering the temperature, primers are attached (annealed) and new strands are synthesised with the participation of the enzyme DNA polymerase, while a fluorescent dye is incorporated into the product. These steps are also repeated in 30-40 consecutive cycles. In contrast to standard PCR, in RT-PCR the fluorescence signal is evaluated after each cycle and its intensity is recorded.

It is obvious that for this reason an ordinary thermocycler is not sufficient for RT-PCR, but a special type is needed that can record the fluorescence signal - light of different wavelengths. We then see the PCR reaction as an amplification curve formed by combining the measured fluorescence intensities after each cycle. In RT-PCR analysis, the key is the so-called C_T value, i.e., the cycle number at which the fluorescence intensity in the sample exceeds a well-defined value that is higher than the background fluorescence. If we analyse different samples in the instrument simultaneously, each may have a different amplification curve and thus a different C_T value (Figure 4.4). Here, the less input DNA present at the beginning, the longer it takes for the fluorescence to exceed the threshold. Thus, by precisely analysing the course of the reaction, the amount of DNA used can be determined with relative accuracy. For this reason, RT-PCR is sometimes also referred to as quantitative PCR (qPCR).

Figure 4.4 RT-PCR amplification curve of samples with different CT values.

Sequencing determines the order of nucleotides in DNA.

Knowledge of the exact sequence, i.e., the order of the DNA nucleotides, is not only important from a research perspective, but also forms a very important part of the diagnosis of many diseases or disease predispositions, is used in criminalistics, and is an important aspect of evolutionary studies to determine the relationship of organisms. The very first molecule sequenced was the tRNA for alanine (this was back in 1965), and a few years later (1977) the two most famous sequencing methods were published. The first, named after its authors Allan Maxam and Walter Gilbert, was the Maxam-Gilbert method. It is also called "chemical sequencing" because various chemical compounds were added to the reaction that changed the nitrogen bases, and based on this change, the analysed DNA segments were then cleaved. Since it is more complicated and has several shortcomings compared to the second successful sequencing method, it is no longer widely used. The second method, which is still widely used today, is the Sanger method, which in turn is named after its author Frederick Sanger. For the discovery of the sequencing methods, both authors - Gilbert and Sanger - received the Nobel Prize in Chemistry in 1980.

The key component of the Sanger sequencing reaction is special variant of nucleotides – dideoxynucleotide triphosphates (ddNTPs). These differ from ordinary nucleotides in that they do not have a free hydroxyl group (-OH) on the 3'-carbon of deoxyribose, so DNA polymerase cannot attach additional nucleotides to them (see chapter 3 for an explanation). If such a special nucleotide is added to the DNA chain, the DNA polymerase gets stuck at this position and the synthesis is terminated. This is why they are also called dideoxyterminators. Within the sequencing reaction, there are the following main components: DNA template (the DNA segment we want to sequence), dideoxynucleotide triphosphates (each of the four, A,T,C, G, labelled with a different fluorescent dye), primer, DNA polymerase and standard nucleotide triphosphates. After the primer binds to the DNA template, synthesis begins, and if we have a suitable ratio of dideoxynucleotide triphosphates to deoxynucleotide triphosphates, a dideoxyterminator is occasionally incorporated at each site, depending on complementarity with the template. When this happens, synthesis is stopped at that DNA molecule. In the end, a mixture of synthesis products of different lengths is obtained, depending on when termination occurred. Since the last nucleotide is a dideoxynucleotide, each product is also fluorescently labelled. This is followed by electrophoresis in a special device (called a sequencer), in which the individual fragments are divided according to their length. The shorter the fragment, the faster it moves. The device is able to split DNA molecules that differ in length by even a single nucleotide. A special detector then reads a fluorescent signal when these fragments pass a certain point. The result is called electrophoretogram, from which we can determine the sequence of nucleotides in the analysed DNA. The entire analysis is performed with the aid of software (Figure 4.5).

Figure 4.5 Process of Sanger sequencing. ddATP = dideoxy-ATP, ddTTP = dideoxy-TTP, ddGTP = dideoxy-GTP, ddCTP = dideoxy-CTP, dNTPs = deoxynucleosidetriphosphates.

Sanger sequencing can sequence about 1,000 nucleotides per reaction, which is why second-generation sequencing methods - NGS (Next Generation Sequencing) - are already being used to sequence entire genomes, but often also in diagnostics. These technologies came onto the market between 2005 and 2007, the best known of which include Illumina, GS FLX system, Ion Torrent or SOLiD. Their common feature is that they are based on the principle of initial amplification (synthesis) and consequently differ in approaches such as "reading" the DNA sequence. This type of sequencing is much more efficient than the Sanger method because NGS uses micro-nanotechnologies, minimizes the amount of input sample, and allows parallel sequencing of a large number of DNA sequences at once.

Did you know that...

...DNA analysis methods are an everyday part of diagnostics in modern society. We could already see this during the pandemic COVID-19. After the global spread of the SARS Cov2 virus, it was necessary to quickly and efficiently track down people who were carriers of the virus to isolate them and prevent further spread. Since symptoms of illness are not sufficient evidence of the presence of the virus in the body (other diseases have similar symptoms), more sophisticated diagnostic methods had to be used. Nucleic acid and its primary sequence of nucleotides are characteristic of a particular organism and also of a virus, so methods aimed at identifying a section of nucleic acid can detect the presence of specific virus with high accuracy. In addition to accuracy, the speed of detection is also important in the case of a spreading infection. The PCR reaction, the result of which can be evaluated within 1 to 2 hours, is a tremendous advantage over long-term cultivation of cells. Thus, RT-PCR immediately became the predominant method in the diagnosis of COVID -19. The Sars Cov2 virus has its genetic information stored in an RNA molecule on which no PCR reaction can be performed, so it must first be transcribed into DNA in a process of reverse transcription. This step is a standard part of the RT-PCR reaction. As you have learned in this chapter, the RT-PCR method allows us to determine the amount of input nucleic acid in the reaction, so it also gives us very valuable information when diagnosing the virus. Individuals with a higher amount of virus in their body may be more contagious and therefore pose a greater risk to those around them.

Want to read more?

When trying to understand how to work with DNA, it can be helpful to watch videos. Below are some short YouTube videos to help explain the concepts.

A video on PCR

A comparison of PCR and qPCR on video

A video on Sanger Sequencing

Chapter 5: Mutations - how they arise and what to do with them

The classical experiments of Johann Gregor Mendel demonstrate how hereditary traits are passed on from generation to generation. Such experiments dealt with the inheritance of genetic information and led to the emergence of a new scientific field - genetics. However, further research has shown that genetic information can change, and this observation has led geneticists to begin studying the variability of living things.

Variability can be divided into heritable or nonheritable types. In non-heritable variability, the differences between individuals are caused by the influence of external environmental conditions. This type of variability is usually not passed onto offspring because it does not change the genetic information itself. Here, the environmental factors affect the structure of the proteins that the DNA wraps around (histones) or change how molecules bind to DNA. These are called epigenetic changes and sometimes these can be passed on to the next generation. You can learn more about epigenetics in Chapter 6 - How the Environment Can Affect Our Genes. Heritable variability is about changes at the level of the organism's genetic information, i.e., changes in the nucleotide sequence of the DNA molecule, which we call mutations. The term mutation was introduced by one of the rediscoverers of Mendel's laws, Hugo de Vries, who observed the appearance of new phenotypes in evening primrose plants (Oenothera lamarckiana). Initially, as offspring were clearly different from their parents, de Vries assumed that they were new species which were a result of spontaneous changes in traits. To describe their origin, de Vries introduced the term mutation in 1901 and postulated the “theory of mutation”, which, however, turned out to be not entirely correct. He assumed that mutations occur in batches and discontinuously, in contrast to Darwin's idea of gradual accumulation of new deviations from which selection chooses the most reproductive variants (you can learn more about Darwin's work in Chapter 14 - The principle of evolution). Experiments conducted in the following years showed that the Oenothera species were not the origin of a new species, but that the changes resulted from major rearrangements of chromosomes leading to new phenotypes, and the formation of new species in this way is rare.

Mutations can be deliberately induced

The work of Hermann Joseph Muller in 1927 represented an important milestone in the study of mutations. Muller was a member of the laboratory of the well-known geneticist Thomas Hunt Morgan, and his experiments showed that the frequency of mutations could be increased by the application of an external factor, in this case ionizing radiation. His experiments were of ground-breaking importance, because thanks to them it was possible to obtain new mutants necessary for studying the inheritance of various traits. The organism of choice was the fruit fly Drosophila melanogaster.

In the fruit fly, sex is determined by the presence of the sex chromosomes X and Y, with the female carrying two X chromosomes and the male carrying one X chromosome and one Y chromosome (XY). It should be noted that the Y chromosome in flies is not involved in sex determination, but in sperm formation, and is only found in male flies. Due to the balance of X chromosomes in sex determination, it is ensured that after crossing a female (XX) with a male (XY), there will be a 1:1 sex ratio in the offspring (Figure 5.1). Muller developed a simple method to determine the frequency of recessive lethal (incompatible with life) mutations associated with the sex chromosome X. A recessive mutation occurs only if there is no standard allele of the same gene (A) in the genotype, i.e., in males who have only one X chromosome with a recessive allele (a), because this gene is not present on the Y chromosome. If there is only one recessive lethal mutation in the genotype, the individual in question will not develop, and so the gender ratio will be different. The principle of Muller's experiment was that a recessive lethal mutation (a) on the X chromosome leads to a reduction in the proportion of viable males, i.e., there is a change in the sex ratio (Figure 5.1). The genius of this experiment is in its simplicity: to determine the frequency of recessive lethal mutations, it is sufficient to count the resulting offspring from a cross and determine if males are missing.

$Figure 5.1 The principle of the test for sex-linked recessive lethal mutations. Sex in flies is determined by the presence of XX sex chromosomes in females and XY in males. In a normal cross, there will be a 1:1 ration of male:female flies. If a recessive lethal mutation (a) occurs in a female in the parental generation, this is passed onto the male, which as it only has a single X chromosome, will not have the dominant (A) gene. Therefore, this male will not survive and the result is a reduction in the fraction of males, i.e. the change in the gender ratio, in the next generation.$

Figure 5.1 The principle of the test for sex-linked recessive lethal mutations. Sex in flies is determined by the presence of XX sex chromosomes in females and XY in males. In a normal cross, there will be a 1:1 ration of male:female flies. If a recessive lethal mutation (a) occurs in a female in the parental generation, this is passed onto the male, which as it only has a single X chromosome, will not have the dominant (A) gene. Therefore, this male will not survive and the result is a reduction in the fraction of males, i.e. the change in the gender ratio, in the next generation.

With his experiments, Muller drew attention to a fact that may seem trivial to us today, but was revolutionary at the time: in evolution, heredity and variability do not play a role separately, but their combination is essential - inherited variability. It is also important to note that Mueller's work described the basic properties of the gene in great detail, at a time when the chemical basis of genes and DNA were unknown.

Although the material that carried genetic information was not known at the time, scientists assumed that the macromolecule in which the genetic information is written must be extraordinarily stable in order to fulfil its function - to carry the information for the creation of an individual and to be passed on undamaged to the next generations. At the time when Muller conducted his experiments, it was already clear that genetic information was somehow linked to the chromosomes. However, it was not yet known which chemical compound in the chromosome - proteins or nucleic acid - would be the physical carrier of the genetic information. To scientists, nucleic acids appeared to be too chemically simple and not variable enough to store complex genetic information. It was therefore assumed that genetic information was found primarily in the structure of proteins, for which DNA forms only a scaffold. Until the 1940s, most scientists considered the protein portion of chromosomes to be the basis of heredity. This is because in contrast to the four nucleotides of DNA, proteins consist of up to 20 different amino acids, the combination of which offers far greater possibilities for storing genetic information. Convincing the scientific community of the importance of DNA as a carrier of hereditary information was difficult and lengthy for this very reason, and required much experimental evidence. However, the discovery of the DNA structure in the mid-20th century by Watson and Crick was of crucial importance for mutation research. Watson and Crick described the DNA molecule as a double helix and proposed a semiconservative method of replication based on specific base pairing that ensured the accurate transmission of genetic information from generation to generation. As already mentioned in Chapter 3, this discovery represented an important milestone in genetics and the scientists were jointly awarded the Nobel Prize in 1962.

DNA damage - a potential source of mutations

The discovery of the structure of DNA and the subsequent detailed knowledge of this molecule have revealed an indisputable fact: DNA is a very dynamic molecule that is constantly undergoing changes. These changes result from its chemical nature but are also caused by the environment in which DNA resides within the cell. In addition, changes to DNA can also be caused by external factors (Figure 5.2), which include ionizing radiation as used by Muller in his experiments with flies. These changes are collectively called DNA damage and are a potential source of mutations. The process by which DNA damage becomes a mutation is usually a multistep process, and cells have many ways to reverse it and maintain the integrity of the DNA molecule.

Figure 5.2 Sources of DNA damage. DNA can be damaged by many external environmental factors, including ROS (Reactive Oxygen Species), ionizing and UV radiation, as well as chemical agents. The result is that DNA is broken or forms different structures (crosslinks or dimers).

Endogenous causes of DNA damage

Among the changes that occur spontaneously due to the chemical properties of DNA are tautomeric rearrangements and errors in DNA replication. Tautomeric shifts are spontaneous rearrangements that involve the transfer of hydrogen atoms within a base, resulting in a non-standard base that will have altered pairing. For example, it is usual for cytosine (an amino) to pair with guanine (a keto), but when a tautomeric rearrangement occurs within the cytosine molecule this results in the formation of the less abundant imino form of cytosine, which means it can pair with adenine (Figure 5.3). Such a change in the base pairing cannot be considered a mutation because the cell can repair it, but such pairing presents a problem that must be resolved before the next replication. If the alternate form of the base cannot be solved, a mutation already occurs after replication, where the original C-G pair is replaced by a T-A pair (termed a transition mutation).

Figure 5.3 Altered pairing properties of nucleotides when a tautomeric rearrangement has occurred. Keto forms of guanine and thymine become enol forms (G' and T') after a tautomeric shift. Adenosine and cytosine shift from the standard amino form to an imino form (A' and C'). The modified bases have altered pairing, with T' pairing with G, C' pairing with A, G' pairs with T and A' will pair with C.

Similarly, a mutation can also occur during DNA replication when the enzyme DNA polymerase inserts an incorrect nucleotide, adds an extra nucleotide or even omits one. In all of these cases, a mismatched base pair is created, which the cell can still repair – but only if the mismatch is recognised quickly. Since DNA damage is a very serious change, the enzyme DNA polymerase itself is able to recognize and repair mismatched/damaged bases. However, if the cell fails to recognize a mismatch base pair, a mutation called a base substitution will result after the next round of replication (Figure 5.4). A base substitution can have different effects on the cell depending on which substitution has occurred. As mentioned in Chapter 3 – Get to know DNA as the carrier of genetic information, DNA is transcribed into an mRNA sequence during transcription, which is subsequently translated into the amino acid sequence for the protein during translation. Each nucleotide triplet in the mRNA, called a codon, defines one of the 20 standard amino acids that can be part of a protein chain. However, an amino acid can be encoded by more than one codon, so for example serine can be coded by the codon AGU and AGC. In our example (Figure 5.4), phenylalanine is coded by UUU, but a single base swap mutation that turns the codon to UUC has no obvious effect, as both codons result in phenylalanine being added to the sequence. We refer to such a mutation as a silent, or synonymous, mutation. In the case of a non-synonymous mutation, the original amino acid is replaced by another. If this is an amino acid with similar properties to the original one, the resulting effect may not have serious consequences - then it is a neutral mutation. However, if an amino acid with different properties is incorporated, such as a mutation from UUU to UUA resulting in leucine in place of phenylalanine, the resulting protein may not function properly, which is called a missense mutation. A substitution, or base insertion, that results in the formation of a premature STOP codon (a so-called nonsense mutation) causes premature termination of translation and the formation of a truncated protein. Another error that can occur during DNA replication is the insertion or deletion of one or more nucleotides. In this case, it is the so-called shift mutation because it shifts the reading of the genetic code (Figure 5.4). Such a change has more severe effects on the cell because the shift in the reading of the genetic code at the site where the nucleotide was inserted results in a completely different protein.

Figure 5.4 Types of mutations and their appearance. When a base is altered, it can have different effects on the cell. In the case of a synonymous mutation, or silent mutation, the same amino acid is incorporated into the protein chain during translation. In some cases, a mutation results in one amino acid being replaced by another, which can change the properties of the newly formed protein, a missense mutation. If the mutation results in a STOP codon, transcription is prematurely terminated and a truncated protein is formed. This protein is not always function and the type of mutation is called a nonsense mutation. If a nucleotide deletion or insertion occurs, the genetic information changes due to a shift in the reading order of the nucleotides resulting in an alternative protein sequence.

Sickle cell aneamia

Sickle cell anaemia is a disease that is caused by a substitution mutation in the DNA sequence which results in a change to the morphology (shape) of red blood cells. Haemoglobin is a molecule in the red blood cell that is required to bind oxygen to deliver it around the body. A healthy human produces haemoglobin A, which is composed of two α- and two β-subunits, with each subunit encoded by a different gene. The essence of this disease is the substitution of a single nucleotide in the gene for haemoglobin A, resulting in sickle-shaped red blood cells containing haemoglobin S instead of red blood cells with the classic sponge shape containing haemoglobin A. These two haemoglobins differ at the DNA level only by a single substitution of thymine for adenine, which results in the original amino acid, glutamic acid, being replaced by valine, consequently haemoglobin A is changed to haemoglobin S (Figure 5.5). The consequence of this change is the altered properties of the sickle-shaped blood cells. Normal shaped red blood cells are able to move through even the smallest blood vessels, whereas in sickle cell anaemia the cells change shape, becoming crescent, or sickle, shaped. Sickle cells block the blood vessels and prevent oxygen from reaching the organs and tissues. Sickle cell anaemia is a recessive disease, meaning that two mutant alleles of the same gene are required for symptomatic expression. In recessive homozygotes, i.e., individuals carrying both mutant versions of the gene, severe haemolytic anaemia (a low number of red blood cells) with frequent mortality develops by the age of 20. In heterozygotes (individuals carrying one normal and one mutant copy of the gene), the symptoms of the disease are less severe and manifest mainly in stressful situations, resulting in a reduced quality of life.

Figure 5.5 A mutation in the molecule of haemoglobin causes sickle cell disease. A base pair substitution from adenine to thymine, results in a missense mutation, where the amino acid glutamic acid is replaced with valine. The result is that haemoglobin S is formed, not haemoglobin A and so the red blood cell is distorted into a sickle, or crescent, shape.

Exogenous sources of DNA damage

In addition to endogenous (internal) sources of damage, DNA damage can also be triggered by exogenous factors, which can be divided into three main groups: physical, chemical, and biological. Physical factors that cause DNA damage include UV and ionising radiation. Chemical factors that damage DNA form a very large group of substances – including those that are used in industry and agriculture, as well as some that are used in medicine. An example of such a substance is cisplatin, which leads to the formation of interchain crosslinks (Figure 5.2). If a crosslink is present, it blocks DNA replication. It is for this reason that cisplatin is used as a chemotherapeutic agent, as it prevents rapidly dividing tumour cells from replicating. In addition, some chemical substances that occur in the environment are in the form of pro-mutagens, which are inactive by themselves but become mutagens when activated following cell metabolism. These substances include polycyclic aromatic hydrocarbons, which are formed in the lungs as intermediates from the chemicals inhaled from cigarette smoke. Biological factors also have mutagenic potential, such as mobile genetic elements (transposons) and some viruses. These have the ability to insert themselves at any location in the genome, and if they integrate into a gene region, the gene is disrupted and produces a truncated protein with altered properties.

DNA damage occurs relatively frequently in cells - some sources report 10,000-20,000 occurrences of damage per cell per day. In some cases, this damage results in mutations (mutagenic effect), while in others, the damage blocks replication and transcription (cytotoxic effect). It is therefore not surprising that the cell has evolved several mechanisms over the course of evolution to remove such damage.

DNA damage is a serious condition that the cell must repair

The cell's response to damage depends on its nature and extent. If the DNA damage is so severe that the cell cannot repair it, programmed cell death (apoptosis) occurs (Figure 5.6). However, if the damage is repairable, the cell cycle is halted, giving the cell time to repair itself. At the same time, repair mechanisms are activated and genes whose products are involved in repairing the damaged DNA are transcribed. After successful repair of the damage and restoration of DNA integrity, the cell cycle can be resumed.

Figure 5.6 Cell response to DNA damage. If the damage is severe, the cell will undergo apoptosis. However, if the damage is less severe, the cell cycle will be paused to allow changes in transcription to occur and ultimately DNA will be repaired.

DNA repair mechanisms play an important role in maintaining the integrity of the cell's genetic material and ensures the genetic stability of species by allowing intact DNA to be passed from parents to offspring. As DNA damage has been occurring since the beginning of life on Earth, cells have developed an elaborate network of systems that can repair the negative effects of DNA damage. The variety of repair mechanisms that have evolved from bacteria to humans demonstrates the importance of keeping the number of mutations low.

There are several ways to repair damaged DNA, and which of the available systems the cell chooses to use depends on the specific type of damage. Among the most accurate and commonly used repair methods are excision repairs (Figure 5.7). In this case, the cell must first recognize that DNA damage has occurred and identify the location of the damage. Then the cell machinery must remove either only the damaged base (in what is known as base excision repair) or a short section around the damaged base (in a process called nucleotide excision repair). In both cases, a short gap in the DNA strand is created which is filled by synthesizing a new DNA strand by the enzyme DNA polymerase, using the undamaged complementary DNA chain as a template. Finally, DNA ligase connects the newly synthesized DNA with the rest of the strand.

Single-strand and double-strand breaks are very serious results of DNA damage. If they are not immediately repaired in the cell, the cell cannot function properly and will die. Single-stranded breaks are relatively easy to repair because the broken segment needs to be found, the missing DNA synthesized by DNA polymerase using the unbroken DNA strand as a template, and the new strand is connected by DNA ligase. In contrast, double-stranded breaks are more difficult for the cell to repair. The cell has two ways to repair this damage - non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is a faster, less demanding, but also relatively inaccurate repair method in which the cell removes the remnants of the damaged bases from the broken ends of the DNA and joins them together using DNA ligase (Figure 5.7). While some short segments of genetic information may be lost in this type of repair which can have significant consequences, it is even more dangerous to leave the DNA unrepaired. Homologous recombination is a difficult but highly accurate repair method that requires a sister chromatid or homologous chromosome. In this repair mechanism, the cell first finds the ends of the break and creates a 3' overhang by degrading a piece of the 5' strand from the break itself. Such an end is called recombinogenic and is very important because it can insert into the DNA strand of the homologous chromosome. It then moves along the homologous chromosome looking for a complementary sequence of DNA, whereupon DNA polymerase synthesizes the broken part and brings back the strand to its original location. This repaired strand acts as a template for repairing the complementary DNA strand (Figure 5.7). Whether a cell uses HR or NHEJ to repair a break depends not only on the presence of a homologous chromosome or sister chromatid, but also on the organism itself. Human cells prefer the less accurate NHEJ when repairing this type of damage because it is faster and more efficient than HR.

Figure 5.7 Methods of DNA damage repair. A) Removal of the damaged section of DNA using excision repair, which is replaced with new bases. B) DNA double-strand break repair by non-homologous end joining (NHEJ) with the participation of DNA ligase (green triangles). C) Homologous recombination (HR) also repairs double stranded DNA breaks, but requires the presence of a sister (homologous) chromatid as well as DNA ligase (green triangles).

In some cases, the cell also has mechanisms that allow it to tolerate some damage. These include, for example, what is known as translation synthesis. This is a mechanism that allows the cell to replicate its DNA despite damage that could act as a block to replication and thus allows the cell to complete the cell cycle.

Disorders of DNA repair mechanisms lead to increased accumulation of mutations that can result in the development of severe syndromes, such as Xeroderma pigmentosum (causes extreme sensitivity of the skin to UV radiation). They also manifest in severe disorders leading to tumour formation and premature aging, such as Cockayne syndrome (read more in Chapter 9 - When Cells Go Crazy: how a healthy cell becomes cancerous).

Mutations and their effects on organisms

As mentioned previously, DNA damage, if not repaired, can lead to mutations, which is a change in the nucleotide sequence of DNA. The effect of the mutation then depends on several factors: If the mutation originated in a non-coding region (outside the region of a gene that codes for a protein) its effect is usually not present. Such a mutation can only be detected by DNA sequencing. However, if it has arisen in the gene region, its effect may still be seen, depending on whether it is a substitution or a displacement mutation (Figure 5.4).

The effect of the mutation also depends on the cell in which the mutation arose. If it originated in a somatic (body) cell, the mutation cannot be passed onto future generations, and manifests itself only in that cell and its daughter cells. Thus, such a mutation affects only the organism in which the cell is located. In contrast, a mutation in a germ cell (a sperm or egg) is passed onto the next generation and will affect the offspring. An example of such a mutation is the mutation causing haemophilia that originated in a member of the English royal family, Queen Victoria, and was passed down through generations.

The term "mutation" has a negative connotation for most people. However, mutations can also lead to the emergence of new traits that are beneficial to humans, especially when it comes to food, which can give fruit and vegetables new size, taste or colour. An example of this is the somatic mutation in apples that led to the creation of the ‘Delicious’ variety, which can be red or yellow in colour. The possibility of vegetative propagation of plants makes it possible to preserve and use this mutation. The mutation that leads to sickle cell anaemia also cannot be considered only a negative mutation, since people who are heterozygous for the mutation (i.e., are carriers of the disease) have resistance to malaria (since the mutant red blood cells cannot be attacked by the Plasmodium parasite that causes this disease. You can read more about this in Chapter 11 - Plants as inspiration in biomedicine. For this reason, sickle cell anaemia is very common in some populations, especially in areas heavily affected by malaria.

So far, we have only talked about mutations that occur at the level of one or more nucleotides. However, a large group of mutations consist of changes at the level of the chromosomes, where one or more can be multiplied (so-called chromosomal aberrations). These changes occur mainly as a result of cell division disorders, when chromosomes are incorrectly distributed into daughter cells. The result is germ cells with an extra chromosome or, conversely, a chromosome missing from the germ cell. The likelihood of incorrect chromosome division increases with the age of the mother, since germ cell embryos are formed during intrauterine development of females. However, in humans only trisomies of some chromosomes (13, 18, 21 and X) are known, as others have such severe effects on the organism that foetuses with such anomalies die during embryonic development. A well-known example of duplication of a chromosome in humans is trisomy of chromosome 21, known as Down’s Syndrome.

In addition to the change in the number of individual chromosomes, the duplication of entire sets of chromosomes (polyploidy) can also occur under certain circumstances. This occurs mainly in plants and can also be achieved specifically through breeding. One of the consequences of polyploidy is an increase in the volume of the cell and thus of the entire organism, which is why polyploid species are often used as food in agriculture (e.g. wheat, banana, etc.). In animals, polyploidy occurs less frequently however examples can be found in some amphibians and fish.

Mutations and Evolution

Mutations are fundamental to biological evolution - they are one of the mechanisms that provide the genetic variability that forms the basis to expand the repertoire of phenotypes. In this process, natural selection chooses variants that are better adapted to the given environmental conditions and thus produce more offspring. Thus, they are a source of new genetic variability that enables organisms to adapt to environmental changes. You can read more about the principle of evolution in chapter 14.

Did you know that...

...mutations in some genes cause a disease that is already present and obvious at birth, while other mutations do not directly cause the disease but can increase the likelihood that you will develop it later in life? Breast cancer is the most common cancer in women and the second leading cause of death, with its incidence steadily increasing worldwide. About 10-15% of cases are associated with a clustered occurrence in a particular family, so there is a direct link to heredity. Several studies have confirmed that mutations in the BRCA1 and BRCA2 genes are directly involved in the development of breast cancer. The product of the BRCA1 gene is a protein that suppresses tumour formation by participating in the repair of damaged DNA, when mutated this checkpoint does not occur and the cells undergo rapid replication with lots of DNA errors. A woman diagnosed with breast cancer should therefore always undergo a genetic examination to check if she has the BRCA1/2 mutations. If she has already succumbed to the disease, her closest female relatives should also go for a genetic examination. Mutations in these genes are considered genetic "markers" that indicate a woman's lifetime risk of developing cancer. While screening can reveal a genetic predisposition, a positive test result does not mean that an individual will absolutely develop the disease, but the risk is much higher. However, it is important that the carrier of such a mutation be under increased medical supervision.

Want to read more?

DNA Repair and Mutagenesis, Friedberg, E. C., Walker, G. C., Siede, W., Wood, R. D., Schultz, R. A., Ellenberger, T., ASM Press (2006).

DNA damage, mutagenesis and cancer A.K. Basu (2018) Internation Journal of Molecular Sciences 19)4):970

The Nobel Prize in Chemistry 2015 for "mechanistic studies of DNA repair", NobelPrize.org

Chapter 6: How the environment can affect our genes

At the beginning of the 20^th Century, scientists did not yet have an exact idea of how genes cause certain phenotypes. While the existence of DNA was already known, it was not yet linked to the task of transporting genetic information, and it was debated whether proteins fulfilled this task. Later, after the discovery of the gene as the basic unit of heredity, there were efforts to describe its function, but this could only be done on the basis of the analysis of phenotypes. The result of these observations was the conclusion that genes do not tend to act in isolation, but that they perform their task in harmony with other genes and, moreover, in the context of the environment surrounding them. Thus, the scientists realized that the external appearance of an individual - the phenotype - is created by the interaction between the genotype and the environment.

There are traits encoded by a gene that manifest themselves at the phenotype level with relatively low variability (i.e., a low number of possible manifestations). In this case we talk about qualitative traits, which includes, for example, a person's blood type or a hereditary disease, the occurrence of which can be assumed based on the presence of the disease in the family, such as the blood disease haemophilia A. However, there is also a second and very numerous group of genes, the so-called polygenes. Here, the effect of a single gene on the phenotype is too small to be observed, but when acting with other genes, together they produce an effect that is seen. The phenotypic manifestation is dependent on this group of genes, characteristic of the quantitative type of inheritance, since every single allele contributes to the formation of the phenotype. As a result, continuous phenotypic variability can be observed, e.g. in the case of human height. All the people of the world are not divided into two or three height categories, but there is a wide range of different heights, while the resulting height of each of us depends on the number and types of alleles of individual genes, each with a small effect. In addition, the overall phenotypic manifestation of a trait is also influenced by the environment in which the organism lives, which is why the inheritance of these traits is sometimes referred to as multifactorial inheritance. If a person has a predisposition to be tall, but did not have sufficient nutrition throughout childhood, then their height potential cannot be manifested to its full extent. Quantitative genetics studies the influence of specific polygenes and the influence of the environment on the formation of the phenotype. The distribution of phenotypic classes of quantitative traits in the population is characterized by the so-called Gaussian curve (Figure 6.1), with the highest number of average individuals and the lowest number of individuals with extreme phenotypes (too short or too tall individuals).

Figure 6.1 Gaussian curve of quantitative traits. The graph shows the distribution of phenotypic classes of quantitative traits in the population, using height as an example. The course of the curve shows that most of the people in the studied group are of average height (170cm) and only a small number of people are very short or very tall.

The environment allows the signs to manifest

One example that demonstrates how the environment can influence the resulting phenotype is the disease phenylketonuria (PKU). In affected individuals (recessive homozygotes for the mutated allele), toxic compounds accumulate in the brain and consequently damage its development, leading to mental retardation. PKU is a recessive disorder of amino acid metabolism, resulting in the inability to convert phenylalanine to tyrosine. Phenylalanine, which is commonly found in our diet, is not toxic in itself, but it is metabolized to other compounds that are toxic. Infants with this disease who are fed a normal diet thus ingest phenylalanine, and this causes the development of symptoms of the disease. Infants diagnosed with PKU who are given a diet low in phenylalanine usually develop without severe mental impairment. Screening for PKU, the frequency of which ranges from 1:4,500 in Ireland to 1:100,000 in Finland, is therefore done immediately after birth, and early implementation of a modified diet makes it possible to alleviate the clinical manifestations of this disease. This example illustrates how the influence of an external factor, in this case diet, can affect a change in phenotype.

Epigenetics is the interaction of DNA and the environment

The expression of the genes in each organism undoubtedly responds to the stimuli of the environment in which the given organism lives. It is a mechanism that helps the organism adapt to environmental conditions, and this adaptation can to a certain extent be transferred to the next generation as hereditary information. As discussed in chapter 3 - Mutations: how they arise and what to do with them, modifications to DNA that cause a change in genetic information were mentioned. In some cases, this can be manifested by the creation of a protein that has different properties or function. If a change in the DNA is also transferred to the daughter DNA molecule, it becomes a mutation. However, the change caused by an epigenetic modification has a different character, especially since it is not a change to the DNA sequence. It does not change the information in the DNA itself, but it changes the way in which this information is realized. To put it simply, epigenetic modifications can turn genes on and off. The set of epigenetic information affecting gene expression is called the epigenome.

The epigenetic landscape illustrates the importance of epigenetics

The first indications that the fate of cells may depend on the environment in which they live was highlighted in the 1950s by developmental biologist Conrad Hal Waddington, who studied the development of multicellular organisms. He based his model, called the epigenetic landscape, on his observations (Figure 6.2A). In the epigenetic landscape model, the cell represents a ball that changes its direction during the development of the organism depending on which path it takes. In fact, the direction of the cell depends on which metabolic pathways will be turned on, which themselves depend on the environmental conditions, and based on their combination, the resulting phenotype is formed. As a result, in a multicellular organism, all cells do not perform the same function, but are specialized for specific tasks (Figure 6.2B).

Figure 6.2 A model of the epigenetic landscape. A. Waddington landscape - the metaphor for the development of a cell depending on the conditions to which it is exposed. B. In a multicellular organism, cells undergo differentiation, which allows the emergence of various specialized cells from the original unspecialized cell.

According to estimates, the adult human body contains 37 trillion (3.7x10¹³) cells. After the cells have gone through the complete process of differentiation, there are approximately 200 types of cells in the body, which differ from each other in their function and structure. Each of the body's cells has the same DNA, but not all genes are transcribed in individual types of specialized cells. After all, why would it be necessary to synthesize liver enzymes in brain neurons? In addition, in female mammals, including humans, one entire chromosome is inactivated - the sex chromosome X. This inactivation is necessary to compensate for the gene dosage, because females have two X chromosomes, unlike males, who have only one X chromosome (their second sex chromosome is the Y chromosome). The need for a mechanism to turn genes on and off is therefore obvious.

Gene imprinting switches off an allele from one of the parents

People inherit two copies of each gene - one from their mother and one from their father. In accordance with the common Mendelian notion of genetics, both inherited copies are expressed and contribute to the phenotype. However, there are also cases of genes that are active only when inherited from the father and others that are active only when inherited from the mother. How is this possible if there is no difference in the DNA sequence?

To explain this phenomenon, we can use as an example a cross between a horse and a donkey. If a male horse is crossed with a female donkey, an individual called a mule is created. If a male donkey is crossed with a female mare (horse), a mule is also produced. Interestingly, the offspring in both cases looks more like the mother - the mule looks more like a donkey if the mother is a donkey while the mule from a mare looks more like a horse. This is a striking morphological, anatomical and etiological difference. Such a crossbreed is sterile, i.e., it cannot have offspring, which is due to the fact that a donkey has 31 pairs of chromosomes (2n=62), while a horse has 32 (2n=64). As a result, the crossbreed (the mule) will therefore have 63 pairs of chromosomes, and with this odd number of chromosomes, it is not possible to properly divide into sex cells. Similar examples illustrating that the direction of crossing matters can be found in nature, e.g. a liger is a cross between a lion and a tigress, a tigon is a cross between a tiger and a lioness (Figure 6.3).

Figure 6.3 The emergence of hybrid individuals. When a lion and a tigress are crossed, a liger is created (left) and when a tiger and a lioness are crossed, a tigon is created (right). Hybrid individuals resemble the mother more than the father.

What is gene imprinting in humans? One gene (either from the mother or from the father) is not expressed because it is silenced and is called imprinted. The second gene, which is not silenced, is thus expressed (Figure 6.4). Imprinting occurs through the action of DNA methylation along with the modification of histones by removing acetyl groups, which changes the structure of chromatin. Germ cells are characterized as having only one set of chromosomes, and form a diploid zygote after fusion with a gamete of the opposite type. During the formation of gametes, the information about which allele originally came from the mother and which from the father is removed, and they undergo modification depending on whether the given DNA is currently in the egg or in the sperm. Therefore, in the case of an imprinted allele of paternal origin, the offspring always inherits only the allele on which the genes subject to imprinting are silenced (Figure 6.4).

Figure 6.4 Principle of gene parental imprinting. For genes subject to imprinting, one allele is always imprinted during the formation of gametes, in this example the allele of paternal origin. Then, it does not matter which allele a man inherited from his father or mother, because he will pass both of his alleles on to the offspring as imprinted.

Interestingly, genes that undergo imprinting are clustered in groups located on the short arm of chromosome 11 and on the long arm of chromosome 15. As for the function of imprinted genes, they play an important role during embryonic development, in the regulation of cell division, but they also influence the formation of behaviour. Possible errors in these genes can cause human diseases, and the same mutation can cause two different diseases with different manifestations, depending on which parent the mutation comes from. In the case of a deletion on the 15th chromosome, if such an allele is inherited from the father, a disease called Prader-Willi syndrome occurs. The syndrome is characterized by insatiable hunger caused by damage to the hypothalamus, leading to obesity. It is also accompanied by short stature, atrophied muscles, weak crying and mental retardation. Typical facial features for this disease are almond-shaped eyes, a narrow upper lip and a high forehead. Other problems are also related to obesity, e.g. type II diabetes and cardiovascular diseases. If the fault is on the allele inherited from the mother, the child will develop Angelman syndrome, characterised by fits of unexplained laughter and jerky movements. This syndrome is also called the Angel syndrome precisely because of the impression of happiness that the patient creates by smiling and laughing. Patients suffer from microcephaly, severe mental retardation, have very limited ability to speak and epileptic seizures. People with this condition also have typical facial features (wide mouth, protruding tongue, widely spaced teeth) and a small head that is flattened at the back. It is not yet known what causes the frequent laughter, but it is known that this manifestation is related to changes in the structures of the brain, as most of the brain centres responsible for laughter are located in the left hemisphere.

You are what you eat

It was Waddington's understanding that the epigenetic landscape is formed by the organism itself, so it is not the external environment of the organism, but rather the environment around the developing cell. However, let us try to think whether the external environment can also influence genes in the sense of turning them on or off. Bees can serve as an excellent example as they are social insects that are very interesting for geneticists for several reasons. One of them is the method of sex determination, since in bees diploid females arise from fertilised eggs (they carry two sets of chromosomes (one from the mother and the other from the father) and haploid males develop parthenogenetically from unfertilised eggs (they have only one set of chromosomes obtained from the mother). Another interesting feature of bees is that it is possible to distinguish two phenotypically different groups of females: workers and queens. Although both groups arise from fertilized eggs and are genetically equivalent (they have the same genes in their genome), there is an obvious difference between them at first glance, which lies not only in size, but also in physiology and social behaviour (Figure 6.5).

Figure 6.5 Differences between a queen bee and a worker bee. When comparing the western honey bee, the most fundamental difference is life expectancy and the number of ovaries between the worker (drones) and queen bee.

The question is what causes a diploid larva to develop into a worker in one case and a queen in another. The answer is clear - diet. The fate of the larva is determined by the food it receives during the early stages of development. If she is fed regular food, she develops into a worker, but if she is fed royal jelly, a milk-like secretion of honey bees, she develops into a queen. The development of a queen requires different genes than the development of a worker, and in this case the royal jelly is able to cause the "reprogramming" of these genes. It was also found that the genes that lead to the development of the queen remain switched off (they are not transcribed) in the worker bees. Different genes can therefore be transcribed in individuals with the same genotype. This was a very interesting finding, and as a result, scientists have decided to clarify the mechanism by which royal jelly causes such a change.

How do epigenetic changes work?

Three main types of mechanisms are involved in the regulation of gene expression depending on epigenetic changes. The first of them is DNA methylation, which, among other things, is also responsible for switching off genes important for the development of the queen bee. It involves adding a small chemical tag – a methyl group – to DNA. Methylation of the fifth carbon of cytosine occurs most often, resulting in the modified base 5-methylcytosine. In eukaryotes, cytosines are almost exclusively methylated, which are adjacent to guanines and are connected to them by a phosphate group. Such a pair of nucleotides is called a CpG dinucleotide. Most CpG dinucleotides are found in gene promoters and help their expression by maintaining an open chromatin structure, which in turn allows access to the appropriate transcription factors that trigger the transcription of the gene in that region. However, the presence of methyl groups prevents regulatory proteins and transcription factors from accessing the DNA sequences necessary for the gene to be expressed. DNA methylation is a reversible process, as the methyl groups can be removed, a process of demethylation, and the gene is turned back on (Figure 6.6). Specific enzymes are responsible for the process of binding and removing the methyl group, while in most cases DNA methylation turns genes off and demethylation turns them on.

Figure 6.6 Epigenetic regulation of transcription by DNA methylation. Adding a methyl group to a gene promoter turns off expression/transcription. Demethylation is the opposite process in which methyl groups are removed, resulting in the initiation of transcription.

Histones are proteins whose task is to package DNA in the nucleus by wrapping it around itself. Such a structure of histones wrapped around DNA is called a nucleosome. Histones themselves can also undergo chemical modifications, if methyl groups are additionally attached to the histones, the nucleosomes are more tightly packed next to each other and prevent other proteins and enzymes from accessing the DNA. DNA packed in this way is called condensed chromatin - heterochromatin. Another chemical modification of histones is acetylation, which generally has the exact opposite effect. When an acetyl group is attached to histones, DNA is more accessible and genes can be expressed. Then we talk about relaxed chromatin - euchromatin (Figure 6.7). However, there are also places and situations where the effect of modifications on histones is opposite and acetylation turns genes off and methylation has the ability to turn on their expression. The changes in histone modification is more dynamic than DNA methylation, which is why heterochromatin can change to euchromatin and vice versa more often during the life of a cell.

Figure 6.7 Histone modification. This process causes a change in chromatin packing. Acetylation causes loose packing of euchromatin in most cases, and methylation mostly causes tight packing of heterochromatin.

Genetically identical mice can differ from each other

One of the favourite mammalian models for studying the influence of the external environment on the genome are mice, specifically their Agouti gene, which determines coat coloration. In this model, coat coloration correlates with epigenetic marks established early in development, and therefore allows investigation of the influence of nutritional and environmental factors on the foetal epigenome. The Agouti gene is transcribed only in a certain phase of mouse development and even then only in certain tissues, which is why such mice have dark fur. However, Duke University scientists led by Professor R. Jirtl obtained mice in which the Agouti gene was transcribed continuously, resulting in yellow mice which had a tendency towards obesity and a predisposition to cancer and diabetes (Figure 6.8). Using such mice, they then monitored the effect of adding food supplements to the mothers' diet.

If Agouti mice were fed normal food, obese yellow males mated with obese yellow females and produced obese yellow offspring. However, if obese yellow females were fed food fortified with nutritional supplements containing vitamin B12, folic acid and choline two weeks before mating and during pregnancy, after mating with obese yellow males, they gave birth to slender brown pups. These offspring were not predisposed to cancer or diabetes and were agile and alert until old age. A big surprise was the discovery that the lean mice had the same genes (no new mutations appeared) as their obese parents. It turned out that the added nutritional supplements were sources of methyl groups that were used to methylate DNA around the Agouti gene. By making a small change in the diet, it was possible to turn off the Agouti gene without changing the sequence of nucleotides in the DNA.

Figure 6.8 The Agouti mouse model. Individuals are genetically identical and of the same age. The expression of the Agouti gene is made possible by the fact that the promoter of the gene is not methylated and causes yellow and obese mice.

Another interesting finding was that not only diet, but also maternal care can cause epigenetic changes that affect the development of the organism for life. Moreover, these changes do not only concern physical characteristics, such as coat colour and height, but also psychological characteristics. As follows from another series of experiments, rats that received sufficient maternal care in their youth grew up to be calm and courageous individuals. Rats that were not cared for by their mothers after birth became very shy later in life. Epigenetic changes in the brain were confirmed in these rats. Thanks to the chemical substance trichostatin A, which inhibits enzymes that remove acetyl groups from histones, scientists even managed to increase stress resistance in shy rats. On the contrary, L-methionine, which serves as a source of methyl groups, succeeded in inducing timidity in courageous rats. The results of these experiments show that epigenetic markers can be changed even in old age.

How far does epigenetics go in humans?

As already indicated by the example of mice and their Agouti gene, an epigenetic change can be hereditary and passed onto offspring. It is therefore appropriate to distinguish between two types of inheritance of epigenetic modifications: intergenerational and transgenerational types (Figure 6.9). In the case of intergenerational inheritance, this is a manifestation of epigenetic change and its inheritance only to the extent of individuals who were directly exposed to the factor that changed the epigenome. In this case, the factor acts on a person whose epigenetic state changes and other genes begin to be expressed (P0 generation), or on the fetus that is currently developing in the womb (F1 generation) or on the germ cells of the parent or this foetus (the basis of the F2 generation). This can be illustrated using cigarette smoke as an example. If a man smokes during puberty, when his sperm are starting to form, the effect of the chemicals from the smoke will not only affect his health, but will also be written into the sex germ cells and manifest in his sons, who may have a higher tendency to obesity. This type of inheritance is observed in humans and is described in more detail and illustrated with many examples in Chapter 6 – From epigenetics to human diseases.

Figure 6.9 Intergenerational and transgenerational transmission. The difference lies in the fact that intergenerational transmission is conditioned by the action of the factor on the individual and its gametes (P0), the developing fetus (F1) or the gametes of the developing fetus (F2).

However, there is also longer-term transgenerational transmission. It has not yet been described in humans, but it can be observed in some model organisms (especially rodents). During transgenerational transmission, changes in gene expression caused by epigenetic modifications are inherited and manifested even in individuals that have never been exposed to the given factor (the F3 generation and onwards), not even at the level of the germ cells from which they originated. An example is a study on the nematode worm Caenorhabditis elegans (see chapter 20 - the small but mighty nematode) in which imprinting was analysed representing an early experience during a critical period that can permanently change an individual's behaviour. The stimulus for imprinting was olfactory sensation, specifically the volatile substance benzaldehyde, to which the offspring were exposed during the first 24 hours after hatching. In adults, chemotaxis was then observed, which is the movement of the individual in the direction from which the given smell comes, as it was familiar to the nematode since childhood. Olfactory imprinting was dependent on the expression of the SRA-11 gene, which encodes one of the chemoreceptors. If benzaldehyde acted on only one generation at an early age (P0 generation), chemotaxis was still evident in the F1 generation, but not in the F2 generation. However, if four consecutive generations were exposed to the presence of benzaldehyde, chemotaxis became heritable and stable for at least 40 consecutive generations. This suggests that somewhere between the first and fourth generations exposed to benzaldehyde, there was a change from a temporary effect to a stable transgenerational transmission. However, similar studies in humans are still lacking. The problem is the need for a large statistical sample and the monitoring of several successive generations, which is logistically challenging, time-consuming as well as ethically and financially demanding.

Genetics or epigenetics - which is more?

The fundamental discoveries brought about by epigenetics have shaken the classical understanding of genetics. Until now, it has been generally accepted that not only our appearance, but also to a certain extent our behaviour, intelligence and predisposition to diseases are dependent on the genes we have inherited. The study of epigenetics has shown that the statement “our destiny is written in genes“ needs to be changed. It seems that we can influence our genetic inheritance to some extent and this influence can be passed onto future generations. Epigenetic changes are thus necessary for the proper development and functioning of the organism, but they can also be responsible for the development of many diseases. Disruption of the proper function of epigenetic mechanisms can mean abnormal activation or silencing of genes. In humans, epigenetic changes can cause the development of cancer, but also schizophrenia and autism. Although epigenetic changes are heritable, unlike genetic ones, they are also reversible – so there is a possibility to reverse such modifications and thereby restore the normal state in the cell.

According to the current idea, the DNA of an organism serves as a universal building plan, but at the same time, a different set of genes is expressed in each type of cell, so that different parts of this plan are implemented in different places in the body. The differences in the expression of individual genes are also regulated by epigenetic modifications, so it is necessary to take into account that each of the many different types of cells has its own epigenome. Currently, as part of the Human Epigenome Project, scientists are trying to identify all the chemical changes and relationships that mediate the realization of the genetic information stored in DNA. Thanks to this information, in the future it will be possible to better understand processes such as natural development, aging, carcinogenesis and the development of other diseases, as well as the impact of environmental factors on human health. So if we were to think of the human genome sequence as a map, the goal of the human epigenome will be to find out which pathways are open and which are closed.

Did you know that...

...The agouti mouse model, in which coat coloration is correlated with epigenetic changes made during intrauterine development, has also been used to monitor the effects of nutritional and environmental factors on the foetal epigenome? Using this model, scientists observed the effect of the plant phytoestrogen genistein on the epigenome of future offspring. Genistein is a substance present in soy and belongs to the class of phytoestrogens (plant hormones), which are active in several biological systems. Addition of genistein to the diet of pregnant females resulted in delayed coat coloration of the offspring, and this significant phenotypic change was caused by increased DNA methylation of the Agouti gene. Genistein increased methylation during early embryonic development, and this excess methylation persisted into adulthood, reducing Agouti gene expression and protecting adult offspring from obesity. The observed effects of genistein on the epigenome could provide a plausible explanation for the lower incidence of certain cancers in Asians (they consume more soy than, for example, Europeans) compared to Western countries, as well as the increased incidence of cancer in Asians who immigrate to the United States.

Want to know more?

Obesity, epigenetics, and gene regulation. Adams, J., Nature Education 1(1):128 (2008).

Epigenetics, 2nd Edition, C. David Allis, Marie-Laure Caparros, Thomas Jenuwein, Danny Reinberg,CSHL Press (2015) ISBN-13: 978-1936113590

Chapter 7: From epigenetics to human diseases

The human organism is not an isolated, constant system, because it lives in a close relationship with the environment which influences it. The conditions of the environment in which it lives affect many processes, including gene expression. As mentioned in Chapter 6 – How the environment affects our genes, various types of changes in gene expression that are not caused by mutations are being studied by the relatively young scientific field of epigenetics.

Identical twins are a model for studying epigenetic processes

Epigenetic changes occur not only during intrauterine development, but also throughout the life of the organism. This is evidenced by research results in which scientists analysed epigenetic modifications in the genome of identical twins. In the case of identical twins, they both develop from one egg fertilized by a single sperm and therefore have the same genotype. At the level of genetic information, such twins are completely identical, and in the first years of life they are almost indistinguishable at the level of phenotype as well. Before birth, they shared a common space in utero, and since their childhood experiences were more or less the same, they will also have the same epigenetic traces. However, this is true only as long as they live under the same conditions and are affected by the same factors. The older the twins get, the more their epigenomes differ because they are exposed to different conditions. A different environment, different relationship experiences that evoke different emotions, and a different attitude towards their health mean that although they still have the same DNA, it may change which genes are expressed, and which are not. This is also proven by a study of older identical twins, where a comparison of the total content and distribution of modified bases and histones showed that the less time the twins lived together, the more their epigenomes differed.

For this reason, identical twins are not likely to develop each genetic disease with the same probability. For example, in the case of schizophrenia, if it has developed in one of the twins, there is less than a 50% chance that it will develop in the other. Similarly, the risk of developing the autoimmune systemic disease lupus erythematosus is also 50%, while for Alzheimer's disease, the probability is 40%. The question arises to what extent heredity is responsible for health and to what extent the environment in which a person lives. The results of the studies concluded that genes are responsible for only about 49% of the studied traits, characteristics and the environment is responsible for the remaining 51%.

Epigenetic modifications play a role in the development of human diseases

Many studies suggest that changes in the epigenetic profile may disrupt biosynthetic pathways in cells and play a key role in the development of some human diseases. Currently, there are already known examples of certain diseases whose development or progression depends to some extent on epigenetic changes. The mechanism of carcinogenesis involves alteration at the genetic level, primarily through the activation of oncogenes (genes whose mutations lead to the development of cancer) or the inactivation of anti-cancer tumour suppressor genes as a result of various mutations (explained in detail in Chapter 9 – When cells go crazy). However, recent research shows that extensive epigenetic modifications also occur in tumour cells that can alter the expression of genes critical for cancer development, namely global hypomethylation (insufficient methylation) of DNA and hypermethylation (excessive methylation) of tumour suppressor genes. An altered state of the epigenome contributing to carcinogenesis has been observed in many types of oncological diseases. In acute myeloid leukaemia, alterations in the epigenome have been found to be caused in part by mutations in genes encoding enzymes responsible for DNA methylation/demethylation. An example is lung cancer, where DNA hypermethylation has been shown to occur in early stages of the disease as well as in advanced stages, with up to 3% of all functional genes containing CpG-rich regions being deactivated by methylation. Epigenetic alterations have also been studied in childhood gliomas, gastric cancer, and skin melanoma. Intensive research is therefore being conducted in the field of cancer therapy to use new knowledge in diagnosis and, at the same time, to find suitable therapeutic agents that can correct defective epigenetic marks in tumour cells. Azacytidine is a substance that causes the removal of methyl groups from DNA (demethylation). The drug is one of the therapies also used in clinical practice in the treatment of patients with myelodysplastic syndrome, a hematopoietic disorder often referred to as preleukemia.

In addition to cancer, however, there are other diseases in the development of which the epigenome plays an important role. One group of such diseases is autoimmune diseases, including systemic lupus erythematosus, which affects the skin, joints, kidneys or nervous system, rheumatoid arthritis (inflammatory joint disease) or multiple sclerosis, which affects the brain. The link is also observed in endocrine diseases such as type II diabetes, respiratory diseases (asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis), but also skin diseases (psoriasis or systemic sclerosis). The role of the epigenome has already been demonstrated in diseases of the cardiovascular system, manifested by atherosclerosis, hypertension, and heart failure. Digestive tract diseases associated with epigenome changes include inflammatory bowel diseases (Crohn's disease, ulcerative colitis) and liver cirrhosis.

Age is not a negligible factor from an epigenome perspective

The study from 2018, based on more than 40 million births, aimed to find out whether there is a correlation between the age of the father and the health of his children. Women are born with fully developed eggs (they are formed during intrauterine development and mature gradually throughout life), while men's sperm are produced only from puberty and then throughout life. This poses the risk that diet, toxins, lifestyle, stress, and eventually male aging will be inscribed in the epigenome of the developing sperm. It was assumed that the father's role in fathering the offspring was paramount, but it turns out that the father also has an impact on the course of the pregnancy. Men's age turned out to be not negligible in this aspect. The health status of the offspring of 25-34-year-old fathers and fathers aged 45 and older was compared. Older age in fathers was associated with up to a 14% increased risk of preterm birth and was also associated with up to a 28% increased probability of gestational diabetes during pregnancy. The children of older fathers also have a higher risk of developing schizophrenia and autism. In the case of schizophrenia, the odds are 1:144 for fathers younger than 25, 1:99 for fathers between 30 and 35, and 1:47 for fathers older than 50. Of course, responsible mutations in DNA are known and these mutations arise in the germ line of sex cells. However, new research suggests that the development of this disease is also related to an altered DNA methylation pattern that developed during the father's lifetime.

Both famine and food surplus have consequences

The Dutch are said to be very meticulous people, so it's not surprising that their medical records correspond to this. In the past, when a Dutch woman became pregnant, doctors would record detailed information about the pregnancy, the birth, and many important details about the newborns. These records were destroyed after 15 years, but thanks to a lucky coincidence, one set of these - the records from the clinic of Wilhelmina Gasthuis - was kept in the attic and has survived to this day. These records are a rarity in that they were created in the very specific historical context of World War II. At that time, the Dutch were accustomed to a complete and balanced diet, as they had a high-quality agriculture. However, in 1944, realizing that it would soon be defeated by Allied forces, the German military leadership ordered railroads to be bombed, making it more difficult for the enemy to advance. The loss of a functioning railroad led to the food shipments blockage to some parts of the Netherlands, including Amsterdam, and famine spread through the region. The embargo lasted for 7 long months, until the liberation of the Netherlands in May 1945, at which point the energy intake for a person was less than 1,000 calories per day, and as low as 400 calories per day at the peak of the famine. The recommended number of calories for an adult is between 1,800 and 2,200 calories, depending on age and gender. The famine forced people to eat tulip bulbs and, combined with the harsh winter, mental strain, stress, and infectious diseases, led to a high mortality rate. Detailed medical records of this difficult period later provided scientists with very valuable information, especially since it lasted only a few months, affected a well-defined population, and all these people were malnourished at the same time. This combination of factors made it possible to study in detail how the given conditions affected the health of people born during this period. The result, compared to children born under normal conditions, the offspring of starving parents had twice the risk of developing cardiovascular diseases, obesity, diabetes, hypertension, and high cholesterol. In many cases, the inheritance of epigenetically modified gene expression was evident within the family line - from mothers to daughters and from fathers to sons.

The opposite of famine, is a food surplus, which occurs when the supply and availablilty of food is greater than the demand for it. Food surplus is linked to fertile years, and was more common in the days before the creation of a network of stores and supermarkets. Knowledge of these famine and surplus periods as well as the family situation was used in a 2014 study. The authors of this study analysed the causes of death of 239 people born in a small Swedish village in 1890, 1905, and 1920. They also obtained historical data on how the parents' families lived and their access to food during childhood. If the paternal grandfather (inheritance through the male line) had an excess of food during the pre-adolescent period, his grandchildren were four times more likely to die from diabetes-related complications than if he had a standard amount of food available. Surprisingly, the grandchildren had a lower risk of dying from heart disease if their father or paternal grandmother was starving during the pre-adolescent period. The explanation for the influence of diet is its effect on metabolic adaptation, which is written into sperm DNA and transmitted to offspring as an epigenetic modification.

Toxins are all around us, including you

An English study dealing with the impact of smoking on people's health, worked with the data of 5,451 fathers who smoked. Among them was a group of 166 fathers who smoked just before puberty, at age 11, the period when sperm begin to form in males, which later transfer from the father's genetic material to their children. They left their sons an undesirable inheritance - obesity. The sons were more obese than average and even more obese than the sons of men who smoked but not at that critical stage of puberty. The impact on the daughters has not yet been confirmed. Cigarette smoke, both from active smoking and the passive inhalation of cigarette smoke, is one of the best-described toxic substances affecting pregnant women. The effects of cigarette smoke result in an increased risk of low birth weight, asthma, obesity, and type II diabetes. A study including a large number of existing studies, called a meta-analysis, has demonstrated a direct association between smoking and the incidence of cancer of the nervous system (brain and central nervous system) in children. However, in addition to cancers, smoking is also associated with placental insufficiency and preterm birth as well as being a risk factor for sudden infant death syndrome. The fact that a woman's smoking has an undeniable negative effect on the child is also evidenced by the fact that the by-products of tobacco combustion have been detected not only in the breast milk of smoking mothers but also in the blood of the foetus and the mother of the newborn.

Life experiences are reflected in the epigenome

In addition to the already mentioned, easily measurable factors that affect the human organism (type and amount of food, exposure to chemical substances, biological causes of aging), there are other factors influencing gene expression, but they are rather subjective and result from the individual's reaction to a particular situation. The influence of these factors, namely life experiences of a social nature, on the human epigenome is studied by a separate branch of the scientific discipline – behavioural epigenetics. One of the studies conducted in mice demonstrates that trauma experienced can be transmitted to at least the next two generations. The authors of this study used the olfactory (smell) sensation, which they associated with fear of pain (Figure 7.1). The volatile substance acetophenone, reminiscent of the smell of cherry blossoms, with the olfactory receptor Olfr151 responding to acetophenone. Males of the P0 (see Chapter 6 - How environment can affect our genes) were placed in an environment with the odour of acetophenone and, simultaneously with the activation of the sense of smell, received an electrical discharge in a limb that caused pain. After several repetitions, the males with this experience were crossed with a female that had not had this experience. When the offspring of the F1 and F2 generations were exposed to the odour of acetophenone, these individuals showed a high level of nervousness compared with the control group, although they never received an electric shock. Strikingly, even in the F2 generation, which had been created by artificial insemination and rearing by surrogate parents. This experiment served as evidence that sensitivity to acetophenone is a consequence of heredity and is transmitted based on the behaviour of the P0 generation. Evidence of reduced methylation of the Olfr151 receptor in the tested mice also confirmed that epigenetic transfer had occurred.

Figure 7.1 Transfer of the association of olfactory perception and pain to children and grandchildren. The olfactory sensation of the presence of acetophenone was associated with pain from an electrical stimulus in male mice and inscribed in the epigenome of their gametes. Reduced methylation of the olfactory receptor Olfr151 caused the offspring of the F1 and F2 generations to be hypersensitive to the olfactory perception of acetophenone, even though they were never exposed to electric shock.

In examining human behavioural epigenetics, we must go back to the time of World War II. Part of this war was the so-called "Winter War" (1939-1940) and the "Continuation War" (1941-1944) between the Soviet Union and Finland. Using demographic data of Helsinki children born in 1934-1944, a study was conducted to determine whether wartime experiences could be transferred to the next generation, who did not experience the war. During the war, the government offered the voluntary opportunity to evacuate children from Finland to Denmark and Sweden to protect them from the horrors of war. Most children, with the average age of 4.6 years, were evacuated without a parent, with the average duration of separation from their parents was 1.7 years. Children of women who were evacuated as infants were found to have a twofold increased risk of developing one of the psychiatric diagnoses and a fourfold increased risk of developing bipolar disorder or depression compared with the control group. It is interesting to note, however, that psychiatric problems occurred only in the daughters of the evacuees and not in the sons. A similar association was also not observed in children of men who were evacuated in childhood. A negative impact on offspring was also found in the context of prison camps during the American Civil War around 1864. The study was based on the medical records of approximately 4,600 children of fathers interned in overcrowded camps with poor sanitation and inadequate nutrition, and on records of 15,300 children whose fathers were war veterans but not prisoners. Observation of these two groups revealed that the sons and grandsons of ex-captives had an 11% higher mortality rate due to cerebral haemorrhage and an increased risk of dying from cancer. Overall, this appeared to be more of a male line of inheritance, and the health of the daughters and granddaughters was not affected.

On September 11, 2001, there was a terrorist attack on the World Trade Centre in New York, known as the "Twin Towers". Two hijacked planes crashed into the Twin Towers, bringing down both towers. A group of 38 pregnant women who survived this terrorist attack participated in the research of Rachel Yehud, a professor of psychiatry and neuroscience who specialises in post-traumatic stress disorder. PTSD is an anxiety disorder that some people develop after experiencing an extremely stressful event. The triggers for this disorder can vary in severity depending on psychological resilience and experience, which is why not all 38 women developed it. Cortisol is a stress hormone whose level increases in a person in difficult situations, but in women it decreases rather than increases in PTSD. In addition, the expression profile of 17 other genes involved in immune response, nerve signalling, and brain function was altered. The researchers also measured the cortisol levels of the children born to these women. If the women were in their second or third trimester when the Twin Towers fell, the newborns also had lower cortisol levels. The exact explanation of the mechanism is still being studied, but it is more than certain that the traumatic event experienced by the mother during pregnancy had an impact on changing gene expression in the next generation. And because cortisol also plays a role in food metabolism, the offspring of people with PTSD are more sensitive to stress as food deprivation, and vice-versa, they have a higher risk of obesity in an environment where they have enough food.

Another phenomenon over which humans have little influence is the occurrence of natural disasters such as volcanic eruptions, earthquakes, tornadoes, floods, fires, or landslides. One of the studies that examines the effects of a natural disaster during pregnancy focuses on the ice storm that occurred in Quebec in 1998. This storm killed 28 people and injured 945. It was a 6-day period of freezing rain that formed layers of ice up to 11 cm thick on power lines and poles, leaving more than four million people without electricity, and in some places, it took a month to restore power. The 1998 ice storm is ranked as the costliest natural disaster in Canada's history, affecting more people than any other natural event in Canada. Women who were pregnant at the time underwent an assessment of the extent of suffering they experienced, both objectively and from their subjective perspective. The children born were examined for their DNA methylation levels at age 8 and then at age 15. Indeed, there was a change in methylation levels in 957 genes whose functions were mainly related to the immune system. This study showed that prenatal stress can affect the epigenome, with potentially long-lasting effects.

Economic and social background has an influence on the development of intellect

A study on the effects of child bullying on gene expression involved 28 pairs of identical twins, one of which was always a victim of bullying and the other was not. Serotonin is a neurotransmitter that helps transmit nerve impulses, and as a hormone, it causes vasoconstriction and affects sleep and thermoregulation. Serotonin is generally considered the hormone of happiness and well-being. Deficiency of serotonin manifests itself in depressed mood, sleep disturbances, and depression. For this research, the gene studied codes for the serotonin transporter (SERT). The result of the study was that the DNA methylation level of the SERT gene was increased in the bullied twin (in comparison with the non-bullied sibling). The consequence of the increased methylation is the weak production of the transporter that has to mediate the entry of serotonin into the cells (Figure 7.2). At the same time, a change in the response to cortisol during periods of stress was also observed. This is another of the studies demonstrating that early stress exposure has effects on the state of the epigenome, alters stress tolerance, and can significantly affect child development.

Figure 7.2 Bullying alters serotonin receptor expression. Bullying of one of the identical twins was observed to increase methylation of the promoter of the serotonin receptor gene, reducing the amount of serotonin in cells.

Economic background can also have an impact on the epigenome. In 2017, a published study focused on a sample of 1,000 girls who were divided into "low-income families" and "high-income families" groups based on the household in which they lived. Differences in brain development were found, that was most evident in poorer girls who lived in neighbourhoods where they were exposed to wealthier families. Such differences manifested as a thinning of the cerebral cortex, which is a commonly measured indicator of brain tissue development. The cerebral cortex is where conscious activity takes place. Expression of genes encoding the glucocorticoid receptor (NR3C1) and the androgen receptor (AR) was suppressed in girls from poorer backgrounds. These two receptors respond to stress hormones and are associated with the occurrence of depression. Interestingly, boys showed no such disparity, which may be explained by the fact that they have higher levels of testosterone, which can block hormones activated by social stress.

The epigenome can also be influenced positively

Diet can also have a positive effect on the epigenome – for example, positive effects have been observed in colon cancer cells after administration of cruciferous vegetables. Broccoli, Brussel sprouts, and cabbage contain a high concentration of a sulfphur-containing compound called sulphoraphane. This compound was found to affect histone acetylation, which alters the packaging of DNA into chromatin and thus regulates gene expression. In tumour cells, increased activation of genes that can suppress tumour growth was observed after sulphoraphane administration. It is not an isolated case that a substance contained in vegetables shows a positive effect in the form of donors of chemical groups for epigenetic modification. DNA methylation can be influenced by the intake of folic acid (vitamin B9, folate), which is contained in leafy vegetables, legumes, broccoli, and eggs. The intake of folic acid is important especially before and during the first months of pregnancy, where it also serves to prevent foetal spina bifida.

Just as gene expression is affected by a negative factor (electroshock associated with the smell of acetophenone, smoking, or a traumatic experience), some scientific teams have focused on studying a positive emotion - laughter. The researchers used young rats as a model, exposing them to tactile stimulation, namely tickling, which triggered their positive emotions. Gene expression was compared in the hypothalamus of the tickled and non-tickled (control) groups. After four weeks of tickling the rats, 321 out of 41,012 genes showed a change in expression. Of these, the expression of 136 genes was increased more than 1.5-fold and 185 genes were expressed less. The genes with altered expression were mainly involved in neuronal signalling and behaviour related to the feeding process.

The effect of laughter on the epigenetic state of cells was later observed in humans. Type II diabetes is a chronic metabolic syndrome in which the body is less sensitive to insulin due to insulin resistance, resulting in high blood glucose levels. In one study participants with type II diabetes were divided into two groups - one listened to a lecture, the other to a humorous skit. An hour and a half after the lecture or skit, samples were taken to compare gene expression. Of the 18,716 genes analysed, the group that had listened to the humorous skit was found to have a change in the expression of 23 genes, 8 of which were expressed more strongly and 15 less strongly. The change occurred in genes involved in immune processes, cell division, or programmed cell death. Another study examining the effect of laughter on diabetics showed that the expression of the gene encoding the protein which is responsible for the occurrence of complications in diabetes, was reduced in laughing patients. In addition, an increase in the expression of 15 genes involved in the immune system was found, 14 of which are related to the natural killer cells of the immune system. These genes were further researched in mice, where it was shown that these genes could reduce glucose resistance.

Did you know that...

...individual studies describing the effects of the environment on the health of humans or their offspring through epigenetic changes should be taken with a grain of salt? Research on epigenetics is relatively difficult and comes with many limitations, such as the need for a large statistical sample, observation of multiple generations in succession, and the need to better understand individual interactions between epigenetic systems. Epigenetics is still a relatively young scientific discipline that has already yielded some success in understanding the effects of the environment on human health or disease. However, it holds great potential for the future which is waiting to be discovered. While epigenetics began with the identification of specific changes that can be used as biomarkers to detect a particular disease, in practice, this approach still encounters low sensitivity and specificity of these biomarkers in most cases. It is not an isolated occurrence and effect of DNA methylation on specific sequences, but a complicated combination of all epigenetic mechanisms. However, in terms of the current state of knowledge, it is perhaps best described by the thought, "We have enough evidence for humanity to do further and more detailed research, but we probably do not have enough results yet to say exactly how it works ." Well, from what is known today, we can conclude that it does not matter what a person is, where they live, or what they experience. There are mechanisms, although they are still poorly understood, that can transmit certain traces of the parents' lives to their offspring, perhaps even beyond. This is also the reason why, given today's knowledge of epigenetic regulations, it is more important than ever to maintain a healthy lifestyle.

Want to know more?

Website & video: Insights from identical twins

Epigenetics and Human Disease Zoghbi & Beaudet (2016) Cold Spring Harbour Perspectives in Biology 8)2):a019497

Website and information: Dutch Hunger Winter study

Epigenetics and Lifestyle Alegría-Torres, Baccareli & Bollati (2011) Epigenomics 3(3):267-277

Chapter 8: Your cells are stressed too

Stress is a problem for many people nowadays because it can cause serious health problems and complications. However, stress affects not only the whole organism, but also individual cells. At the cellular level, stress can be defined as a set of biochemical reactions that are activated when internal or external conditions change. If the cell does not react correctly and quickly enough to these changes, it can even lead to death. Among the main factors that trigger stress in cells are mechanical damage (cell wall damage or damage of cytoplasmic membrane, pressure exerted on cells) and the action of biological (viruses), chemical (toxic substances, for example alcohol) or physical (UV radiation) agents.

In the study of cellular stress and cellular response, temperature stress has played a key role because it is easily induced in laboratory conditions. Temperature stress is generated by a change in the temperature of the environment, which can affect many intracellular processes. Optimal temperature is crucial to ensure the proteins fold correctly, i.e. achieve the correct conformation, to function properly. During folding, the parts of proteins called active domains, which are crucial for interaction with other proteins or with DNA, are located on the outer parts of protein to make them accessible for reactions. The correct conformation is ensured by chemical bonds and interactions between the functional groups of individual amino acids. However, these bonds are sensitive to changes in temperature, and as soon as there is an increase in temperature, they are disrupted. The disruption results in changes of protein conformation and this protein cannot perform its functions correctly. For example, transcription factors are proteins whose main function is to interact with DNA and regulate transcription. In order to be able to bind to DNA, they have DNA-binding domains in their structure. For proper folding of the transcription factor, the DNA-binding domains must be located on the surface where they can interact with target DNA sequence. However, if there is a change in conformation caused by, for example, temperature stress, these domains can be covered by another part of the protein, which prevents them from binding to DNA. Due to the incorrect conformation, the transcription factor is subsequently unable to fulfil its function and cell must quickly resolve the situation. There are two possible ways to deal with this situation. Either the cell marks the protein for degradation (by a ubiquitin molecule), or the cell can repair the damaged protein. For repair, special proteins called "heat shock" proteins (Hsp) or chaperones are used. Chaperons bind to the targeted damaged protein, break the wrong bonds and restore the original conformation of the protein. During the response to stress, cells preferentially use chaperones, because the degradation of a damaged protein and the synthesis of a new one is an energy-intensive process. However, such correction of protein conformation cannot take place in cells under all conditions. If the temperature in the cell is elevated for a long time or constantly rises, the chaperones themselves may be damaged, resulting in cell death. In addition, Hsp proteins are not only involved in the response to temperature stress, but also in the response to other agents, such as UV radiation or starvation. In cells, they have their physiological function even outside of stressful situations, for example, they have a role in the mutual interaction between proteins, in which they ensure the correct conformation and also prevent protein aggregation.

Changes in conditions cause oxidative stress in cells

In addition to temperature changes, other factors can cause stress in the cell too. Oxidative stress, which is defined as a disruption of the balance between the concentration of oxidants and antioxidants in the cell, is very common and dangerous for the cell. Oxidative stress is caused not only by changes in external but also internal conditions, for example by damage to various cellular structures. Oxidants or reactive particles are molecules that, if they are presented in excess in the cell and at the same time this excess is not removed quickly enough by antioxidants, will cause serious damage to macromolecules, especially proteins, lipids and DNA. The main source of reactive particles in the cell are mitochondria, which are created during cellular respiration when adenosine triphosphate (ATP) molecules are formed on the inner mitochondria membrane (Figure 8.1). ATP is the main source and storehouse of energy for cells and can be released at any time by splitting the so-called high-energy bonds.

Figure 8.1 Adenosine triphosphate (ATP) molecule. High-energy bonds between phosphate groups are represented by a red wavy line, the splitting of which release energy in the cell.

During the ATP synthesis, a large number of electrons are released, which must be stored somewhere, and most often the location for storage is the oxygen molecule. Since oxygen has a high electronegativity (in the periodic table of elements, it is located in sixth group, so it lacks only two electrons in the second valence layer to reach the full complement of eight electrons). Oxygen can bind electrons relatively strongly to form unstable particles known as reactive oxygen species (ROS) (Table 8.1). The most dangerous ROS for cells are the these with extremely short half-life, namely superoxide (O₂•-) and hydroxyl (OH•) radicals. The superoxide radical is produced in cells naturally during cellular respiration and the hydroxyl radical by the Fenton reaction, when in the presence of metal ions (Fe²⁺ or Cu⁺) hydrogen peroxide is degraded into a hydroxyl radical and a hydroxide anion.

Reactive species		Average time of occurence
Radicals	Superoxide (O2^·-)	10^-6 seconds
	Hydroxyl (OH^·)	10^-10 seconds
	Peroxyl radical (ROO^·)	17 seconds
	Nitric oxide (NO^·)	a few seconds
Non-radical compounds	Hydrogen peroxide (H₂O₂)	Stable
	Singlet oxygen (¹O₂)	10^-6 seconds
	Ozone (O₃)	a few seconds
	Nitrogen trioxide (N₂O₃)	a few seconds
	Nitrous acid (NHO₂)	a few seconds

Table 8.1 Examples of some reactive oxygen species (ROS) with the average time of occurance in the cell.

Since the processes that cause the formation of reactive particles occur naturally in cells, there are of course mechanisms for their regulation. These are referred to as antioxidant mechanisms and are provided by molecules called antioxidants. Depending on how antioxidants perform their function, they are classified as enzymatic and non-enzymatic antioxidants. Enzymatic antioxidants are protein complexes that ensure the breakdown of reactive particles into stable, safe molecules. The best-known enzymatic antioxidants are superoxide dismutase, catalase, and glutathione peroxidase.

Superoxide dismutase is an enzyme that ensures the decomposition (dismutation) of the superoxide radical into hydrogen peroxide and molecular oxygen. Since the superoxide radicals are produced primarily in mitochondria, superoxide dismutase also is primarily located there in order to eliminate superoxide as soon as possible. Activity of superoxide dismutase produces hydrogen peroxide, which is also a reactive particle. While hydrogen peroxide is not as dangerous for the cell as the superoxide radical, if it is not eliminated, it can enter the Fenton reaction and creates an extremely reactive and dangerous hydroxyl radical. To prevent this, the cell eliminates hydrogen peroxide with the action from another antioxidant catalase, to form a water molecule and molecular oxygen. In addition to catalase, the cell can also activate glutathione peroxidase, which also eliminates hydrogen peroxide with the help of the non-enzymatic antioxidant glutathione. Which enzyme the cell chooses depends on the conditions inside the cell. If the concentration of peroxide is low, the cell will primarily use glutathione peroxidase because it eliminates hydrogen peroxide relatively slowly, even at low concentrations. However, as soon as hydrogen peroxide concentration rises sharply, glutathione peroxidase cannot effectively degrade it, thus catalase is activated, which eliminates the toxic hydrogen peroxide more quickly.

The second group of antioxidants that the cell uses to eliminate reactive particles are non-enzymatic antioxidants. They are small molecules that can remove or give an electron to reactive particles and thus return them to their original stable state. Such "used" antioxidants can either be degraded in the cell or recycled and used to eliminate other reactive particles. The advantage of non-enzymic antioxidants compared to enzymes is their size - they are much smaller than enzymatic antioxidants, so they can move relatively quickly to places that are inaccessible to the enzyme. The best-known non-enzymatic antioxidant is glutathione. It is a short peptide that contains the amino acid cysteine with a thiol functional group (-SH) in its structure. Under standard conditions, a hydrogen cation is bound to sulphur. However, in the presence of reactive particles, oxidation of the thiol group occurs, i.e. the transfer of an electron and formation of a disulphide bond (S-S) between two glutathiones (Figure 8.2). Such cysteines can no longer donate more electrons, so they are returned to their original state by the enzyme glutathione reductase.

Figure 8.2 Mechanism of reactive particles elimination by glutathione. Glutathione is a small peptide composed of the amino acids glycine (Gly), cysteine (Cys) and glutamic acid (Glu). In case of reactive particles presence in the cell, oxidation of thiol groups (-SH) of cysteine occurs and disulphide bonds (S-S) are formed between two glutathiones.

To cause oxidative stress in the cell, the concentration of reactive particles must be so high that antioxidants cannot remove them effectively. This condition can occur either due to the influence of the external environment, i.e. reactive particles can enter the cells by passing through the cytoplasmic membrane, or they are created in excessive amounts directly in the cell due to the influence of foreign substances, or by processes inherent in the cell (for example, during cellular respiration). If the reactive particles are not eliminated quickly enough, damage of various macromolecules can occur. Oxidation of macromolecules is random, taking place based on which are located near reactive particles.

One of the most frequently oxidized macromolecules in cell are proteins. Their oxidation can cause a change in conformation, overall charge, fragmentation of the amino acid chain or aggregation. The most common oxidative damage of proteins is carbonylation. In this process, the carbonyl group (-C=O) binds to amino acids in the protein. Carbonylation can occur in several places of the protein, which can significantly change its conformation (Figure 8.3A). The cell is unable to repair such damage, so the protein have to be degraded. The second relatively frequent damage is the formation of disulfide bonds, like in glutathione. Disulfide bonds may not only occur between two proteins, but also within one protein (Figure 8.3B). With such damage, the active site may be covered, or another part of the protein may be exposed, which may react with other proteins in the cell, thus causing protein aggregation. However, this modification can be easily repaired so protein does not have to be degraded.

Figure 8.3 Oxidative damage of proteins. A. Protein carbonylation which cause a change in conformation. B. Formation of disulfide bonds within the protein.

The second often target of reactive particles are lipids. Lipids are the main component of membranes, placed right next to each other to form a phospholipid bilayer. They are composed of a hydrophilic head, which is oriented to the environment (toward the intracellular and extracellular space) and a hydrophobic tail composed of higher fatty acid tails (Figure 8.4A). The main task of lipids are to form a partially permeable membrane between the environment and the inside of the cell. However, reactive particles presented in the cell can oxidize fatty acids chains, causing them to bend. The oxidized chains change conformation leading to the movement of lipids in the bilayer and therefore creating a hole in the membrane (Figure 8.4B).

Figure 8.4 Oxidative damage of lipids. A. Lipids composed of a hydrophilic head and a hydrophobic tail placed next to each other in the membrane. B. Oxidation of the lipid tail causes a change in its conformation and the appearance of a hole in the membrane.

A third macromolecule that can be targeted by reactive particles is DNA. Although DNA is protected in the nucleus, covered by nuclear proteins and nucleus membrane, reactive particles still manage to reach and cause damage to DNA. Oxidative damage of DNA is relatively frequent, the cell must repair 10,000-100,000 instances of them per day. The most common are oxidized DNA bases, of which guanine is the most susceptible to this type of damage. Oxidation of guanine by reactive particles produces a modified 8-oxoguanine. It differs from standard guanine only by the presence of oxygen on the eighth carbon. Although this change is not noticeable at first glance, it causes 8-oxoguanine to pair with thymine instead of cytosine. If the cell does not repair the damage, a mutation will occur after DNA replication.

Another type of DNA damage that can result from oxidative stress are strand breaks, where the sugar backbone of DNA is oxidised and thus is broken, or interrupted. Primarily, single-strand breaks are formed (Figure 8.5A), because reactive particles can disrupt only one strand at a time, but if co-oxidation of both strands in close proximity occurs, the hydrogen bonds between the bases are not able to hold both strands together and a double-strand break occurs (Figure 8.5B). For cells, double-strand breaks are a serious damage, because they cause chromosome fragmentation, and the cell cannot replicate or transcribe such damaged DNA (more in chapter 5 - Mutations: how they arise and what to do with them).

Figure 8.5 Formation of single-stranded and double-stranded DNA breaks.

Reactive particles have important functions in cells too

So far, we have only mentioned the negative properties of reactive particles (the various types of damage they cause), but these molecules also have their physiological functions in the cells. They are used, for example, as signalling molecules, which means that they ensure communication between cellular structures, but also between individual cells. Reactive particles are ideal to act as a signal molecule for several reasons: they are small, so they can relatively easily pass through membranes and thus reliably transmit a signal, their concentration in the cell can increase sharply if necessary (thus the cell can immediately react to various stimuli), and they are very quickly eliminated as soon as the signal was recorded and the cell began to react.

The importance of reactive particles as signal transmitters can be illustrated by the example of mitochondria, which naturally generate them. If the cell has functional and undamaged mitochondria, it generates reactive particles of a certain concentration and subsequently degrades them by antioxidant mechanisms. If any component of the respiratory chain is damaged, the concentration of reactive particles starts to sharply rise. The cell also notices this change due to the increased occurrence of oxidative damage. To reverse this condition, various repair mechanisms are activated to either repairs or degrades the damaged component of the respiratory chain. In extreme conditions, when the damage is severe or extensive, the entire mitochondrion can be eliminated.

A very interesting example of reactive particles role in cellular signalling is the role of nitric oxide (NO) in the relaxation of blood vessels. Excretion of this substance is the body's natural reaction to increased blood pressure and therefore increased blood flow in the vessels. In order to ensure normal blood flow, the communication of up to three types of cells is necessary - nerve, vascular and endothelial (forms the inner lining of blood vessels) cells. The nerve cell is the main signal messenger from the brain to the blood vessels. With the help of small molecules of acetylcholine, the nerve cell sends a signal to the endothelial cell to relax the vessels. As a response to the presence of acetylcholine, the endothelial cell begins to form nitric oxide, which quickly and efficiently passes through the cell membrane and the intercellular space to the blood cell. In this case, nitric oxide is not generated by the endothelial cell randomly when some process is damaged, but generated purposefully by cell to transmit a signal. The result of such effective communication is the relaxation of the blood vessel structure, which allows better blood flow. The discovery of the physiological function of nitric oxide, a reactive particle occurring in an influent state, as a signal transmitter was so significant that the Nobel Prize was awarded in 1998 for this discovery.

Exposure to stressful conditions that do not kill the cell can help it survive

A phenomenon known as adaptive response is closely related to stress and the response to it. It can be described as cellular "immunity", where a stress that was not lethal to the cell, nor did it cause serious damage, helped prepare the cell for the action of a more severe stressor. This more severe stressor could kill the cell by its action, if cell was not already prepared thanks to the first impulse. The explanation of this phenomenon is that the first stress impulse activates several repair and defence mechanisms. Subsequently, with the second impulse, the cell no longer had to wait for the necessary proteins to be synthesized and could defend itself immediately.

The adaptive response works under different forms of stress. It was discovered during the action of ionising radiation, when scientists found that after the cells were exposed to ionising radiation, DNA damage was lower in the case that the cells were previously affected by lower doses of radiation. The adaptive response not only manifests when both impulses are the same, but it can also be triggered by another impulse, which must activate appropriate defense mechanisms in the cell. The cell will then use them during the second impulse. However, the condition is that the first impulse must not be too strong, otherwise it could weaken the cell, nor too weak so it is unable to activate all the necessary mechanisms.

Tumour cells are stressed too

You can read more about the transformation of a healthy cell into a tumour in chapter 9 - When cells go crazy, but at this point we will mention that in the process of tumour transformation, when an initially healthy cell undergoes a large number of changes that fundamentally affect many aspects of its functioning, this cases the cell stress. A direct consequence of the rapid division and accumulation of mutations is the stress of DNA damage. Rapid division is also accompanied by errors in the division of chromosomes into daughter cells, which results in the formation of cells with a defected karyotype, and the cell thus finds itself in mitotic stress. The instability of the genome is further manifested by changes in the level of proteins produced in the cell, which causes proteotoxic stress. Non-functional proteins or the accumulation of proteins can cause their insufficient removal and possible aggregation, which has a toxic effect on the cell. The stress of lack of oxygen also has a significant impact, as the imperfect vascular network that forms in the tumor cannot cover the demands of the growing tumour. There are also changes in the expression of genes which could help the cells deal with this condition, but these changes give them aggressive properties. The consequence of adaptation to lack of oxygen is the suppression of breathing and the transition to metabolism without the need for oxygen, which is accompanied by unnatural metabolic stress. And last but not least, there is also the stress of damage to immune control. Cells (not only tumour cells) can thus experience a large number of different stressful situations, which they have to deal with during their lifetime and thus ensure the proper functioning of the organism even in adverse environments.

Did you know that...

...the increased concentration of reactive oxygen species and the subsequent occurrence of oxidative stress in cells are associated with many serious human diseases, for example leprosy or the development of neurodegenerative diseases such as Alzheimer's and Parkinson's disease? A team of scientists from Mexico studied the use of melatonin in patients with Parkinson's Disease. Melatonin is an antioxidant that can absorb reactive particles in cells, thereby preventing serious damage to cells. In this study, the authors monitored markers of oxidative stress, namely protein carbonylation, oxidative damage of lipids, activity of antioxidant enzymes and activity of respiratory chain complexes in mitochondria in two groups of patients. The first group was given melatonin twice a day (at lunch and approximately 30 minutes before bedtime), while the second (control) group received a placebo. The experiment lasted 30 days and the results were that patients who were given melatonin showed a reduction in markers of oxidative stress compared to the control group and recovery of the respiratory chain complex activity. These results suggest that melatonin could be part of the treatment of Parkinson's Disease.

Want to know more?

Reactive Oxygen Species in Plant Biology, Bhattacharjee, S., first edition, Springer (2019).

Antioxidant Compounds and Their Antioxidant Mechanism, Santos-Sánchez, N. F., Salas-Coronado, R., Villanueva-Cañongo, C., Hernández-Carlos, B., v Antioxidants, IntechOpen (2019).

Oxidative stress and antioxidant defense, Birden, E., Sahiner, U. M., Sackesen, C., Erzurum, S., Kalayci, O., Worlds Allergy Organization Journal (2012).

Adaptive response: some underlying mechanisms and open questions, Dimova, E. G., Bryant, P. E., Chankova, S. G., Genetics and Molecular Biology (2008).

Function of reactive oxygen species during animal development: passive or active?, Covarrubias, L., Hernández-García, D., Schnabel, D., Salas-Vidal, E., Castro-Obregón, S. Developmental Biology (2008).

Effect of melatonin administration on mitochondrial activity and oxidative stress markers in patients with Parkinson's Disease Jiménez-Delgado et al. (2021) Oxidative Medicine and Cellular Longevity 2021:5577541

Chapter 9: When cells go crazy

The human body is a multicellular organism composed of cells whose number changes according to age, height, weight, and state of health, but also due to environmental factors. The average number of cells is said to be 37 trillion, and they can be divided into about 200 different types. Just like human beings, the cells of their bodies do not live forever. Most cell types can be replaced by new cells after their death, through cell division, which has already been discussed in Chapter 3 - Meet DNA, the bearer of genetic information. The division is a cyclical process without which life would not be possible. The problem arises when cell division gets out of control.

Cancer is a disease caused by uncontrolled cell division

The term cancer is used as a collective term for the entire group of cancers. The essence of cancer is uncontrolled cell division, where cells can divide faster, at the wrong time, in the wrong place, or despite damage. Tumour cells are our human cells, but due to a change in regulatory mechanisms, they no longer follow the instructions of the rest of the body. They become their own "organism" that behaves in a way that allows them to survive.

The cause of cancer is a failure of the mechanisms that control healthy cell division. It is called a solid tumour when the dividing cells begin to form solid and compact foci located in an organ, for example in the liver or brain. A second type of tumours are hematologic tumours, such as leukaemia, which do not form a solid foci of dividing cells. Although such tumour cells may temporarily clump together, they normally circulate in the blood and lymphatic system, which is why they are called liquid tumours.

Tumour types differ in their ability to invade surrounding tissue

The types of tumours are distinguished according to their ability to spread into the surrounding tissue (Figure 9.1). A non-malignant (also called benign) tumour is usually small and likely to grow for years. A benign tumour has a regular shape and does not interfere with surrounding tissue because it is separated from it by a fibrous capsule that often allows its complete surgical removal. At the same time, the molecules that provide the connections between the individual cells hold the tissue together and prevent it from migrating from its place of origin. It generally poses relatively little risk to the host as long as its size does not threaten the organ from which it originated or if it does not release a large amount of biologically active substances, such as hormones. What makes a tumour malignant is its ability to invade surrounding tissue. This is due to rapid growth due to accelerated cell division. As a result, a large amount of DNA mutations accumulate, further altering the properties of the cells. Intercellular communication and cellular cohesion are also disrupted. The shape of a malignant tumour is irregular and narrow as it invades and damages surrounding tissue. At the same time, due to the high demand for nutrients and oxygen, a solid malignant tumour begins to form new blood vessels that supply the tumour with oxygen and nutrients.

Figure 9.1 Benign and malignant tumour. A benign tumour (A) is demarcated from surrounding tissue, while a malignant tumour (B) begins to invade and damage healthy cells.

A metastasis occurs when some cells are torn away from the primary tumour and become lodged elsewhere in the body (Figure 9.2). Tumour cells can migrate through the blood and lymphatic systems to very distant parts of the body, where they become established and form another tumour. Approximately one in every 1,000 to 10,000 tumour cells that enter the bloodstream colonizes other tissues. Metastases are the most dangerous in terms of the severity of consequences for the body, as evidenced by the fact that they are responsible for up to 90% of deaths from various types of cancer.

Figure 9.2 Metastatic process. The primary tumour arises in the mammary ducts of the breast. The cells of the mammary gland epithelium are separated from the surrounding ligament by a basement membrane. The tumour cells penetrate this, mechanically enter the blood vessel through the wall, and are carried away by the body. At some point, they attach to the blood vessel wall and migrate the other way to another tissue, for example, the brain. There they attach and form a micrometastasis, in which division continues until a metastasis has formed.

Different types of cancer are classified according to the origin of the cells

There are several types of cancer classification. One of the most common is the classification according to the original tissue type from which the tumour cells originate. According to this classification, a distinction is made between carcinomas which are from epithelial cells (e.g., lung, breast, prostate, pancreas, colon), sarcomas from connective tissue (e.g., bones, cartilage, fat, nerves), germ cell tumours (from pluripotent cells e.g., testis, ovary), blastomas (immature precursor cells of the embryo), lymphomas and leukaemia’s (blood lymph cells - e.g., spleen, thymus, lymph nodes).

However, a tumour cannot be thought of as a mass of identical cells. Rather, it is a very heterogeneous tissue in terms of the presence of different cell types, but also changes at the level of individual cells. The changes in the cells are usually caused by mutations in the DNA, which accumulate during tumour development. Tumours differ not only in their location in the body, the architecture of the tumour, the response of tumour cells to treatment, the effect of the tumour on surrounding tissue, and the ability to metastasize but also in the response of the immune system and the genetic nature of cancer. These are all aspects that are different for each patient. For this reason, the likelihood of finding a single drug in the future that will cure all cancer patients is very low.

Cancer begins at the genetic level

Cancer has accompanied mankind since time immemorial, so it is not surprising that attempts have been made to explain its occurrence. For some cancers, it has been possible in the past to identify a definite cause - a certain aspect of the environment. For example, skin cancer was found to be caused when people were frequently exposed to UV-laden sunlight. Ernest Hemingway, in his 1952 book The old man and the Sea, wrote about skin spots caused by frequent sun exposure or while fishing. Environmental factors that cause or promote tumour growth are called carcinogens. To date, more than 1,000 substances are known to be carcinogens to some degree, and this number is constantly increasing. Carcinogens can be of various natures: biological (viruses - e.g., papillomavirus, responsible for cervical cancer), chemical (smoking, chemicals - e.g., asbestos), or physical (UV radiation, radiation).

Environmental factors may contribute to the development or progression of the disease, but the main cause of tumour transformation is always genetic alterations or changes in gene expression caused by changes in the epigenome (see chapter 7 for more details). These changes can occur even without the influence of the external environment, because of the natural error rate of DNA polymerases that copy DNA before cell division. Essentially, these are "errors" in information, with errors in some genes being much more dangerous for cancer development than others. Mutations in 3 groups of genes are particularly dangerous (Figure 9.3). The first group is the proto-oncogenes, which code for proteins that activate division in healthy cells and prevent premature cell death. The standard proto-oncogene of a healthy cell can transform into an oncogene that promotes cell division and blocks cell death, even if this is not appropriate for the cell. Another group are the tumour suppressors, which ensure that cells do not divide too early. Tumour suppressors also support the cell when it realizes it is damaged, so that the cell dies according to plan and harmlessly, not affecting the surrounding cells. However, when the tumour suppressor gene is damaged, the resulting protein blocks division and promotes the death of the damaged cell. Combined with the ease of division caused by the activated oncogene, this leads to uncontrollable cell division. A third, very important group of genes are those that code for proteins that are important for maintaining DNA integrity. Their job is to correct DNA mutations, and if this is not done, the mutations accumulate.

Figure 9.3 Comparison of normal and cancerous cell division. Loss of function of the mutant tumour suppressor and/or gain of function of the (proto-)oncogene led to a proliferation of tumour cells.

But what is the cause of the deletion of a tumour suppressor gene or activation of an oncogene? The mechanism of conversion of a proto-oncogene into an oncogene is often that the gene is shifted within the DNA and thus enters the domain of another promoter that triggers its expression differently (Figure 9.4). Duplication of the gene into multiple copies may also occur and simultaneous point mutations in which only one nucleotide is changed are not uncommon. A nucleotide substitution in a gene may result in an amino acid exchange. However, a point mutation can also have an effect in a non-coding region that serves to regulate gene expression. If the result is that too much oncogenic protein is produced, the protein is more active, or the resulting protein is resistant to removal, it is enough for a point mutation to occur in just one copy of the gene (in one allele), and the cell has a problem. The effects of such a change are dominant and occur even if the second allele of the gene is perfectly fine.

Figure 9.4 Transformation of a proto-oncogene to an oncogene.

The mechanism of damage to tumour suppressor genes and DNA repair genes differs in part from the activation of oncogenes. These genes are more likely to have point mutations or complete gene deletions, or a change in their epigenetic regulation, e.g., methylation of the promoter, which prevents transcription of the gene information and formation of the protein. The main difference is that mutations in tumour suppressor genes and repair enzymes result in the loss of function of the protein derived from one allele of the gene, but the product of the second (functional) allele is usually sufficient to ensure function. A mutation in both alleles of a gene is therefore necessary for the development of cancer, since the effect of the mutation in this case is recessive. However, it must be said that the body has several safeguard mechanisms in place, so a mutation in only one gene is not sufficient. For the development of cancer, a combination of several mutations in genes are required, with the order in which the changes occur is also important.

A predisposition to cancer can be inherited

Mutations in proto-oncogenes, tumour suppressor genes, and substances that ensure genome integrity can not only be acquired during life (through exposure to carcinogens or DNA replication errors) but in some cases can also be inherited from a parent through the germline. In this case, the affected individual has an increased risk of developing cancer during his or her lifetime, and this risk can also be passed on to his or her offspring if they inherit the mutant allele. An example of a familial form of cancer is retinoblastoma, which is caused by the loss of function of the tumour suppressor gene RB1. The standard tumour suppressor protein Rb prevents the cell from starting DNA replication and dividing under certain circumstances. Since mutations in the tumour suppressor gene have a recessive effect, both alleles must be mutated to trigger cancer development - this is then referred to as the need for two interventions: an inherited mutation turns off one allele, and a mutation acquired during life turns off the other. If mutations occur independently in all alleles of the gene during life, it is a sporadic occurrence of retinoblastoma (Figure 9.5), which is relatively rare and usually affects only one eye. However, there is also an inherited form of retinoblastoma, which is also one of the most common childhood cancers (Figure 9.5). The predisposition to retinoblastoma means that the mutated RB1 allele is inherited through the germline. When one allele is mutated, such patients are born with only one healthy allele. Although it is sufficient for a normal life, this means that a person with only one functional allele of the tumour suppressor Rb is more susceptible to environmental carcinogens, which may also cause mutation of the second allele. In the case of an inherited predisposition, not only one eye is usually affected, but both. An inherited predisposition to cancers can also be the cause of breast cancer, ovarian cancer, or colon and rectal cancer, for example.

Figure 9.5 Inheritance of an allele with a mutation in the RB1 gene results in an increased risk of hereditary retinoblastoma. Development of retinoblastoma requires the deletion of both copies of the tumour suppressor gene RB1. In hereditary retinoblastoma, the damaged gene is inherited from one parent, and a single mutation is sufficient to develop the disease (bottom row). In sporadic retinoblastoma, two independent mutations must occur during life.

Properties of tumour cells

Although different types of tumours may behave very differently in the same patient, certain features are typical for tumour cells in general (Figure 9.6). There are 10 typical characteristics of tumour cells, of which the first 6 features are critical for human cells to undergo a cancerous transformation and form a malignant tumour:

1. Signalling for non-permanent division - In healthy cells, cell division is activated by the binding of a growth factor to a receptor on the cell surface, which triggers a whole cascade of events. Tumour cells have mechanisms capable of constant signalling that drive the cell to divide more and more, either by activating division independently of growth factors (a mutation in the receptor that causes it to always be activated), or the tumour cell can produce many of its own growth factors which it can respond to itself.

2. Resistance to division suppressors - Neighbouring cells normally produce molecules that signal tumour cells to stop dividing. However, the tumour cells ignore these signals, often due to decreased production of receptors that should catch them. The signal to stop dividing should also be contact inhibition, where the cell bumps into other surrounding cells while dividing, so it decides to stop dividing (so they all fit into place). Tumour cells, however, bypass this control mechanism and continue to divide.

3. Resistance to cell death - The surrounding healthy cells notice that uncontrolled division of some cells is taking place, which negatively affects them by depriving them of nutrients and physically limiting them. Therefore, the healthy cells send signals to the tumour cells, prompting them to undergo programmed cell death. However, the tumour cells fight back by activating factors that prevent death or inactivating factors that lead to death.

4. Replicative immortality – With some exceptions, all healthy cells can undergo only a limited number of divisions before they become old. Linear chromosomes loose DNA at their ends (telomeres) and after reaching a certain number of copies DNA is no longer copied. Tumour cells often circumvent this problem by possessing a functional telomerase enzyme (more on this in Chapter 3 - DNA as the bearer of genetic information) that enables them to replace these lost DNA. Apart from tumour cells, only embryonic cells, germ cells, and stem cells have this ability. Thanks to the ability to maintain the telomeres, tumour cells practically do not age and can divide relatively indefinitely. The disruption of the function of proteins involved in the interruption of division also contributes to this.

5. Invasive growth and metastasis – Tissue-forming cells are not free but bound to each other through the extracellular matrix. Tumour cells can break bonds in their environment, break free from the tissue, and migrate through the blood system to the rest of the body where they can metastasize.

6. Induction of angiogenesis - Rapidly dividing tumour cells have a high demand for the supply of nutrients and oxygen. To achieve a more efficient supply, they create their own blood vessels that direct blood into circulation. The newly formed vascular network is functionally and structurally imperfect, which poses risks to the supply of the tumour.

7. Deregulation of cellular energetics - Despite the formation of blood vessels during rapid tumour growth, there is a lack of oxygen for the cells located in the tumour. The cells solve this deficiency by switching their metabolism to another metabolic pathway that is independent of oxygen. This leads to a change in the characteristics of the tumour cells and their environment.

8. Tumour-induced inflammation - It is known that immune cells are also part of the tumour mass, often invading and causing inflammation to destroy the tumour. These include, for example, CD8⁺ cytotoxic T-lymphocytes or natural killer cells. Other immune cells also arrive at the site of inflammation to produce growth factors and promote the formation of new blood vessels to heal the inflammation or injury. However, growth factors can also trigger excessive division of tumour cells. As a result, up to 20% of tumours develop at the site of chronic infection, for example, in a stomach colonized by the bacterium Helicobacter pylori, resulting in long-term inflammation. Another example is the autoimmune disease Crohn's disease, which can lead to colorectal cancer, or a liver infection with the hepatitis B or C virus, which promotes the formation of hepatocellular carcinoma under certain circumstances.

9. Escape from the immune system - The human body can recognize foreign particles such as viruses, bacteria, or even its own cells when they get sick. When the body recognizes them, the target is marked which triggers an immune response that eventually eliminates it. In the case of tumour cells, the tumour controls a mechanism that allows it to evade the immune system's efforts to destroy it. It does this, for example, by trying to hide from the immune system by altering the proteins on the surface of the cell that might cause the immune system to recognize it.

10. Instability of the genome - Point mutations, small or large duplications, deletions, translocations, or chromosomal aneuploidies are very common in tumours, as discussed in Chapter 5 - Mutations: How they arise and what to do with them. Interestingly, DNA mutations in tumour cells are rare in paediatric cases (only about 0.1 bases per million bases), but in tumours such as lung cancer or skin melanomas that are caused by mutagens, the frequency of DNA mutations is 1000 times greater, up to 100 mutations per million bases.

Figure 9.6 Characteristic features of tumour transformation.

Why does cancer die when cells only divide?

Cancer involves actively dividing cells that, in advanced stages, damage healthy tissue, depriving it not only of space but also of the nutrients that healthy organs need. This causes the organ to stop functioning properly. There are many ways a tumour can damage the human body. If the digestive system is affected, the body cannot absorb nutrients and suffers from malnutrition. If the respiratory system is affected, the problem is that the body does not receive enough oxygen. Damage to the bones can result in too much calcium being released into the blood or weakened bones can break and fail to heal. The liver, in turn, has an irreplaceable role in eliminating toxins from the body, and when it cannot perform this function, the human body is exposed to poisoning. Damage to the bone marrow, in turn, does not allow the formation of a sufficient amount of blood cells, which leads to the development of anaemia, in which the red blood cells do not have enough time to supply the tissues with oxygen. If there are not enough white blood cells, the body cannot fight infections, the lack of platelets is dangerous in bleeding because the body cannot stop it. A brain tumour, in turn, can press on the centres of memory, speech, vision, or orientation, as well as vital control systems, such as the respiratory centre. In some cases, when the cancer can no longer be treated, the body gradually weakens, and the organs fail.

Pancreatic cancer is one of the deadliest diagnoses among all cancers. Approximately 95% of patients with this diagnosis die. The pancreas is a relatively small organ, but it performs a variety of functions. Pancreatic juice is secreted into the small intestine and helps digest proteins, fats, and carbohydrates. The organ is also important because it contains the Islets of Langerhans, which produce the hormones insulin and glucagon, which affect blood sugar levels. It is the weakening and loss of pancreatic functions that lead to a dramatic deterioration in the general health of patients. Part of the reason for the high mortality rate is that there are currently no sensitive methods for diagnosing pancreatic cancer, as the symptoms can be too general, so the diagnosis is often made at a late stage.

Each patient is an individual, and there are already many means to relieve the pain, eliminate the side effects of the disease, and, in the case of terminal illness, improve and prolong the quality of life through palliative care. Modern medicine saves lives, but cancer is still a challenge. While the development of modern medicine has had a decisive influence on the treatment of cancer, with its onset, a worsening of cancer incidence could be initially observed. This is the so-called paradox of modern medicine.

Society has undergone many changes in the last century, which have prevented many deaths. Thanks to this, a larger percentage of the population is growing old. And with increasing age, the risk of developing cancer also increases. With each cell division, the number of which is directly proportional to the lifespan, there is a certain number of DNA errors that occur and so the risk of developing cancer by chance increases, which is also different for different tissue types. It is the same as the lottery: someone who plays every week is statistically more likely to win than someone who plays once a decade. Therefore, in the colon, where cells are replaced every 2-5 days, the risk of developing colorectal cancer increases with age, and is greater than the risk of developing a tumour in the bones. Thus, the paradox is that despite the great advances of modern medicine in the treatment of many other diseases, society has not yet achieved the complete elimination of cancer, but on the contrary, more and more people develop it during their lifetime.

Fortunately, modern medicine also has the dream and ambition to eliminate cancer and has effective means for cancer treatment. Classical cytostatic drugs work on the principle of inhibiting cell division by damaging cell structures, damaging DNA and RNA, or altering metabolism. The problem is that these drugs act non-specifically since they target all dividing cells. Therefore, chemotherapy can also cause hair loss, damage to blood formation, or nausea. In addition to chemotherapy, radiation therapy based on ionizing radiation, surgical removal of the tumour, or (for some diagnoses) bone marrow transplantation is often used in combination. But targeted treatments are also on the rise, including biological treatment and personalized medicine.

The scientific organization International Cancer Genome Consortium for Medicine aims to identify the genetic cause of the 50 most common types of tumours. This would enable the personalised treatment of patients based on the genetic cause of cancer rather than whether it is breast cancer or colorectal cancer. For example, a drug that binds to the receptor HER -2 (receptor for human epidermal growth factor 2), which is involved in the development of breast and gastric cancer, is already being used. The target of personalized medicine is also the protein product resulting from the genetic rearrangement of the Philadelphia chromosome (Figure 9.7). In this process, the ends of chromosomes 9 and 22 are exchanged, resulting in a fusion gene consisting of genes for bcr ac-abl proteins. The part of the chromosome encoding c-abl contains the information for the formation of the proto-oncogene, and the other chromosome contains part of the information for bcr to cause its continuous expression. The proto-oncogene thus transforms into an oncogene, activating white blood cell division and causing the development of chronic myeloid leukaemia. The drug can inhibit the activity of ABL so that the cells stop dividing excessively.

Figure 9.7 Philadelphia chromosome. After a break on chromosomes 22 and 9, chromosomal segments are exchanged, resulting in a gene that generates a fusion protein consisting of the proto-oncogene and the part that causes its constant expression.

Prevention is more effective than treatment

Thanks to modern medical research, it is possible to discover the causes of many types of cancer as well as suitable methods of treatment. However, the most important thing for a person's health is to prevent cancer, or at least detect it at the earliest possible stage. Detection at the earliest stage possible is associated with the best prognosis for treatment and survival. Prevention consists of a healthy lifestyle and special medical examinations. The importance of prevention can be illustrated by two examples. The first example is adenomatous polyposis - a rare autosomal dominant disease. It is caused by a mutation in the tumour suppressor gene APC, which normally serves to regulate the renewal of intestinal mucosal cells. However, in the mutated state, when a person reaches the age of 20, it causes excessive cell division and the formation of many small tumours in the intestine and rectum. These growths are called polyps and are benign in the early stages. However, there is a risk that some of them may become malignant over time. With this diagnosis, on average, malignant transformation occurs around the age of 42, with the development of colon cancer. If polyps are removed at a benign stage, there is a high probability that the disease will not develop. However, colon polyps may not only be this hereditary form, but also the accidental occurrence of a mutation at a later age.

An example of the importance of prevention is breast cancer, which is curable in 75-90% of cases if detected early before the lymph nodes are affected. Breast cancer is best diagnosed by mammography and the sensitivity of a mammogram is such that it can detect a tumour as small as 5 mm, about the size of a pea. At this stage, it is likely that the tumour has not yet metastasized. According to statistics, 8 in 10 women will develop breast cancer.

The occurrence of cancer is essentially a natural process due to a failure of the body's regulatory mechanisms. Some factors of the external environment may contribute to its occurrence or development. However, the main cause of this disease is genetic changes over which we have little control. We can reduce the risk of cancer to some extent by adopting a healthy lifestyle, but the most important thing is to pay attention to prevention and take preventive examinations at the doctor's office. These can help detect cancer at an early stage when it is still treatable. We are also helped by the tools of modern genetics and molecular biology - they enable us to find out what kind of tumour it is, to determine the target sites for the action of drugs, and thus to transform cancer from a fatal disease into a curable one.

Did you know that...

...a large number of mutations accumulate in tumour cells during the onset and progression of the disease - they can contain hundreds of thousands, sometimes even more than a million mutations. However, only a small proportion of them are directly responsible for the development of cancer; these are called "driver" mutations. The remaining mutations in DNA, called "passenger" genes, are remnants of the cell's experience before cancer development and may not be in the domains of oncogenes or tumour suppressor genes. However, there is also a large group of mutations that arise during the transformation of cells into tumours. These set of mutations in tumour cells is usually referred to as the mutational signature, which can make tumours of the same organ in different patients significantly different from each other. A new study aim to sequence the genomes of 19 tumour types from 12,000 patients and create a genetic signature for each. Individual mutation types can be used to determine whether a person has had a sunburn or been exposed to cigarette smoke, for example, and then used to clarify the cause(s) of a particular type of cancer. The authors of the study liken the mutation signature to a sandy beach full of footprints. If you know what you are looking for, you can learn a lot about who was on that beach and what happened there. For example, the study was able to uncover 58 new signatures, which means 58 causes of cancer that we do not yet understand. At the same time, by comparing mutations across genomes it was possible to determine the type of tumour, or if the tumour was causes by an inherited mutation or a random type of disease. In addition, mutation signature data can indicate an appropriate therapeutic target and thus be a useful tool for personalized medicine.

Want to know more?

Hallmarks of Cancer (2000) Hanahan & Weinberg Cell 100(1):P57-70

Hallmarks of Cancer: the next generation (2011) Hanahan & Weinberg Cell 144(5):646-674

The Emperor of all maladies: A biography of cancer by Siddhartha Mukherjee

Chapter 10: Gene therapy

The exponential growth of knowledge in the field of biology, as well as the rapid pace of technological development, are opening up possibilities that until recently would have been considered part of the science fiction genre. This naturally also applies to the field of genetics and its application in medicine. Gene therapy is a promising technology for treating diseases caused by errors in DNA. In humans, there are about 4,600 gene mutations of which have an external manifestation. Interestingly, mutations in a gene can manifest themselves differently in different people. Currently, there are more than 7,100 phenotypes whose molecular basis is known. Some of them are classified among diseases, including various forms of cancer, cardiovascular and neurodegenerative diseases, musculoskeletal disorders, and many others.

Viruses are capable of DNA transfer

In the case of hereditary diseases, medical interventions are largely limited to prevention and treatment of symptoms, rather than addressing the cause of the disease, namely the mutated DNA. A key discovery that led to the development of therapies for inherited diseases was the discovery of transduction, a transmission of DNA by viruses, in the early 1950s. Bacteria-infecting viruses, called bacteriophages, have been shown to be able to transfer heritable traits from one bacterium to another. Several traits could be transferred using bacteriophages, such as the ability to grow on different substrates, fermentation as a source of energy, or resistance to certain antibiotics. In this process, the virus injects its own DNA into the bacterium after contact with the cell. The viral DNA is replicated, resulting in many copies of it in the cell. At the same time, the protein components of the viral envelope, called capsid, are produced by the bacteria. The viral DNA is packaged into the capsid, and the viral particle, virion, is completed. A necessary step prior packaging into the protein particle is cutting the viral DNA to a suitable size. During this process, fragmentation (cutting into smaller pieces) of the bacterial DNA also occurs. In rare cases, this may result in the bacterial DNA fragment being packaged into the newly formed capsid instead of the viral nucleic acid. After bacteriophage progeny assembly, the host cell bursts and the released virions can infect other bacteria. Depending on the information contained in the bacteriophage, an infected bacterium may permanently acquire a new trait after the incorporation of foreign DNA into its genome (Figure 10.1).

Figure 10.1 Transfer of DNA from bacterium Salmonella typhimurium by bacteriophages.

The discovery that DNA can be transferred between cells by viruses and subsequently inserted into the host genome was revolutionary. If such transfer is possible in bacteria, surely there must be viruses that can do the same in humans. Indeed, retroviruses such as HIV (human immunodeficiency virus) are able to insert their genetic information into the genome of the host and then force the cell to express their genes (Figure 10.2). Of course, the use of the HIV virus in medicine would be extremely dangerous, but mankind has several methods by which the viral DNA or RNA can be manipulated so that the originally dangerous virus becomes much safer. To do this, the genes whose products ensure the replication and capsid formation are removed from the genome of the virus. A virus modified in this way can infect the host cell, but is unable to replicate and spread further. In addition, the vacated space in the virions can be filled, for example, by a healthy human gene that we want to transport into the cell and potentially repair the mutated one. However, a major risk in using viruses that are able to insert their genetic information into the host DNA is their poor targeting. It is very important to know where the therapeutic DNA will be inserted. For example, the insertion could disrupt a gene that is important for regulating cell division, potentially triggering tumour transformation of the cell.

Figure 10.2 Life cycle of retroviruses. After identification and binding to a receptor, the virus particle is engulfed by the host cell. The nucleic acid (RNA in retroviruses) is transcribed back into DNA in a process called reverse transcription. The DNA is then transported into the nucleus and inserted into the host chromosome. The gene present on the inserted viral DNA are transcribed and translated by the host cell and assembled into a new viral particle that leaves the infected cell.

The origin of cell therapy

The idea of using viruses as carriers – vectors for transferring DNA into human cells was implemented in the early 1990s, when a foreign gene was introduced into the human body for the first time. TIL lymphocytes(tumour-infiltrating lymphocytes), a class of white blood cells found in or in close proximity to tumours, were isolated and then transfected with a retrovirus carrying the NeoR gene. The product of this gene confers resistance to the antibiotic neomycin to the cell, which can subsequently be used for selection of the transformed cells and simultaneously as a marker for detection of cells with NeoR gene. After administration to the patient, the transformed cells were able to migrate in the body and reach their target, the tumour tissue (Figure 10.3).

Figure 10.3 First introduction of foreign gene into the human body.

The promising beginnings triggered a great wave of enthusiasm. This led to the first clinical trials and, unfortunately, also to the first tragedy. The victim was the then 19-year-old Jesse Gelsinger. Jesse suffered from a disease caused by a mutation in the gene for ornithine transcarbamylase (OTC), an important enzyme in amino acid and nitrogen metabolism. In his case, the disease had “mild” symptoms – he lived on supportive care and a strict diet. He volunteered for clinical trials in which he was administered gene therapy in the form of an adenovirus (ADV) carrying a healthy OTC gene. Although it was supposed to be a virus better tolerated by the immune system, an unfortunate combination of circumstances led to tragedy, and Jesse died as a result of the adenovirus infection. Over the course of a lifetime, people usually overcome several infections caused by ADV in the form of respiratory or digestive illnesses, usually with mild symptoms. This means that our immune system is highly likely to be able to respond quickly to repeated infections. This was apparently also the case with Jesse, who was given a very high dose of virus that likely led to an exuberant immune response. Four days after the therapy, Jesse's body collapsed. Because of this and several similar tragedies, the development of gene therapy has slowed down, with the main goal being to identify possible risks and find appropriate solutions.

Vectors used in gene therapy

One of the priorities was and is the development of new, safer vectors. In addition to retroviruses and adenoviruses, adeno-associated viruses (AAV), which are very well tolerated by human immunity, are currently gaining attention. Non-viral vectors, e.g. fat particles – liposomes, gold particles and polymeric micelles, also seem to be promising. Each of the vectors mentioned has its advantages and disadvantages. Some viruses incorporate their DNA into the genome, while others leave their DNA in the form of episomes. Episomes are located in the nucleus, replicated and inherited along with the nuclear chromosomes without inserting themselves into the host genome. In this case, there is no risk that an important gene will be affected by the insertion. In dividing cells, on the other hand, there is a high probability that the episome will be lost over time and the patient will have to undergo repeated gene therapy.

When selecting a vector, it is also important to know what types of cells the virus can infect. This is referred to as viral tropism. Some viruses only infect dividing cells or are tissue specific, meaning they only infect cells of a certain type. All vectors have a limited coding capacity, which means that only a limited size of nucleic acid can be incorporated into the virion. Thus, if a long gene is to be repaired by gene therapy, this must be taken into account when selecting the vector. Here, non-viral vectors, for example liposomes, prove to be very suitable. They have a much greater capacity and also do not trigger such a strong immune response. On the other hand, they are less specific. Despite the advent of new vectors, it is still necessary to use relatively high doses, which contributes to a strong immune response. For this reason, gene therapy is often combined with the administration of immunosuppressants.

Current state of gene therapies

According to the European Medicines Agency (EMA), gene therapy is the name given to medical products consisting of or containing recombinant nucleic acids. These are used or administered with the aim of regulating, repairing, replacing, adding or removing a specific DNA sequence. The therapeutic, prophylactic, or diagnostic effect of the gene therapy is directly related to the sequence of recombinant nucleic acids that the gene therapy contains, or to the expression product of that sequence. According to the mode of administration, gene therapy can be divided into in vivo and ex vivo. In vivo therapy is administered directly to the patient, while in ex vivo therapy, cells are first isolated from the patient and then genetically modified. The cells are then selected, allowed to proliferate, and administered to the patient. The major advantage of the ex vivostrategy is that the modified cells can be selected and only those with the desired characteristics (corrected gene) are kept. However, this approach is limited to the dividing cells of the bloodstream, particularly the hematopoietic cells ensuring the formation of red and white blood cells and platelets. For the other tissues, a different approach must be taken (Figure 10.4).

Figure 10.4: In vivo and ex vivo approaches in the gene therapy.

Ex vivo gene therapy

The first disease treated with gene therapy was so-called severe combined immunodeficiency (SCID). Patients with SCID have a severely weakened immune system. Several cases were described in which patients had to be isolated in ventilated rooms or personal bubbles because otherwise there was a great risk that even the mildest infection could be fatal. Bone marrow transplantation or gene therapy are the only chance for such patients to lead a normal life. So far, two genes are known whose mutations lead to the manifestation of the disease – a mutation in the IL2RG gene, which codes for the gamma interleukin receptor (located on the X chromosome, the disease is called X-linked SCID) and a mutation in the ADA gene, which codes for the enzyme adenosine deaminase (located on chromosome 20, ADA-SCID). In both cases, the mutations lead to the inactivation or complete loss of T and B lymphocytes. The first attempt at gene therapy for this disease was made in 1990. A four-year-old Ashanti DeSilva was diagnosed with the ADA-SCID variant. Blood samples were taken and white blood cells were isolated. These cells were transformed with a retrovirus carrying a healthy copy of the ADA gene. The transformed cells were returned to the patient's body, where they began to produce a functioning enzyme. Ashanti DeSilva thus became the first patient to successfully undergo gene therapy. Three years later, Andrew Gobea, who was only 5 days old, underwent a similar therapy. The difference was that he was administered modified germ cells from the mother's placental blood and umbilical cord. The administration was successful, but in the first years the therapy had to be combined with additional administration of the ADA enzyme. As mentioned earlier, there is a major risk associated with the use of retroviruses in the form of integration of DNA into important parts of the genome. This was also observed with this therapy – nine patients developed leukemia within six years of administration of the drug. Fortunately, a much safer therapy is now available for ADA-SCID, using a modified HIV virus.

An intuitive target for ex vivo gene therapy is blood diseases, such as beta-thalassemia and sickle cell anaemia. In both cases, the disease is caused by mutations in the gene encoding the β-chain of haemoglobin (HBB). Haemoglobin is responsible for binding oxygen and carbon dioxide in red blood cells. There are several mutations that can lead to a disruption of this function, which in the case of sickle cell anaemia is also reflected in an altered structure of the blood cells. Patients suffering from beta-thalassemia or sickle cell anaemia are dependent on frequent blood transfusions or a bone marrow transplant. The disadvantage of a bone marrow transplant is that it may not be accepted by the patient's body and is therefore attacked by the immune system. For that reason, a suitable alternative is therapy with the patient's own hematopoietic germ cells modified with a lentiviral vector carrying the human HBB gene. This is a one-time therapy for beta-thalassemia patients older than 12 years who are dependent on blood transfusions. Another promising example of ex vivo gene therapy is the so-called CAR-T therapy in the treatment of cancer patients (Figure 10.5). In this therapy, T lymphocytes are taken from the patient and then genetically engineered to have a chimeric antigen receptor (CAR) on their surface. CAR enables T lymphocytes to bind to specific sites – epitopes on the surface of target cells. Binding occurs only when the structures of the epitope and the CAR receptor are complementary, i.e. they fit together like two pieces of a puzzle. After the binding of CAR and the epitope, a molecular signal is sent that leads to the activation of the immune response. When the CAR is designed to recognize an epitope specific to cells of a particular cancer type, its activation leads to an attack on those cancer cells. Because the altered cells come directly from the patient, there is little chance that the therapy will not be accepted. However, an important prerequisite is that the patient's type of cancer cells is known and that the right CAR is available. There are several drugs on the market in Europe that use the CAR-T system. They are aimed at treating certain types of leukaemia and lymphoma when conventional therapy has failed or the cancer has returned shortly after the treatment. The shortcoming at present is the small number of known pairs of CARs and epitopes specific for different types of cancer, as well as the observed toxicity of activated T lymphocytes, which slows down their use in practice.

Figure 10.5 CAR-T therapy.

In vivo gene therapy

Some cells of the human body cannot be easily removed and reinserted after modification, at least not without serious consequences. In addition, specialized fat, muscle, or nerve cells have lost their ability to divide, so if their genetic information is to be altered, they must be targeted directly in their place, i.e., in the patient's body. An example of in vivo gene therapy is a drug targeting a rare recessive disease caused by the absence of the enzyme lipoprotein lipase (lipoprotein lipase deficiency – LPLD). The disease is characterized by the presence of high concentrations of triacylglycerides and lipoproteins in blood plasma, leading to other clinical complications such as diabetes, pancreatitis and pancreatic cancer. This drug uses an adeno-associated virus 1 (AAV1) that carries a copy of the human LPL gene, which encodes a functional enzyme. As mentioned earlier, the viral DNA carrying the human gene is not incorporated into the patient's DNA in this case, but remains in the form of an episome. Multiple injections are administered into the thigh muscles. This drug was the first approved gene therapy in Europe; it was launched in 2012. Another prime place was its price: at $ 1 million per therapy, it was the most expensive drug in the world at the time. Although it proved effective, it was administered to only 31 people in total. The high price and low interest meant that the license was not renewed and the drug was thus withdrawn from the market.

Gene therapy has shown promise for treating various forms of vision loss. A specific example is age-related macular degeneration (AMD). Unlike the previous examples, AMD is a multifactorial disease whose cause is not precisely known. The environment, the genetic background of the individual as well as age play a very important role in its occurrence. It is a chronic disease associated with irreversible loss of vision with an onset of symptoms at the age of 50, being most common in people over 70. One of the most important factors in AMD is likely our complement immune system (the non-specific immune response thaht complements the immune system), which can cause inflammation of the eye or permanent damage to vision if it overreacts. A drug with the gene CFI (human complement factor I), whose product is able to alleviate and slow retinal atrophy by regulating the immune response, is in the clinical trial phase. Another example is the treatment of hereditary retinal dystrophy (retinitis pigmentosa), which is caused by the loss of functional photoreceptors. This is a collective term for diseases with similar symptoms but with different causes. In the past, several dozen genes have been identified whose mutation can lead to the manifestation of this disease – one of them is RPE65 (the product of this gene is a key enzyme for the light cycle, in which the light signal in the retinal cells is converted into an electrical signal leading to the brain). There is an approved gene therapy with a healthy copy of the RPE65 gene using adeno-associated virus 2 (AAV2). However, the application of this therapy is difficult as it must be delivered directly to the retinal area. This is achieved by injecting the drug into the space between the retina and choroid (Figure 10.6). Since the damage to the light-sensitive cells of the retina is irreversible, the therapy must be administered as soon as possible. Furthermore, the aim of the two aforementioned therapies is not to restore vision to its original state, but to stop or slow down the progression of the disease.

Figure 10.6 Gene therapy in the treatment of visual impairments. The drug is injected into the space between the retina and the choroid.

Another inherited disease whose treatment has attracted much attention is spinal muscular atrophy (SMA). It is one of the most common autosomal recessive diseases with an approximate incidence of 1:10,000 in the population. It affects the central nervous system, motor neurons, and skeletal muscles. The mutations responsible for this disease were mapped to the SMN1 gene. In addition to the SMN1, the human genome also contains its copy – the SMN2 gene. SMN2 can be present in varying numbers, from one to multiple (> 4) copies, and the SMN2 protein, which is the product of this gene, can compensate to some extent for the loss of SMN1. The more copies present, the milder the disease symptoms in the case of the SMN1 mutation (Table 10.1).

Table 10.1 SMA patient prognosis related to the presence of different numbers of SMN2 gene copies.

type	Age of symptoms onset	Artificial ventilation in birth	Ability to sit		Ability to stand up	Ability to walk	Life expectancy	*# SMA2* copies**
0	prenatal age	yes	no	no		no	< 6 months	1
1	< 6 months	no	no	no		no	< 2 years	2
2	6-18 months	no	yes	no		no	10-40 years	3
3	> 18 months	no	yes	yes		assisted	adulthood	3 - 4
4	> 5 years	no	yes	yes		yes	adulthood	> 4

An important difference between the two genes is that exon 7 of the SMN2 gene is excised in some cases during mRNA maturation (Figure 10.7). The genetic information contained in the sequence of this exon, which is important for the folding and function of the resulting protein, is thus lost. It has also been shown that a single nucleotide substitution in this exon can lead to the pathological condition. Splice modification of the exon 7 of the SMN2 gene has therefore become the target of antisense RNA gene therapy. By binding the antisense RNA (complementary to a natural mRNA), exon 7 is not excised during the maturation of the mRNA. However, the drug does not have a great longevity after the administration. In addition, the blood-brain barrier must be bypassed for the therapeutic RNA to reach its destination. Therefore, the therapy must be administered intrathecally (into the space between the brain and its soft membrane (pia mater) or between the spinal cord and its soft membrane) an average of three times per year, which is painful and technically challenging.

Figure 10.7 SMN1 ans SMN2 gene expression.

An alternative in SMA gene therapy is a drug that uses adeno-associated virus 9 (AAV9), which carries a healthy copy of the SMN1 gene. A major advantage is that the AAV9 viral vector has the ability to reach the brain and thus cross the blood-brain barrier. After entering the neurons, the genetic information of the virus moves to the nucleus, where it exists in the form of an episome. Due to the one-time and less painful administration, this drug seems to be a better choice for patients suffering from SMA.

Genome editing technologies

As mentioned earlier, one of the greatest challenges in gene therapy is the precise editing of the genome. This was very difficult before the discovery of programmable nucleases. The term nuclease refers to a group of enzymes capable of cleaving nucleic acids. Of particular interest for gene therapy applications are nucleases that cause double-strand breaks in the DNA, such as the enzyme FokI, which is sequence-specific, meaning that it first recognizes a particular sequence in the genome and then cuts the DNA in that region. However, there are many such regions in the human genome, so the presence of this nuclease in the nucleus would mean fragmentation of the entire genome. This is obviously undesirable, so only the catalytic domain of this nuclease is retained and further linked to DNA-binding domains such as zinc finger (ZF) and transcription activator-like effector (TALE). What is important here is that we are able to modify the DNA-binding domains in such a way that they recognize a sequence of choice. The FokI nuclease functions as a dimer, meaning that two molecules of the enzyme must be present to form a functional complex. The DNA-binding domains must therefore be designed to bind on both sides of the site where the double-strand break is to be created (Fig. 10.8). This is very useful because the longer the recognition sequence, the lower the probability that the enzyme will accidentally cut the DNA in a different place in the genome as intended. An alternative to the aforementioned genome editing technologies is the CRISPR/Cas9 system. Similar to the examples already mentioned, it is a two-component system of a nuclease (Cas9) and a guiding molecule. These molecules are short RNAs (single guide RNA – sgRNA), which can be very easily modified so that, based on complementarity, they recognize practically any sequence in the genome. Together, Cas9 and sgRNA form a functional complex, which results in a double-strand break upon recognition of the target sequence (Fig. 10.8).

So the programmable nucleases allow us to create a double-strand break in the DNA molecule with high precision. In humans, there are two ways in which cells repair double-strand breaks (see Chapter 5). One of these methods is non-homologous end joining (NHEJ), in which the DNA ends are simply glued together (Fig. 10.8). However, errors in the form of small insertions or deletions occur relatively frequently. This can be useful when interrupting or removing the section of DNA that is the cause of the pathology. When a DNA template is supplied along with the nucleases, the breaks are repaired by a process known as homology directed repair (HDR). HDR enables DNA repair, insertion, and modification with very high precision and efficiency (Figure 10.8). Gene therapy with programmable nucleases therefore has great potential to make this type of treatment more precise and efficient but unfortunately we still have to wait for its entry into clinical practice.

Figure 10.8 Programmable nucleases. The most common systems for inducing a double-strand break at specific sites in the genome are ZFN, TALEN, and CRISPR/Cas9. In all cases, these are two-component systems – the part that recognizes the sequence is linked to the enzymatic part that creates a double-strand break. In ZFNs and TALENs, the DNA-binding domains are responsible for targeting and the FokI nuclease is responsible for enzymatic activity. In the CRISPR/Cas9 system, the sgRNA binds to the DNA based on base complementarity, and the Cas9 nuclease cleaves the DNA at the target site. Double-strand breaks are repaired by non-homologous end joining (NHEJ) or homologous recombination (HDR).

Why is gene therapy not a common part of our lives?

One might think (based on available technology and clinical trials) that the gene therapy is ready for practical use. So why is it still not part of our lives? One of the reasons is that in many cases, safety and efficacy for human use have not yet been studied. In drug development, it is not uncommon for test results to be favourable under laboratory conditions, but later to find that the therapy does not work or is associated with severe side effects when animal models or human volunteers are used. It takes an average of 12 to 15 years from the start of the approval process for a drug to its market launch (Figure 10.9). Negative effects at any stage can lead to the approval process being halted or the drug being withdrawn from the market.

Figure 10.9 Drug approval process.

It is obvious today that the gene therapy will become an important tool of modern medicine. Thanks to it, in a very short time it will be possible to help people overcome diseases for which there is currently no cure. But it is not that simple – gene therapy also has many pitfalls and side effects, the elimination of which is crucial for its further development. One example of such a problem is the insertion toxicity of in vivo gene therapy. Insertion of DNA in the wrong place or its multiple insertions can lead to side effects that endanger the patient's life. In addition, the right tissue must be targeted, for which specially modified viruses or complicated surgical procedures are used. Another challenge is to develop safer and more effective vectors that are better tolerated by our immune system. An alternative is to find a way to reduce the dose administered to patients. Clinical testing of gene therapy itself is also challenging, as many of the diseases mentioned are extremely rare, which limits the number of volunteers for drug testing. Because this type of treatment has only been used for a relatively short time, there is almost no data on its longevity. With integrative gene therapy, the effects can last a lifetime, but with episomes, the effects can only last a few years. The timing of administration is also critical, as damage to some tissues is often irreversible. These include, in particular, diseases of nerve, muscle and pigment cells. Last but not least, the question of price is also important. Currently, the cost of gene therapy is in the hundreds of thousands of dollars or euros, mainly due to the difficult preparation, the high investment in drug development and the small market. However, it is certain that the price of gene therapy will gradually decrease, and it is expected that it will eventually become widely available.

Did you know that..,

... that we can look forward to several new gene therapy drugs in the coming years? This is illustrated by the pie charts with data on various gene therapy products that are in clinical trials worldwide (Figure 10.10). The largest number of these drugs target cancer, with the second largest group belonging to the therapy of various monogenic diseases. Most are still in the first and second phases of clinical testing, but up to 140 such products are currently entering the third phase, which means they are close to approval.

Figure 10.10 Gene therapy products currently in clinical trials. Two pie charts showing the targets of the gene therapy products (left) and the clinical phase they are in (right).

Want to know more?

Gene therapy: A double-edged sword with great powers, Tang, R., Xu, Z., Molecular and Cellular Biochemistry (2020).

A brief history of gene therapy, Friedmann T. Nature Genetics (1992).

Entering the Modern Era of Gene Therapy, Anguela, X. M., High, K. A., Annual Review of Medicine (2019).

Gene Therapy, High, K. A., Roncarolo, M. G., The New England Journal of Medicine (2019).

OMIM: An Online Catalog of Human Genes and Genetic Disorders

Zinder, N. D., Lederberg, J. (1952). Genetic exchange in Salmonella. J Bacteriol., 64(5): 679-99. doi: 10.1128/jb.64.5.679-699.1952.

Rosenberg et al. (1990). Gene transfer into humans – immunotherapy of patients with advanced melanoma, using tumor-inf iltrating lymphocytes modif ied by re-troviral gene transduction. N Engl J Med., 323(9): 570-578

The Story of David Vetter at the Immune Deficiency Foundation

Hacein-Bey-Abina et al. (2008). Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J Clin Invest., 118(9): 3132-42

Gene therapy clinical trials worldwide, provided by the Journal of Gene Medicine

Chapter 11: Plants as an inspiration in biomedicine

We often do not realise in our daily lives how essential plants are to us, and we would probably start to appreciate their significance only in their absence. However, we don't need an apocalyptic vision to understand that the plant kingdom and its members, with their fascinating abilities, deserve our attention. Plants are indeed a part of our everyday life, and industries that process plants are well-known. Still, we can also find that plant biomass provide energy, in the form of biofuels and subsequently heat. Of course, we should not forget the cosmetic industry, where plant-based ingredients and extracts are highly sought after and popular. At the same time, we must remind ourselves that plants are an integral part of art and relaxation. Indeed, each of us has at least one indoor plant at home and likes to walk in a park or forest, representing an optimal form of relaxation for many. Humans have always been connected to nature, providing us not only with food and shelter but also with refuge and a place to rest. In this chapter, we will focus more closely on two areas in which plants play a significant role - basic research and medicine.

Plants and important historical discoveries

In science, plants are often used as model systems for several reasons. Since they are eukaryotic organisms, mechanisms and phenomena observed in plant cells are often found in a similar form in other organisms. Unlike laboratory animals, plants provide a great advantage as they are not difficult to handle and cultivate. They are also easily available, have a short generation time and in many cases provide a large number of offspring, allowing scientists to perform multiple-generation experiments in a relatively short time. However, there are also exceptions where we have to wait several years for the offspring (oak is a good example). In addition, in the case of plants, the ethical dimension of research is usually not a problem.

One of the most significant scientific discoveries was the first-ever observation of a eukaryotic cell by the English physicist Robert Hooke, who analysed cork oak slices. Later, other structures were discovered in plant cells. Robert Brown, a Scottish botanist discovered the nucleus in orchid plants and the process of cell division was described in algae by Barthélemy C. Dumortier, a Belgian botanist. Plants were also part of experiments related to genetics. Gregor Johann Mendel, the founder of genetics, used pea plants in his experiments and the Danish botanist Wilhelm Johansen introduced the terms phenotype and genotype when he experimented with lines of self-pollinating beans. Another important discovery, of which plants were essential (specifically corn), was the identification of mobile genetic elements, which we also call "transposons". These DNA sequences can "jump" in the genome, which is why they are sometimes referred to as "jumping genes". They can be copied, transposed, and incorporated into another place in the genome so that we can find many of them in eukaryotic genomes. In the past, it was assumed that these pieces of DNA had no meaning or may even have had a negative effect, initially referred to as selfish genetic elements. However, it was gradually discovered that transposons can play a role in adaptation and evolution. This phenomenon was later described in other organisms, and Barbara McClintock's work on mobile elements in corn was awarded the Nobel Prize in Physiology or Medicine in 1983.

The specific characteristics of plants

In relation to the environment in which plants exist, they have one major disadvantage compared to other organisms. They have a sessile way of life, which means they are fixed in one specific place and cannot leave in case of unfavourable conditions. Despite their sessile way of life, plants can spread, via seeds, from their original habitat over large distances. However, plants with a sessile way of life have evolved various sophisticated defence mechanisms to cope with the challenges of being located in one place. Thanks to these mechanisms, they can minimise damage caused by stress, maintain intracellular balance, and adapt at the cellular and molecular levels. The diversity of plant species allows us to observe how plants have adapted to various environmental conditions during evolution: Plants can be found in different environments, facing high or low temperatures, intense sunlight or lack of light, extreme weather conditions (strong winds, heavy snowfall), excess water, low atmospheric pressure, increased UV radiation or the presence of salts. In adapting to all these conditions during evolution, these changes are reflected in their genetic information.

Regarding the ability of plants to adapt to the environment, we have to look deeper into the structure of these organisms. Cells of all eukaryotes contain two types of genomes with different regulations - the nuclear and mitochondrial genomes (in the nuclei and mitochondria). Plant cells differ from other eukaryotes because they also have plastids, wich include chloroplasts which themselves contain a chloroplast genome. Due to the presence of the plastids, plants can react relatively flexibly to various stimuli from the external environment and adjust their structure and metabolic activity accordingly (for example, changing white etioplasts to green chloroplasts when exposed to light). All plastids originate from so-called proplastids and are generally divided into chloroplasts, chromoplasts, leucoplasts (amyloplasts, elaioplasts, and proteinoplasts), and etioplasts (Figure 11.1). Some types of plastids can repeatedly differentiate and transform from one type to another depending on environmental changes and the tissue in which they are located.

Figure 11.1 Types of plastids. Proplastids forms different types of plastics, including chloroplasts, chromoplasts, leucoplasts and etioplasts. Leucoplasts themselves form amyloplasts, elaioplasts, and proteinoplasts.

The significance of secondary metabolites

In today's world, plants face even more demanding challenges than before, as in addition to the mentioned stressors, they are also threatened by increasingly intense pollution of the environment, higher levels of radiation especially UV radiation, the presence of chemicals and heavy metals in soil and water, dust particles in the air, and many other factors. How, then, does a plant respond to such high levels of stress? Plants can produce hundreds of different compounds that help it respond to one stress factor or another, tasks mainly carried out by the so-called secondary metabolism. Such metabolism is responsible for the production of specific compounds that, unlike lipids, carbohydrates, and proteins, are not essential for the survival and normal functioning of the plant. However, these secondary metabolites (compounds) benefit the plant, enabling it to survive even in adverse conditions. Interestingly, individual secondary metabolites are not found in the same proportion in all plants or even in all parts of a single plant. They are often specifically localised in specific organs, where they can perform their function. Another interesting fact is that many of these substances are not present in the plant during its entire life cycle. Some metabolites are only present during flowering, others during the germination of the plant, and some are typical of the ageing of the plant, so-called senescence. While the most important reason for plants producing these secondary metabolites is protection against adverse environmental factors, they also increase attractiveness to pollinators and protect the plant. As far as protection is concerned, substances that provide repellent effects inhibit the growth and multiplication of microorganisms but can also be helpful in fighting off other plants. This phenomenon is called allelopathy, where a plant produces metabolites that influence the germination, growth and survival of another plant (this can be positive or negative). An example of allelopathy can be seen on the leaves of a walnut. They contain compounds that block the germination of other plants, and therefore, no other plants or grass often grow under walnut trees.

A crucial function of secondary metabolites is to attract pollinating insects and other animals that help the plant produce fruits and spread through the ecosystem. This ability is provided by the presence of fragrant compounds but also pigments that attract the attention of pollinators. Other metabolites play a role in protecting plants against UV radiation, whether it is layers of cutin on the surface of leaves or various antioxidants. In order to survive, plants must also support symbiotic relationships that help them defend themselves against enemies. For example, if a parasite (often an aphid) feeds on leaves, as a response to this stress the plant will activate several defence systems immediately (Figure 11.2). The first is natural defence in the form of bitter or poisonous substances, which deters the parasite. Another possible reaction is the secretion of attractants for competitive or predatory organisms towards the parasite. Such interconnection of different defence systems allows for higher efficiency and better plant survival.

Figure 11.2 Interactions of plants with different organisms as a part of the defence mechanism against damage. Plants respond to damage by producing volatile chemicals which attract the enemies of the parasite, or repels the parasite themselves. Secondary metabolites might also alter the layers of cutin on the leaves to protect from UV radiation, or produce antibiotic chemicals to prevent attack by plant pathogens.

Plants in medicine

The search for medicinal effects of plants has been a tradition since ancient times. The oldest records of using plants as medicine date back to the Mesopotamian period (2600 BC). Many of these natural substances (morphine, cocaine, tetrahydrocannabinol, and others) were used by folk healers, shamans and later by doctors. However, adverse effects of specific metabolites on human health, including frequent overdoses, mental disorders, and strong addiction, began to appear and were therefore more deeply studied. Ultimately, the use of many of these natural products was abandoned and replaced with synthetic alternatives. Secondary metabolites have evolved to interact with various molecular targets, thus affecting cells, tissues, and their physiological functions. In many cases, they can even resemble human metabolites, ligands, hormones, signalling molecules, or neurotransmitters, which can have a beneficial therapeutic effect on humans. Today, using plants and their metabolites or synthetic analogues is noteworthy in various areas of medicine.

The most well-known secondary metabolites

Morphine, a substance found in poppy heads and the fruits of the opium poppy (Papaver somniferum), was first discovered in 1804 and is named after the Greek god of dreams, Morpheus. It was the first purely plant-based product commercially produced and sold in 1826. Due to its ability to relieve pain, morphine is mainly used for the management of chronic pain and as part of pre-operative preparations for patients. However, tolerance to the drug can develop with regular use, necessitating an increase in dosage. Other opioids, such as codeine and heroin, can also be produced chemically from morphine. Another well-known plant is deadly nightshade (Atropa belladonna), whose extract was often sought by women in the Middle Ages. This is because it is a powerful antispasmodic, which means it relaxes muscles, and one of its effects is mydriasis – excessive dilation of the pupils, which was considered very attractive in the Middle Ages. To this end, it is also named Belladonna, translated from Italian for “beautiful women”, but a more sinister side to the plant is that the berries are thought to be the poison that made Juliet appear dead in Shakespear’s Romeo and Juliet. Another well-known secondary metabolite from the deadly nightshade is atropine, which has hallucinogenic effects and can be a powerful poison, as with most herbal remedies, if misused. It is currently used to treat digestive problems and in ophthalmology, as it facilitates patient examination. Foxglove (Digitalis purpurea) was very popular among herbalists and folk healers who used it to treat heart disease. However, this treatment was gradually abandoned because it was very difficult to determine the precise dosage of the herb. The secondary metabolite, digitalin, is still used as a medical treatment to increase heart contractions and regulate heart rate.

The plant that helped win the Nobel Prize in Physiology or Medicine in 2015, Artemisia annua, commonly known as sweet wormwood, is often used as a medicine for digestive problems. But what makes this plant exceptional? We must go back to the Vietnam War to find the answer to this question. The problem during this period, as well as the war conflict, was the spread of malaria. The situation was so severe that representatives from China and Vietnam met and jointly created a military project called 523, which focused on developing new antimalarial drugs. The project also included chemist Tu Youyou, who studied traditional Chinese medicine texts and, with the help of the 523 project, isolated the substance artemisinin (qinghaosu) from sweet wormwood, a new antimalarial drug. Malaria is caused by the parasitic organism Plasmodium falciparum, transmitted by mosquitoes of the genus Anopheles. It all starts when a mosquito bites a patient with malaria. In the mosquito's gut, the infected blood with parasite male and female gametocytes undergo sexual reproduction, and the parasite passes through several developmental stages. The final infectious stage (sporozoites) migrates to the mosquito's salivary glands. The mosquito then transmits these sporozoites into the bloodstream of a new host, where they reach the liver and undergo a non-sexual life cycle. After 6-12 days, infected liver cells rupture, releasing the parasite (merozoites) into the bloodstream. Plasmodium then attacks red blood cells, reproducing intensively until the cells rupture. This phase is often associated with malaria attacks and fever, as the destruction of many red blood cells and the release of their contents into the bloodstream is toxic to humans. Later, this merozoite form differentiates into sexual cells (gametocytes), and the cycle repeats. Artemisinin can block the proteins of the Plasmodium parasite, preventing them from functioning correctly in the phase of attacking red blood cells (see Figure 11.3).

Figure 11.3 Mechanism of action of the antimalarial artemisinin against the Plasmodium falciparum parasite.

A clinically relevant chemotherapy drug is the metabolite of the western yew tree (Taxus brevifolia) called paclitaxel. It is mainly used in the fight against cancer due to its ability to affect cell division by preventing the shortening of tubulin (a component of the cell cytoskeleton) and thereby halting the cell in a specific phase of division. This way, paclitaxel prevents cancer cells from intense and continuous division and damaging the body. Another metabolite that can be used in various fields of research and medicine is colchicine, a substance isolated from the autumn crocus (Colchicum autumnale). Colchicine can block the formation of microtubules and disrupt the process of mitosis (consequently, it is classified as a mitotic poison). Interestingly colchicine is also used in plant breeding because it can induce polyploidy (duplication of the entire set of chromosomes). By preventing a cell from dividing, the genetic information doubles, which is passed on to the next generation as a polyploid. Compared to humans, who are diploids (two alleles of the same gene), some plants can have many times more sets of chromosomes (for example wheat has six copies of chromosomes). Since colchicine also has anti-inflammatory effects, it is used in medicine to treat gout. During the COVID-19 pandemic, scientists again turned to medicinal plants and using them as sources of new bioactive compounds that could potentially treat this disease. Since many plant metabolites (such as quercetin, colchicine, and emodin) have antiviral or anti-inflammatory effects, they represent potential candidates in the fight against the SARS-CoV-2 virus.

Secondary metabolites of St. John's wort

A plant currently receiving significant attention in research is St. John's Wort (Hypericum perforatum L.) and is one of the most commonly used medicinal plants in the world. It is widespread in Europe, Asia, North America, and North Africa, with its species name derived from the dots on the flowering parts and leaves. These tiny perforations and dark glands are vital because they contain the most effective compounds of St. John's Wort. The effects of these natural substances have been known for more than 2,000 years, which is why St. John's Wort was often used to treat burns, clean wounds, reduce swelling, and alleviate depression. The ancient Greeks and Romans believed that St. John's Wort expelled evil spirits and was called the "balm for the wounded soul". But what is behind these properties of this inconspicuous plant? In experiments that studied the chemical composition of St. John's Wort extracts, the metabolite hyperforin was discovered, which is found in bright glands mainly on the leaves of this plant. This substance acts as a sedative and is psychoactive drug with euphoric effects, therefore it is used in treating neuroses, depression, and migraines. Hyperforin acts similarly to prescription antidepressants by blocking the reuptake of neurotransmitters, such as norepinephrine, serotonin, and dopamine. These substances are naturally present in the brain and help regulate a person's mood and emotions. They are the chemical messengers that transmit information between individual nerve cells, and their imbalance can cause depression, anxiety, or fatigue. In addition to being able to release these messengers, the cell can also take them up again (reuptake). This is where hyperforin works, acting on the sodium transporter, increasing its intracellular concentration and thus indirectly blocking the mechanisms responsible for neurotransmitter reuptake. Thanks to this mechanism, the concentration of neurotransmitters in the brain increases, which causes a feeling of happiness and improves the patient's mood.

Another significant metabolite of St. John's Wort is hypericin, present in the dark glands on the plant's above-ground parts, especially on the flowers. Hypericin has many effects (e.g., anti-inflammatory, antimicrobial, and others) that are useful in various branches of medicine. However, it is always important to remember that the plant does not produce these phytochemicals for us to use in biomedicine but instead tries to protect itself through them. This phenomenon is called hypericism and protects the plant from being eaten by predators. Hypericin is an illustration of hypericism and the protective effect of metabolites: cattle which graze on St. John’s Wort experience adverse health effects specifically inflammation of the skin and sun sensitivity associated with paralysis. However, these processes can be utilised to treat oncological diseases through photodynamic therapy. As its name suggests, photodynamic therapy is based on the change in the dynamics of the drug depending on the light stimulus (and the presence of molecular oxygen). This therapeutic approach is mainly used to treat skin diseases and tumours. Hypericin is injected into the patient's body and preferentially accumulates in the tumour tissue (Figure 11.4) and subsequently the target area is irradiated through optical fibres. After irradiation, hypericin is excited, which in this state interacts more intensively with its surroundings, whether it is individual cell components or oxygen present in cells. This process triggers the formation of reactive oxygen species (for more details, see Chapter 8 - Your cells are stressed too), which ultimately destroys tumour cells. This therapy results in the selective destruction of tumours without excessive damage to surrounding tissue, as hypericin preferentially binds to tumour cells. Currently, several substances are used as photosensitisers, but hypericin still plays a significant role in this type of therapy. In addition, hypericin can also be used in diagnostics, as it can release fluorescence after irradiation, thus helping surgeons mark the boundaries of the tumour and the presence of possible metastases in the patient's body.

Figure 11.4 Scheme of photodynamic therapy in the treatment of tumours. 1: The application of a photosensitiser, such as hypericin. 2: There is selective accumulation of the photosensitser in the tumour cells. 3: Activation of the photosensitiser using light. 4: Finally the tumour is eliminated.

In conclusion, we can state that plants are extraordinary organisms that represent a large part of our biosphere and can be used in several more ways than just as a food source. Since they cannot avoid adverse environmental factors and change their location, they have to deal with the traps of their surroundings during evolution and adapt to them. In this way, they indirectly provide us with various strategic approaches and medicinal compounds, which are also interesting for human medicine. But before that, it is necessary to properly understand these mechanisms of defence and production of phytocompounds at the molecular level so that we can use them as effectively as possible.

Did you know that...

...recent studies suggest that secondary metabolites can have a positive effect not only on overall health but also on human life span? Resveratrol, which is most often associated with the consumption of red wine and the so-called French paradox, can be an example of such a chemical. This phenomenon was described by the Irish cardiologist Samuel Black for the first time in 1819. He found that French people were less likely to die of heart failure compared to the Irish, despite their increased intake of saturated fats, which negatively affects the human cardiovascular system. This unexpected finding appealed to several scientific teams, and their study concluded that the antioxidants such as tannins and flavonoids (e.g. resveratrol and quercetin), abundantly found in red wine, play a protective role in this case.

Want to know more?

Versatile roles of plastids in plant growth and development. Inaba, T., Ito-Inaba, Y. Plant Cell Physiology (2010).

Plant secondary metabolites synthesis and their regulations under biotic and abiotic constraints. Khare, S., Singh, N. B., Singh, A., Hussain, I., Niharika, K., Yaday, V., Bano, C., Yaday, R., Amist, N. J. Plant Biology (2020).

Chapter 12: When the environment changes our hormones

For the human body to function properly, its various parts and organs must communicate to ensure the maintenance of a constant internal environment (homeostasis). Examples of homeostasis include the maintenance of body temperature and preserving the level of salts and minerals in the blood, which must not fluctuate outside established limits. Communication between the different parts of the body is necessary for the organism to respond appropriately to changes in the internal and external environment. Two systems contribute to this communication: the nervous and endocrine systems, which form the neuroendocrine system together. The nervous system generally enables the rapid transmission of information between different body parts, taking only fractions of a second. In contrast, hormonal communication, which is based on the production and release of hormones from various glands around the body and the transport of these hormones through the bloodstream, is more suitable for situations that require more extensive and prolonged regulatory actions. These two communication systems complement each other but also interact with each other so that stimuli from the nervous system can influence the release of certain hormones and vice versa.

Hormones are small molecules produced by endocrine glands. These include the hypothalamus, pituitary gland, thyroid gland, parathyroid glands, pancreas, adrenal glands, and sex glands (testes and ovaries) (Figure 12.1). The term "endocrine" means that the products of these glands, hormones, are released into the bloodstream in response to specific stimuli. The hormones are then transported through the blood to target cells; some hormones have only a few specific target cells, while others affect several types of cells in the body. The target cells for each hormone are characterized by the presence of specific intermediate molecules (called receptors) for the hormone, located either on the cell surface or inside the cell. The interaction between the hormone and its receptor triggers a cascade of biochemical reactions in the target cell that ultimately alter its function or activity. Thus, the binding of the hormone to the receptor is crucial for the initiation of individual regulatory steps that ultimately influence the behaviour of the cell. In general, however, hormones control the growth, development and metabolism of the body, the electrolyte composition of body fluids and reproduction.

Figure 12.1 Glands of the endocrine system. These glands are also called glands with internal secretion. Their main task is the production of hormones, which are then transported via the blood system to the target cells.

An endocrine active substance does not necessarily have to be an endocrine disruptor

In the environment in which we live, there are substances that, after entering the target cell, can alter the reaction cascade. These substances alter the cell's response to the hormone by a specific mechanism and thus acquire endocrine activity. Endocrine active substances are therefore able to interact with the normal function of naturally occurring hormones or interfere with them in a certain way. These substances can be of natural or artificial origin, and many of them are found in food and the environment (e.g. pesticides or industrial pollutants such as bisphenols, polychlorinated biphenyls, dioxins and others). When endocrine-active substance cause an undesirable effect in the body, it is called the endocrine disruptor. So, the definition of an endocrine disruptor could be a substance in the external environment that, after entering the body under certain circumstances, can behave like a hormone, altering intracellular signalling and thereby affecting the cell's response. The result of this process is an undesirable physiological effect on the organism, with such substances termed endocrine-disrupting chemicals (EDCs).

Whether the effect of an endocrine agent is undesirable depends on several factors. The first is the origin of the substance, with synthetic endocrine agents usually more likely to have adverse effects than natural ones. Furthermore, the dose or concentration of the substance to which the organism is exposed is important, as it does not always mean that a higher dose automatically brings worse effects. There are endocrine active substances that act at very low (nanomolar) concentrations and whose negative effects do not increase with increasing concentration. Examples are BPA that is used to make polycarbonate plastic and epoxy resins that are in turn used in plastic bottles and dental devices, and DEHP which is a phthalate used as a plasticizer to add flexibility to plastic. However, there are also substances whose effects increase with increasing concentration, such as per- and polyfluoroalkyl substances (PFAS). An equally important factor is the state of the body, which reflects the body's ability to deal with the possible negative effects of an endocrine-active substance. Here, it is true that an organism in a superior condition is more able to fight the adverse effects of substances and is thus better prepared for their excretion. Decisive for the evaluation of the effect of a substance is the duration of exposure. The longer exposure time to even low doses of a substance with potentially harmful effects could be more devastating compared to shorter exposure time to higher doses.

In the case of endocrine-disrupting substances, the effects of some of them may also be positive. One example is progesterone, a drug used in high-risk pregnancies to protect against spontaneous abortion or premature birth when the natural hormone levels are insufficient for the current pregnancy and the mother's body is unable to ensure the full term of the foetus. Another example of the positive effects of endocrine agents are medications for arthritis prescribed to menopausal women because they have lower levels of oestrogen than women of reproductive age, which leads to the development of this disease. Such drugs are used precisely because of their positive endocrine effects.

Endocrine disruptors act via several mechanisms

A more deep examination of endocrine disruptors revealed that they can act through several mechanisms. The first is to mimic the function of hormones, where an endocrine disruptor causes that the body responses like to a natural hormone. In this case, the body reacts in two ways. Either there is an exaggerated response to the stimulus, e.g., excessive muscle mass is produced when growth hormones are used, or there is a response to the stimulus at an inappropriate time. An example of such a response is the production of insulin by the beta cells of the pancreas when it is not needed, resulting in a deregulation of blood glucose levels. Another way to disrupt the endocrine system is to block the action of a hormone by binding to its receptor (Figure 12.2). Both the hormone and the endocrine disruptor have a certain binding strength (affinity) to this receptor, which can be higher or lower for the endocrine disruptor than for the natural hormone. If the affinity of the EDC is higher than the hormone, the disruptor displaces the hormone from binding to the receptor. However, if the EDC affinity is lower, the endocrine disruptor may bind preferentially because its concentration is high enough to simply "displace" the hormone from binding. For example, bisphenol A, which is the basic building element of many plastic products, can act in this way. The final mechanism of action of endocrine disruptors is the direct stimulation or inhibition of the endocrine system by altering hormone levels in the body. Accordingly, the body's response is strengthened or weakened, cellular signalling is altered and thus the expression of genes is influenced. The consequence of this effect of endocrine disruptors can be the early onset of puberty, changes in brain development, behavioural changes or cancer development.

Figure 12.2 Mechanism of endocrine disruption by natural hormone receptor binding. The hormone is released from binding to the receptor (A), while the endocrine disruptor (EDC) subsequently alters the physiological response of the cell (B).

The defining characteristic of endocrine disruptors is the triggering of harmful effects in the organism. However, it should be emphasised that humans are more susceptible to endocrine effects during important stages of development (namely during in utero development or during early childhood). It is also an important fact that the same amount of an endocrine disruptor can affect and harm a developing foetus but have little or no impact on the mother's body. This is because a foetus is more sensitive to chemical agents due to the intense and ongoing cell division and differentiation processes, and therefore internal homeostasis can be more easily disturbed. An example of a harmful or destructive effect of hormone disruption is diethylstilbestrol (DES). Diethylstilbestrol is a synthetic oestrogen that was used from the 1940s to the early 1970s for hormonal problems in a pregnant woman or women approaching early onset menopause. The drug helped to prevent spontaneous abortions and to support the growth of the foetus. Its use was banned after it was discovered that children exposed to DES during intrauterine development had an increased incidence of post-pubertal and pubertal cancers. In girls, it was vaginal cancer, in boys, to a lesser extent, prostate cancer. However, before the negative effect of this substance was discovered, the drug was prescribed to up to 5 million women around the world.

It is important to emphasise that the consequence of the action of endocrine disruptors is a (hormonal) imbalance or disruption of certain processes that can lead to the development of various diseases (Figure 12.3). In addition to cancer, these diseases include type 2 diabetes and various other metabolic syndromes (e.g., obesity), as well as impaired reproductive ability (which can lead to reduced fertility or infertility), disorders of brain development or impaired cognitive abilities (which can manifest themselves, for example, in the development of neurodegenerative diseases or learning disorders) or disorders of the immune system and congenital defects. The common features that precede the onset of all these diseases are defective cell signalling and changes in the expression of key genes involved in the control of individual processes in the body.

Figure 12.3 Hormonal imbalance can lead to various diseases. Examples include cancer, diabetes, obesity, other metabolic disorders, reduced fertility or infertility, neurodegenerative diseases, cognitive impairment, immune disorders and birth defects.

Endocrine disrupting chemicals may affect sexual differentiation

Sexual differentiation in vertebrates can vary widely across species, with differences in both timing and the specific mechanisms involved. However, steroid hormones consistently play a key role in signalling the differentiation of tissues that define male and female characteristics. Given their crucial role in this process, exposure to external steroids or EDCs that mimic, block, or otherwise disrupt these hormonal signals during critical stages of development is likely to affect later reproductive and neuroendocrine functions. If an organism is exposed to these EDCs during a sensitive stage of its life cycle, it could lead to changes in differentiation of the brain, behaviour, and reproductive organs. In addition, if a sufficient number of individuals are impacted effects at the population level would be seen. For example, if EDCs result in altered oocyte maturation, in the individual this could result in difficulties to conceive, infertility or miscarriage. Consequently, with more infertile women in the population, there will be a reduction in birth rates and an impact on society in terms of increased healthcare requirements (a need for IVF) and a shift in the age demographic of the population that could have economic impacts.

There are few very specific examples of EDCs – natural or human-made – that could contribute to sex changes. The feminisation caused by estrogenic chemicals was observed in alligators living in some lakes in Florida, or in fish living in English rivers near sewage-treatment works.The most causative chemicals in effluents were the natural steroid estrogens 17ß-estradiol (E2) and estrone (E1), as well as the synthetic steroid estrogen ethinyl estradiol (EE2), which is the active ingredient of the contraceptive pill. As EE2 is used by many people it is therefore excreted by humans and, unfortunately, is incompletely degraded in sewage-treatment works. Subsequently, as EE2 can be present in water, it can be absorbed by the human body, where the chemical is able to interact with estrogen receptors. This interaction can disrupt natural hormone balance, potentially leading to various disorders. EE2 is an example of an xenoestrogen, an estrogen mimicking compound that is not produced by the body. They may be synthetic, like EE2, plasticizers or pesticides but natural compounds also have similarity to estrogen. Such phytoestrogens include genistein which can be found in soy.

While most research has concentrated on estrogens and their estrogenic effects, such as feminisation, there is also substantial evidence that chemicals with androgenic and anti-androgenic properties present in aquatic environments may affect sexual determination. This became apparent when it was discovered that certain effluents from paper and pulp mills were causing masculinisation in fish living downstream from where the effluent entered the river. Typical examples of chemicals that can interfere with androgen actions, and thus interrupt the normal masculinisation of male reproductive organs, are antiandrogens – a persistent DDT metabolite (p,p'-dichloro-diphenyl-trichloroethane) and vinclozolin.

In addition, some other chemicals, phthalates, can interfere with the synthesis of testosterone and thus disrupt normal masculinisation. These effects would not be possible without affecting androgen binding to androgen receptors caused by EDCs, subsequently leading to adverse physiological responses.

Taken together, sexual differentiation of several species can be affected by the binding of human-made chemicals to hormone receptors (especially estrogen and androgen receptors), which can subsequently lead to the changes in relevant cell signal pathways.

Toxicological studies assess the potential risk of substances in the environment to human health

Since substances from the external environment can affect the processes taking place in our bodies, it is important to monitor their effects. Toxicological studies are of central importance and their task is to assess the potential danger posed by substances to human health and other living organisms. Currently, this area of activity is also covered by large organisations or authorities. In Europe, this is the European Food Safety Authority (EFSA), while in the USA, it is the Food and Drug Administration (FDA). At the international level, the Organisation for Economic Co-operation and Development (OECD) approves, among other things, the series of tests (a test battery) that substances are required to undergo if they are thought to have a practical use, or that are found at elevated levels in the environment. A test battery is a series of tests that, according to the OECD, all substances with potentially toxic effects must pass. The results of the individual tests indicate whether, to what extent and on which organisms the substance has an adverse effect.

The spot test is used to determine the toxicity of a substance on prokaryotic cells (Figure 12.4). Bacteria (Salmonella typhimurium) are used, which are inoculated onto a complete medium containing all the nutrients necessary for growth. After soaking the cells in the medium, a sterile circle of filter paper is placed in the centre of the dish. Next, the substance to be tested is dropped onto this circle at the desired concentration and can pass freely from the filter paper into the medium. After an incubation period, the so-called growth inhibition zone is determined, i.e., whether the substance caused the cells around the ring to die. If the inhibition zone is not present, the substance has no toxic effect. If there are dead cells around the ring, the larger the inhibition zone, the stronger the effect of the substance and thus the substance is more toxic.

Figure 12.4 Scheme of the toxicity test on bacterial cells. The zone of growth inhibition is assessed, a larger zone corresponds to a higher toxicity of the substance.

The Ames test is used to determine the mutagenicity (ability to induce the formation of mutations) of the substance under investigation on bacterial cells (Figure 12.5). Here, S. typhimurium bacteria are used which have been modified to have an interrupted gene for the synthesis of the amino acid histidine, so that these bacteria cannot grow on the medium without the addition of this amino acid. The bacteria are exposed to the potentially toxic substance and then allowed to grow on a medium without histidine. If the bacteria grow, it is because of what is called a reverse mutation (either point-substitution or frameshift mutation – the insertion or deletion of a short section, more about mutations in Chapter 5 – Mutations how they arise and what to do with them). This means that due to the mutation, the cells ability to synthesise the amino acid histidine has been restored and the bacterium is therefore able to grow on this medium. Since the tested substance caused the formation of (reverse) mutations, we can speak of its mutagenic effect. When evaluating the result, the more reverse mutants that have grown, the higher the mutagenic effect of the substance. If the tested substance is not added to the cells (such a sample serves as a negative control in the experiment), only a small amount of the so-called spontaneous revertants grow. The reason for this is that the ability to synthesise an amino acid is always restored in the cells with a certain low frequency (spontaneous mutations).

Figure 12.5 Scheme of the Ames test. The test is used to determine the mutagenic effect of various substances. The number of colonies growing on the medium is evaluated. The higher the number of colonies, the higher the ability to trigger the formation of mutations.

The comet assay is used to detect primary DNA damage (DNA strand breaks) that can be repaired in the cell but, if not repaired, will lead to mutations (Figure 12.6). In this assay, the substance tested is applied to different types of eukaryotic cells at a specific concentration for a specific time. Then the cells are lysed (the cytoplasmic and nuclear membranes are destroyed) to release DNA. In the next step, an electric current is applied which causes the DNA fragments to migrate from the head (intact DNA) to the tail – a comet is formed. Short DNA fragments migrate faster in an electric field than longer fragments, so they cover a greater distance in the same amount of time. The longer the tail of the comet or the more DNA it contains, the more DNA is damaged. This assay is evaluated microscopically after the DNA has been stained with a suitable dye.

Figure 12.6 Scheme of the Comet assay. The test is used to detect primary DNA damage. It is evaluated microscopically. The larger the comet tail (which contains more DNA fragments), the more DNA damage is detected.

In addition to the tests mentioned above, there is also a screening programme for the detection of endocrine disruptors developed by the United States Environmental Protection Agency (the Endocrine Disruptor Screening Program, EDSP). This screening is a two-step process, with the first step focusing on identifying compounds that interact with the endocrine system. Subsequently, all compounds found to interact in this way are also tested in the second step, the aim of which is to identify the adverse effects of these endocrine active substances. In addition, it is important to uncover the relationship between the dose of a substance and its effect and, if necessary, to adopt regulatory measures to guide the use of the substance so that there is no risk to human health.

The CALUX® and REA assay As many of the EDC chemicals have adverse outcomes in humans, it is important to monitor the levels of estrogen and other compounds in food to reduce the risk of contaminations. To do this quickly and effectively receptor-based assays have been designed.

CALUX® assays are a family of bioassays using mammalian cells. The cells are genetically modified so that when a chemical binds the receptor, it results in the transcription of a reporter gene, luciferase. Translation of luciferase results in an enzyme that produces light when its substrate, luciferin, is added to the cells. The amount of light produced is related to the activity of the chemical to which the cells are exposed and can be quantified. Commercial CALUX® assay kits are available for rapid, sensitive and cost effective screening for dioxins, one of the most toxic human-made compounds. Similarly, estrogen levels can be detected using the CALUX® assay as well.

Another reporter gene assay is the REA assay. This is a yeast estrogen bioassay, which is a fully validated method to measure the effects that are directly mediated by the estrogen receptor, as binding to and activation of the estrogen receptor. The REA is a reporter-gene bioassay making use of Saccharomyces cerevisiae yeast cells which were transfected with two DNA constructs (Figure 12.7). The first construct contains the gene sequence for the human estrogen receptor alpha (hER-α), which enables the binding of chemicals containing estrogenic activity. The second construct encodes for the yeast enhanced green fluorescence protein (yEGFP) gene, which is under transcriptional control of a hER-α responsive promotor. If the yeast cells are exposed to a sample containing estrogenic compounds, they will bind to the estrogen receptor. This complex will bind and activate DNA at a specific estrogenic response element (ERE), which will cause a response. In the case of the REA, binding of hER-α to the ERE allows transcription and translation of yEGFP ultimately resulting in a green fluorescent signal that can be measured by fluorescence spectrometry in intact, living, cells. As such, this fluorescent signal is a direct measure for the total estrogenic activity in the sample to which the yeast cells were exposed.

Figure 12.7 Principle of the RIKILT Estrogen Assay (REA), a reporter gene assay based on yeast cells responsive to estrogen receptor (ER) activation. Upon activation by estrogenic chemicals, the hERα-estrogen complex is translocated to the cell nucleus where they activate an estrogen responsive element in the promotor region of the reporter gene yEGFP. Consequent transcription and translation of this gene causes an increase in the yEGFP protein, which produces a green fluorescent (GFP) signal.

In practice, the REA test has been successfully applied to detect 17beta-estradiol in the urine of calves treated with the hormone in vivo. The findings suggest that this technique could serve as an effective laboratory method for veterinary monitoring of illegal steroid use. In addition, the estrogenic activity observed in rodent feed was attributed to high levels of genistein, daidzein, along with trace amounts of zearalenone. The REA test has also demonstrated its ability to detect 17β-estradiol in animal feed at low concentrations, ranging from 1.15 to 2 μg/kg^-1. Another study confirmed that the REA assay is specific for the detection of estrogenic compounds in water. These and similar studies hold significance for human health, as they contribute to predicting and identifying potential risks to public health.

Bisphenol A is a potentially hazardous substance

One of the many endocrine disruptors is bisphenol A (BPA), which is a polyphenolic, synthetically produced substance used in the manufacture of many plastic products and epoxy resins, from which it can enter the environment. For example, BPA can be found in some plastic bottles, the inner lining of cans, disposable plastic products, certain sports protection equipment, medical aids or dental fillings. Epoxy resins are used as industrial adhesives or building materials. However, there are several restrictions on the use of BPA in the European Union. In 2011, the European Commission banned the use of BPA in the manufacture of polycarbonate infant feeding bottles. Then, in 2018, BPA was also banned in all plastic bottles and packaging containing food for babies and children under three years, and since 2020, the use of BPA has been banned in thermal paper receipts (https://www.efsa.europa.eu/en/topics/topic/bisphenol). In addition, recent 2023 EFSA regulations have reduced the tolerable daily intake (TDI) of BPA from 4 µg per kilogram of body weight per day (kg/bw/day) to 0.2 ng/kg/bw/day, which represents a 20,000-fold lowering of TDI.

Nevertheless, since BPA can be released from the material, it is detected in all environmental matrices (soil, water, air) as well as in food and drinking water. Therefore, BPA is considered an “ubiquitous compound” and is an environmental pollutant. We can be exposed to BPA from various sources such as food (especially food wrapped in plastic film or cans), heat-sensitive paper used to make cash receipts, as well as some toys, cosmetic products or dust particles. BPA can also enter the body through the digestive system, the skin and by inhalation. Other sources of exposure are from industry (the chemical industry, which produces BPA, and the textile industry, which produces synthetic fibres) as well as waste and sewage. People from the chemical and textile industries and salespeople who handle hundreds of tills every day are therefore particularly exposed to the effects of BPA. Furthermore, it should be considered that not only humans, but also microorganisms, plants and animals that are part of our food chain are exposed to BPA.

After BPA enters the body, there are several ways it can get into the cells themselves. Probably the easiest way for BPA to enter the cell is by free passage through the lipid bilayer of the membrane. This is possible because of the lipophilic character of the chemical, i.e., the ability of BPA to bind to lipids. BPA is also able to bind to oestrogen receptors on the surface of cells, of which there are several types. Binding to each of these receptors affects signalling pathways and the expression of genes involved in regulating the cell cycle, growth and development. Consequently, the presence of BPA alters the cascades of biochemical reactions in the cell, modulating normal cell behaviour. This leads to various undesirable reactions that do not occur in the cell under physiological conditions. As described in the following subsections, scientific evidence of BPA's harmful effects on humans is accumulating.

BPA may promote the development of some cancers

BPA can trigger the transition of cells from an epithelial to mesenchymal form (Figure 12.8), which is characteristic of the progression of cancer. This is a process in which the epithelial cells of the primary tumour lose cell polarity and intercellular contacts, change the structure of the cytoskeleton (cell skeleton) and take on a mesenchymal form. Cells that have undergone such a transition have increased motility and can migrate into the blood or lymph vessels where they take on the typical characteristics of tumour stem cells. Furthermore, it was found that BPA can be responsible for triggering the process of tumorigenesis in ovarian cells, even at low (environmental) doses.

Figure 12.8 Epithelial-mesenchymal cell transition characteristic of cancer development. Under the influence of BPA, the cells gradually lose their orientation and become more mobile, taking on the typical characteristics of tumour stem cells.

BPA promotes metabolic changes in the body

BPA is also known as an adipogenic substance, which can cause or increase the risk of obesity and metabolic syndrome. This is because BPA, due to its lipophilic nature, is deposited in adipose tissue where it binds extensively to the nuclear hormone receptor (Figure 12.9). This receptor is a protein that enters the nucleus from the nuclear membrane and binds to the regulatory regions of genes. It then triggers the expression of genes responsible for the formation and development of adipocytes (fat cells), the regulation of blood glucose levels and the formation of proteins that bind fatty acids. Thus, on the one hand, BPA deregulates the formation of fat cells, but on the other hand, it also has a negative effect on sugar and fat metabolism in the blood.

Figure 12.9 BPA binds to the nuclear hormone receptor (shown in blue in the figure) and thus influences the expression of genes responsible for the development of fat cells. It also has a negative effect on the regulation of sugar and fat metabolism in the blood.

Recently, BPA has been shown to decrease testosterone production in the testes, with the effect depending on the dose and duration of exposure. At the same time, BPA increases the production of the hormone INSL-3 (insulin-like factor 3) in the Leydig cells of the testes, which is responsible for the descent of the testes from the abdominal cavity during intrauterine development of the foetus. Premature descent of the testes at a time when they are not yet sufficiently developed can contribute to reproductive disorders in adulthood.

In young men (aged 18-23 years old) BPA levels were measured in their urine, and this was found to correlate to a reduced amount of luteinising hormone, which stimulates the production and maintenance of testosterone levels. In addition, higher levels of BPA in the urine of these men also corresponded with reduced sperm concentration and total sperm count. These results thus support the hypothesis that BPA can reduce the capacity of Leydig cells, which are responsible for testosterone production, which in turn can reduce fertility.

In females, BPA can impact fertility via multiple pathways. The chemical is able to alter the levels of reproductive hormones, as well as impacting the ovary and there is evidence that exposure to BPA causes follicle loss and decreased oocyte survival. More recent evidence has suggested a link between BPA and PCOS (polycystic ovarian syndrome), although a causal link has yet to be proven.

BPA can be removed from the environment

The first way to remove BPA (as well as other environmental pollutants) from the environment is through the process of biodegradation. Here, microorganisms can use BPA as a carbon source i.e., a food source, which breaks BPA down into less toxic and simpler compounds. Since the entire organic world is made up of carbon and its compounds, microorganisms (e.g., some bacteria from the genera Pseudomonas and Methylomonas) degrade BPA into basic components of organic chemistry and benefit from this process through the production of energy. It is not so important what molecules are made up in the process of BPA degradation, but that bacteria can do it relatively efficiently and in a relatively short time.

Phytoremediation is generally a sustainable method of removing pollutants from the environment. Phytoremediation is based on the fact that plants absorb substances from the environment through their root systems, and some plants can also extract substances that are toxic to us (heavy metals, bisphenols, etc.) from the soil in this way. Examples include Cannabis sativa (hemp) or Panicum virgatum (millet), which can remove even relatively high concentrations of BPA from the soil. In this way, a specific pollutant can be removed relatively quickly and efficiently from the affected area, and the soil can then be used for agricultural purposes without concern of contamination.

BPA is not the only EDC with harmful effects

Nowadays, humans live in an environment where thousands of human-made compounds are increasingly present due to human activity. Many of them are considered potentially dangerous for human health. Special attention should be paid to EDCs, which, by influencing the function of the hormonal system, can cause the emergence of several (also civilizational) diseases. This chapter will briefly address some of them.

Phthalates are plasticizers used in the manufacturing of various products and are regarded as EDCs due to their anti-androgen effects. Phthalates are often added to vinyl products to make them more flexible but are often added to personal care products, such as perfumes, deodorants, shampoos, soaps, hair sprays, hair gels, nail polish, or body lotions, to help lubricate other substances in the formula and to carry fragrances. Despite their widespread use, phthalates can cause in vitro disruption of steroidogenesis by inducing increased estradiol synthesis. The induction of early (precocious) puberty in girls and altered gene expression in the testis caused by phthalates was also observed.There is evidence that also postnatal exposure is associated with significant decreases in sperm quality via anti-androgenic activity. Moreover, phthalates can contribute to metabolic disorders and are suspected to be associated with obesity in older European women.

Parabens are a class of synthetic chemicals commonly employed as preservatives in food, pharmaceuticals and cosmetic or personal care products. Since these products contain biodegradable ingredients, parabens are added to inhibit the proliferation of harmful bacteria and mould, thereby extending the product's shelf life. Similar to phthalates, parabens may also cause reproductive disorders. Long-term exposure of female mice and rats led to a significant decrease in serum estradiol and thyroxine concentration, a significant delay in the date of vaginal opening, and a decrease of corpora lutea, a temporary endocrine organ in female ovaries that is involved in the production of sex hormones. It is also possible that parabens may act as obesogens and contribute to the obesity epidemic.

Polychlorinated biphenyls (PCBs) were predominantly utilized as electrical insulating fluids in capacitors and transformers, as well as hydraulic, heat transfer, and lubricating fluids. Additionally, PCBs were combined with other chemicals to function as plasticizers and fire retardants, leading to their incorporation into various products such as caulks, adhesives, plastics, and carbonless copy paper. It was found that PCBs bind with relatively high affinity to the aryl hydrocarbon receptor (AhR), which is a ligand-activated transcription factor.The AhR responds to environmental, dietary, microbial, and metabolic signals to regulate complex transcriptional programs, with specificity depending on the ligand, cell type, and biological context. Exposure to PCBs results in reproductive disorders or testicular cancer, as was proven by Klenov et al., (2021) and Hardell et al., (2003). The production of PCBs was banned in many countries in the late 1970s due to their environmental persistence, bioaccumulation, and adverse health effects. However, PCBs can still be found in older equipment and materials produced before the ban, and managing these legacy sources remains an ongoing concern for environmental and public health.

Per- and polyfluoroalkyl substances (PFAS) are widespread environmental contaminants, often found in drinking water supplies. These chemicals are environmentally-persistent, human-made compounds that are used in the manufacturing of paper, textiles, pesticides, leather, medical aids, oil, minerals, metal plating, food packaging, cosmetics and personal care products, paints, inks, non-stick cooking utensils, surfactants, firefighting foams, and several other waterproof products (reviewed in detail in Lenka et al., 2021). The PFAS family consists of two main classes which are either fully fluorinated (perfluoro) or partially fluorinated (polyfluoro). The extreme persistence of PFAS lies in the nature of the carbon-fluoride bond because of which PFAS exhibit a high resistance to degradation and heat. PFAS are well known for being non-stick as well as having both, lipophilic and hydrophilic properties. PFASs also have diverse effects on human health, particularly as endocrine disruptors. They influence multiple aspects of male and female reproductive health, alter thyroid hormone synthesis, and contribute to dyslipidemia. However, human studies often yield contrasting results, likely due to the distinct effects of individual PFAS compounds and exposure to mixtures of PFASs in the environment.

Can endocrine disruptors cause epigenetic changes?

Epigenetics is the study of heritable changes in gene function that occur without altering the DNA sequence (more information is in chapter 7). Thus, epigenetic mechanisms control long-term transcriptional regulation, with epigenetic modifications defined as lasting alterations in gene function that persist after the initial trigger has disappeared, without involving changes to the gene sequence or structure. If EDCs are discussed, they are able to affect epigenetic markers such as DNA methylation, histone modifications or non-coding RNAs.

The most commonly studied mechanism of epigenetic changes is the effect of EDCs on the levels of enzymes that regulate epigenetic patterns, for example, DNA methyltransferases (DNMTs), which catalyse the addition of a methyl group to DNA (see chapter 6 – How the environment can affect our genes). Some studies also describe the effects of EDCs on DNMT expression, at both gene and protein levels. On the other hand, only a few studies examine the mechanism behind these changes. It is assumed, that the mechanism lies in the regulation of mRNA or miRNA expression. However, although there are certain assumptions about epigenetic regulation caused by the influence of endocrine disruptors, we still have relatively little information in this area and therefore further intensive research is needed.

Did you know that...

...microplastics, i.e. fragments invisible to the naked eye that are produced when plastics decompose, were recently detected in human blood by a team of Dutch scientists? Microplastics contain similar chemical additives as the original plastic containers i.e., BPA and other EDCs. Plastic microparticles are ubiquitous pollutants that litter the environment and the food chain, but no study has yet confirmed their presence in bodily fluids. Microplastics have been detected in various parts of the world, including the most inaccessible and mostly uninhabited places, such as Mount Everest, Russian Siberia and even Antarctica. This time, however, the scientists analysed blood samples from 22 healthy adult volunteers and found plastic microparticles ≥700 nm in 17 of them. The amount and type of plastics from which these particles originated varied considerably between the volunteers. In half of the volunteers, particles were found from PET (polyethylene terephthalate) plastics and polyethylene in about a quarter of participants. However, the researchers caution that the differences between individuals could also be due to whether they were exposed to plastic just prior to blood collection. At the same time, they note that it is not yet clear what effects plastic microparticles have on human health. However, this does not change the fact that this is a unique study that has demonstrated internal exposure to microplastics in humans for the first time.

Want to read more?

Leslie et al. (2022). Discovery and quantification of plastic particle pollution in human blood. Environment International, 163, 107199.

Alavian-Ghavanini & Rüegg (2018) Understanding epigenetic effects of endocrine disrupting chemicals : from mechanisms to novel test methods. Basic & Clinical Pharmacology & Toxicology 122(1):38-45

Zhang et al. (2021) Phthalate metabolies: Characterisation, toxicities, global distribution and exposure assessment. Enviornmental Pollution 291:118106

Reddy et al. (2022). Xenoestrogens impact brain estrogen receptor signaling during the female lifespan: A precursor to neurological disease? Neurobiology of disease 163:105596

Sumpter, J. P. (2005). Endocrine disrupters in the aquatic environment: an overview. Acta hydrochimica et hydrobiologica 33(1):9-16

Moche et al. (2021). Comparison of in vitro endocrine activity of phthalates and alternative plasticizers. Journal of toxicology 2021(1):8815202

Legler et al. (2015). Obesity, diabetes, and associated costs of exposure to endocrine-disrupting chemicals in the European Union. The Journal of Clinical Endocrinology & Metabolism 100(4):1278-1288.

Nowak et al. (2018). Parabens and their effects on the endocrine system. Molecular and cellular endocrinology 474:238-251.

Hu et al. (2013). Effects of parabens on adipocyte differentiation. Toxicological sciences 131(1):56-70

Erickson, M. D., & Kaley, R. G. (2011). Applications of polychlorinated biphenyls. Environmental Science and Pollution Research 18:135-151.

Gaillard et al. (2024). Per-and polyfluoroalkyl substances as persistent pollutants with metabolic and endocrine-disrupting impacts. Trends in Endocrinology & Metabolism 23:S1043-2760

Gore et al (2015). EDC-2: the Endocrine Society's second scientific statement on endocrine-disrupting chemicals. Endocrine reviews 36(6):E1-E150.

Land et al. (2022) The effects of endocrine disrupting chemicals on ovarian- and ovulation-related fertility outcomes. Molecular Reproduction and Development 89(12):608-631

Chapter 13: Etiquette in our genes

"For behaviour, men learn it, as they take diseases, one of another."

The author of the quote, the English statesman and philosopher Francis Bacon, described a phenomenon that we commonly observe in the course of our lives. Children's behaviour often reflects the behaviour of their parents, siblings or other members of society (you can find an example of such learned behaviour in Chapter 2 – How does a scientist work), but genetic predisposition also plays a role here. But which of these factors is decisive?

The answer lies somewhere in the middle. In general, we can say that the resulting phenotype of an individual is influenced by its genes and at the same time by the environment. The same applies in this case. The influence of genes on the behaviour of an organism is studied by the scientific field of behavioural genetics. Its founder is Francis Galton, cousin of the famous Charles Darwin. Galton lived in the 19^th Century and was a polymath, which means that he was involved in practically all branches of science at the same time. In one of his observations, Galton dealt with the heredity of human abilities and mental qualities. He analysed the family trees of the English nobility, focusing on the social and intellectual achievements of individual members. He published the results in the book "Hereditary Genius" in 1869, exactly 10 years after Charles Darwin published the book "The Origin of Species". In his results, Galton outlined the possible influence of the environment on the exceptionality of individuals of the English nobility. Although he interpreted his results vaguely and spoke only of a possible influence, his study started a debate about the role of genes and environment in shaping behavior. Within the field of behavioural genetics, several model organisms are used in research. Sometimes they are unicellular microorganisms, other times insects or multicellular vertebrates - most often mice or rats, for which there is a very well-developed system of studying various forms of behaviour.

Even molecules and microorganisms show signs of behaviour

At the level of molecular biology, we can think about behaviour in terms of cooperation. This happens, for example, between genes that make up complex genomes. For the functionality of metabolic pathways, the cooperation of several enzymes, which are coded by individual genes, is necessary. Another example of coordinated behaviour at the molecular level is protein subunits that must work together to form a functional enzyme. However, in these cases it is still difficult to talk about "behaviour" as we know it in ordinary life. We also observe cooperation between individual cells. One example are microorganisms that show signs of social behaviour by sharing molecules secreted into the environment, including enzymes that break down food. At the same time, they can communicate with the help of various signalling molecules, which alert community members to the presence of danger in their surroundings. As communities in which microorganisms occur, we can consider cell colonies or more complex biofilms, which are formed by various types of microorganisms, including yeast or bacteria. With their metabolism, they can mutually compensate the needs of the various "inhabitants" of the biofilm, and this cooperation also helps in the defence against antimicrobial substances. Similar communication takes place between the cells that make up multicellular organisms, and their cooperation at every level is essential for the functioning of a complex organism.

Thieves among microorganisms

An example of the social behaviour of microorganisms can be observed in some types of yeast, which are often referred to as social cheaters. We are talking about liars who take advantage of the social community while contributing less than average to its functioning. There are types of baker's yeast, Saccharomyces cerevisiae, which cannot produce the invertase enzyme due to a mutation in the SUC2 gene. The latter breaks down the extracellular disaccharide sucrose into molecules of simple sugars, glucose and fructose, which are subsequently transported into the cells as a source of energy. This means that yeast with a mutation in the SUC2 gene cannot independently process such a disaccharide. However, if they are in a community with other yeasts that do not have the mutation, the wild type yeast will produce invertase, and shortly after breaking down sucrose, the mutant yeast "steal" glucose and fructose molecules from the environment and use them for their own metabolism (Figure 13.1). At the same time, they save energy that would otherwise have to be spent on enzyme production. Thus, social cheaters are favoured in the environment until the environment is full of yeast capable of producing invertase. The rogue behaviour of mutants resulting from the non-functionality of a single gene can change the default behaviour into an exploitative way of functioning in the social group of these yeasts.

Figure 13.1 Social cheaters in the yeast culture Saccharomyces cerevisiae. Yeast known as social cheaters have a mutation in the SUC2 gene that prevents them from producing a functional invertase enzyme. By default, this enzyme ensures the breakdown of sucrose present in the environment into glucose and fructose molecules, which cells use for their metabolism. Social cheaters do not have this ability, so they use the invertase produced by standard cells for their own consumption.

Lazy and drunk flies

Fruit flies, Drosophila melanogaster, which are one of the most frequently used model organisms in genetics, does not bypass behavioural genetics either. Some regions of human genes that have been associated with various neurobiological disorders have related genes present in the genome of these flies. Therefore, Drosophila have become a valuable tool for the analysis of various diseases and disorders. An example is a mutation in the gene for the protein kinase G enzyme, which is believed to be related to the increasingly common neurodevelopmental disorder of attention associated with hyperactivity, known as ADHD (attention deficit hyperactivity disorder). The protein kinase G enzyme is encoded by the DG2 gene in fruit flies. Depending on the specific type of allele located at the respective locus, different production levels of the protein occurs, which subsequently affects the behaviour of flies in search of food. Individuals with rover alleles are able to cover greater distances in search of food than individuals with a phenotype conditioned by sitter alleles (Figure 13.2). Although this pattern of behaviour is conditioned by an allele of one gene, as in many other cases, there is an interaction with other genes and a polygenically conditioned phenotype.

Figure 13.2 Different foraging behaviours of the fruit fly Drosophila melanogaster. Mutations in the gene encoding protein kinase G (PKG) cause differences in the production of this enzyme. With an excess of PKG, flies exhibit a rover phenotype, meaning they cover greater distances during foraging. Conversely, the sitter phenotype is manifested by limited movement.

An interesting example of genetically determined behaviour is the relationship of male fruit flies to alcohol. This relationship is influenced by courtship, which consists of dancing, singing or even gentle touches on the females. If the male's efforts fail, he loses interest in females and compensates for the failure with increased alcohol intake. This is due to low levels of NPF, which is a neuropeptide also found in the human brain. A decrease in the level of NPF can also occur in females, in which it also causes an increased preference for ethanol. The reason is not a loss of interest in mating, but a reaction to the appearance of a parasitic wasp in the environment. In its presence, female flies begin to prefer laying eggs on alcohol substrates, thereby providing the future larvae with the necessary protection against wasp infection. Exposure to such predators can thus lead to epigenetic changes, which you learned more about in Chapter 7 - From epigenetics to diseases. The epigenetic state causing reduced NPF formation, can be inherited in up to five generations. Such studies provide valuable information that can be used in the investigation of hereditary preference for drug or alcohol use.

The researchers also identified several different mutations in genes related to alcohol consumption. The hangover mutation is associated with low tolerance to alcohol. Flies with this mutation do not need to increase doses to achieve the same effect as standard flies without the mutation, so they are less likely to become addicted. It is the same with the krasavietz mutation, which leads to decreased tolerance and taste for alcohol. Flies with this mutation do not show pleasure nor stimulating effects after consuming alcohol. The happyhour mutation has the opposite effect, which manifests itself in low sensitivity to alcohol, and the mutant flies thus experience pleasant feelings after consuming it.

How mosquitoes changed their preferences

Female mosquitos search for a host search using a combination of different stimuli, including chemosensory, thermal or visual impulses. It is smell (chemosensory) that is the critical signal that identifies the presence of the host to mosquitoes. The host odour is a complex mixture of chemicals and volatile compounds produced by skin microflora that induce electrophysiological and behavioural responses in mosquitoes. These compounds include, for example, lactic acid, ammonia, various ketones, sulphides, or carboxylic acids. The female mosquito perceives the odours of the host through olfactory receptors that can be found on the antennae or in the area of their mouthparts. A preference for human hosts, which we refer to as anthropophily, was developed by some mosquito species only later in evolution. This was made possible by changes in their genetic information, precisely at the level of olfactory receptors - anthropophilic mosquitoes are characterized by an increased expression of genes encoding these receptors. The identification and further study of specific genes responsible for the expression of olfactory receptors could in the future lead to the invention of such protective means that would once again change the preference of mosquitoes to other hosts and thus limit the spread of various infectious diseases, which mosquitoes are the carriers of.

Models of human behaviour disorders

Knowledge is a psychological concept that includes the processes of learning, memory and perception. Cognition, i.e. the ability to know, is one of the psychological processes that, from an experimental point of view, can be derived from changes in the behaviour of an organism. Testing of cognitive functions and their genetic background is often investigated using rodents that are subjected to behavioural tests. The strategy for this type of experiment may involve the targeted switching off of a certain candidate gene and then testing and recording changes in the individual's behavior. These experiments are also used in the preclinical investigation of the effect of new drugs targeting the central nervous system.

One such test is the open field test (Figure 13.3), which is used in the study of locomotor activity, anxious behaviour and willingness to explore the surrounding environment in rodents. The most common manifestation of interest in the environment is movement, therefore the test records the distance travelled, time spent moving, or changes in behaviour over time. All these parameters can be affected by the anxiety of individuals. The open field test is also used as a standard when evaluating the toxic and stimulating effects of various compounds. Motor coordination is assessed in rodents by the rotator test (Figure 13.3), in which the animal is placed on a horizontal bar that rotates around its long axis. Only individuals with functional motor skills can coordinate their movements so that they stay on the pole and do not fall.

Some neurodegenerative diseases, such as Alzheimer's or Parkinson's disease, manifest as changes in motor skills and movement, which we refer to as ataxia. The reason is changes at the level of neurons, which are also related to the reduced mental ability of individuals. Depressive behaviour is assessed by the forced swim test (Figure 13.3), in which the rodent is placed in a container of water from which it initially attempts to escape. After a while, however, he gives up his efforts. Individuals showing depressive behavioural tendencies take less time to give up compared to healthy rodents. The parameters of these types of tests can be measured, but they still leave some doubts about their subjective evaluation.

Figure 13.3 Behavioural tests used in research. The tests are focused on monitoring the association of specific genes with the behaviour of individuals, or on the effect of various substances or drugs. Individuals with a non-functional gene or individuals exposed to the effect of the studied substance are subjected to several behavioural tests with a different focus.

From twins to whole genomes

The heredity of certain behavioural characteristics was already known to mankind in prehistoric times which manifested mainly in the domestication of animals. Subsequently, the transmission of various human characteristics have also came to the attention. In the 19^th Century, the idea of heredity explained the blood theory, according to which a child is created by the fusion of the parents' blood, thereby acquiring not only their characteristics, but also the characteristics of previous generations. However, the exact mechanism of inheritance was not known. The turning point occurred at the end of the 19^th Century, when it was first thought that animals could be a model of human behaviour. The connection between the animal and human minds was described by Charles Darwin, who at the same time decided to test the theory of blood. Together with Francis Galton, they transfused blood into several species of rabbits in order to observe the transmission of various behavioural characteristics. However, their experiments always ended with a negative result, so they did not confirm the popular theory at the time.

From a historical point of view, studies of identical twins provide important data related to biomedical, psychiatric or behavioural questions (differences arising between identical twins were described in Chapter 7 – From epigenetics to human diseases). During the 20^th Century, classic twin studies were used extensively to distinguish genetic and environmental sources of variation in human populations. Adoption studies were also widespread, which demonstrated that the cognitive abilities of siblings living in the same household and growing up separately are approximately the same. Thanks to these studies, scientists subsequently began to pay more attention to genetic factors that influence human intelligence. Research in the field of behavioural genetics was (like all other scientific disciplines) greatly influenced by the discovery of the structure of DNA. The polymerase chain reaction became part of the new era of molecular genetics, which enabled the amplification of selected regions of DNA, and other genetic engineering tools became available. Behavioural genetics initially focused on known and available genetic polymorphisms (blood groups or regions of human leukocyte antigens), but the discovery of microsatellite markers, which are distributed along the entire genome at intervals of approximately every 10 million bases, subsequently made it possible to carry out more extensive linkage studies. These provided an approximate localisation of variants responsible for individual differences. Currently, this type of research is mainly focused on analyses of genetic variants within the entire genome, which we call genome-wide association studies, or GWAS for short. When comparing genomes, scientists mainly focus on the possible influence of SNP polymorphisms (you can read more about polymorphisms in Chapter 15 - DNA as evidence) and various other DNA characteristics, which means that not only data from related individuals need to be analysed. The advantage of such studies is that it is not necessary to have pre-selected candidate genes for a certain phenotype. As of 2017, more than 3,000 such studies have been conducted.

Is intelligence, criminality or sexuality coded in our genes?

As early as 1904, the English psychologist Charles Spearman noticed that the level of the ability to react to certain sensory perception and the overall intelligence of a person are closely related. He saw the connection between the ability to orientate in space, the perception of colours, directions or solving mathematical problems. He verified his observations for the first time on 24 pupils who attended a small village school. Spearman tested how well students performed on various intelligence-related tasks and concluded that individuals who did well in one area usually scored higher in other areas. Based on this, he defined general intelligence, which, according to him, represents the central factor of our cognitive abilities.

Later studies of twins and adopted children pointed to the possible heritability of some factors that determine our intelligence. Behavioural geneticists have used genome-wide studies to identify specific genes. They identified genes in several candidate areas in which the individuals differed. In this way, one of the first genetic markers associated with intelligence was also identified - the IGF2R gene, which encodes a receptor for mannose-6-phosphate and participates in the transport of phosphorylated lysosomal enzymes from the Golgi network to lysosomes. This gene is located on chromosome 6 and one of its variants is more common in children with higher IQ. Despite this, it cannot be said with certainty that it is an intelligence gene, because the role of this receptor in the cognitive and analytical processes taking place in the human brain is not known. Later, another study identified six more genes related to intelligence, and upon closer examination, each of these genes was shown to have very little effect on its own. In the case of intelligence, it is therefore a polygenically determined phenotype that is largely influenced by the environment (for example, place of residence, physical activity, family income, parents' occupation and education).

The possibility of the influence of genes on criminal behaviour was first publicly presented in 1991 in the state of Georgia, USA. The central character was, paradoxically, one of the thieves who wanted to justify his act by saying that he comes from a long family line of criminals, and thus his behaviour is conditioned by the genetic mutation he inherited. His appeal was rejected, but the link between genes and crime began to be studied. Since the transmission of this type of trait initially appeared to be female-dependent, scientists turned their attention to the X chromosome. One of the first candidates was the MAOA gene identified during an extensive analysis of a Dutch family with several generations of violent criminals. The MAOA gene encodes the catabolic enzyme monoamine oxidase, which removes amino groups (-NH2) from biogenic amines. It is part of the mitochondrial membrane and is also found in neurons forming presynaptic connections. Disruption of the function of this enzyme also interferes with the activity of the brain. The scientists managed to identify the specific region in which the gene is located and also the point mutation that occurred in the monitored individuals. Such a mutation created a premature stop codon in the gene, which led to the creation of a shorter and therefore non-functional enzyme (to clarify the term stop codon, you can return to Chapter 3 - Meet DNA, the bearer of genetic information). Scientists also confirmed the connection between a mutation in the MAOA gene and aggressive behavior in rodent animal models. The introduction of the MAOA genes increased the aggressiveness of the monitored individuals, which was evaluated based on the number of wounds caused by bites from aggressive individuals. Although it was possible to demonstrate a link between genes and the potential for criminal behavior, as with intelligence, the effect of the mutation alone is clearly not sufficient to be considered the cause of aggressive behavior.

Homosexuality is also a common phenomenon that we observe not only in humans, but is also known from the animal kingdom. The genetic basis of this sexual behavior has been the subject of research for a long time and is still poorly understood. Like intelligence or aggressive behaviour, sexuality is conditioned by polymorphic genes. In the 1990s, Dean Hamer discovered a gene that he believed was responsible for homosexual behaviour in men. He attributed heredity to the X chromosome, as the kinship of homosexual men on the mother's side of the family was more often observed. By comparing the results with heterosexual individuals, he was able to identify a deviation in the section called Xq28. However, it is only a variant in this area, not a "homosexuality gene". Sexuality is related not only to genes, but also to hormonal influences, the course of pregnancy and cultural conditions. The proportion in which these factors contribute to an individual's phenotype is individual. In general, the influence of genes on behaviour can only be estimated from a probabilistic point of view, and we cannot talk about their determining influence.

The main focus of contemporary behavioural genetics is the analysis of variations in behaviour with an emphasis on mental state or cognition. Many medical and military agencies are making huge investments to understand the origins of behavioural differences, since many of the most pressing health problems in modern cultures are linked to pathological behaviour—for example, obesity, certain cardiovascular diseases, cancer, drug use, and psychopathology. Therefore, the goal of behavioural genetics is to identify the genetic sources of individual differences in behaviour and susceptibility to specific environmental influences.

Did you know that...

...genetic background can also affect our success at work? In 2022, behavioural geneticists addressed the question of the extent to which genes might be related to socio-economic status, which is considered a prerequisite for psychological well-being and health. By incorporating information from available employment sources into a genetic database, scientists were able to perform a genome-wide association study on over 200,000 people of European descent. With such an approach, they obtained a large sample, which solved the frequent problem of association studies with data availability. They focused on six main characteristics that can influence success – work complexity, independence, innovativeness, informational, emotional and physical demands. They managed to identify 16 loci and some polymorphisms that could be related to the relevant characteristics, as well as their connection to education, employment or salary achieved. While the complexity of work, innovativeness and IT skills were associated with higher income, on the contrary, emotional or physical demands were mainly associated with health and a sense of well-being. But the authors point out that genetic variants cannot miraculously affect our success in employment. As was often mentioned in the chapter, genetic influences are also mediated and guided by environmental factors.

Want to read more?

Cases, O. et al. (1995). Aggressive behavior and altered amounts of brain serotonin and norepinephrine in mice lacking MAOA. Science. 268(5218):1763-1766.

Song, Z. et al. (2022). Genetic basis of job attainment characteristics and the genetic sharing with other SES indices and well-being. Sci. Rep. 12(1):8902.

Principles of Behavioral Genetics, Anholt, R.R.H., Mackay, T.F.C., Elsevier Science & Technology (2009).

Foundations of Behavior Genetics, Stoltenberg, S.F., Cambridge University Press (2022).

50 Genetics Ideas You Really Need to Know, Henderson, M., Quercus Publishing (2009).

Handbook of Behavior Genetics, Kim, Y.-K., Springer (2009).

Chapter 14: The principle of evolution

The theory of evolution and the field of evolutionary biology are most closely associated with the work of the English biologist Charles Darwin. However, ancient philosophers were already dealing with questions about the origin of life. These philosophers believed that living beings arose from the offspring, or individual parts, of organs that randomly joined together. In the 18^th Century, Carl von Linné, one of the most important systematists, argued that individual species differ in many important characteristics, while individuals within a species differ in only a few important characteristics. However, at that time, various ideas about the origin of life persisted, including for example, that organisms arise from non-living matter and the idea of creationism, which says that all organisms were created as we see them today, and only some of them have already become extinct, was prevalent. The possibility of evolution, the gradual development of a species, was not accepted then.

The first biologist to develop the theory of evolution was Jean-Baptiste Lamarck. In 1809 he published his most important work "La philosophie zoologique". Lamarck assumed that organisms are created repeatedly and gradually improve (transform). He suggested that by using a certain organ its function improves, and if a certain organ is not used, then stunting occurs. An important thesis of his theory was that these acquired characteristics are passed on to offspring. Although Lamarck's reasoning about the mode of change was shown to be incorrect, he was the first major scientist to conclude that evolution occurs at all.

The foundations of the theory of evolution, the principles of which persist to this day, were laid by the famous English scientist Charles Darwin (Figure 14.1). Darwin was a very perceptive pedant, and despite the fact that he did not have a biological education, and we consider him one of the most important biologists of all time. Darwin made his most important records and observations during a voyage on the ship Beagle in 1831-1836, the purpose of which was to map the coast of South America. He was fascinated by the various forms of ocean life and on land he explored many exotic areas (Patagonia, the Andes, the Galapagos islands and Australia). Darwin collected a large amount of material, which he continuously sent to London. After returning from the voyage, he began to write down his observations and formulate the ideas of evolutionary theory. The acceleration of the publication of his main work was the responsibility of Alfred Russel Wallace, who also developed a theory based on his observations, which did not differ in principle from Darwin's ideas. The manuscripts of both scientists were published at a meeting of the Linné Society in London in 1858. However, Darwin accelerated the preparation of the publication of his main work "On the Origin of Species", which was published in 1859.

In this work, he explained the basic principles of the theory of evolution, which we can summarise as follows:

• Existence of species evolution – species change over time, that is, evolution takes place.

• There is a common origin of all species – species separated (diverged) from a common ancestor in the course of evolution.

• Gradualism – species change and diverge gradually, by the slow accumulation of small changes.

• Natural selection – this is the main mechanism driving evolution, selecting the fittest individuals from populations.

Figure 14.1. Charles Darwin (1809 – 1882).

Of course, Darwin's theory of evolution met with many supporters, but also with great critics. The genetic knowledge of heritability, introduced by experiments of Gregor Johann Mendel, helped at the beginning of the 20^th Century to understand some parts of the theory of evolution, which until then were considered as its shortcomings (e.g. that alleles are inherited from parents to offspring, but do not mix).

Although in the first half of the 20th Century while heredity and the existence of mutations were known, many scientists still thought that, for example, bacteria behave differently and can create hereditary properties depending on the environment. An important experiment that contributed to solving this question was the so-called fluctuation test carried out by Max Delbrück and Salvador Luria in 1943 (see also Chapter 2 - How does a scientist works). These scientists worked with cultures of bacteria sensitive to bacteriophage T1. Delbrück and Luria took a part of the bacterial culture, cultivated it for a certain time without the presence of bacteriophage, and then spread the bacteria on media with phages. They found that resistant colonies of bacteria grew on the dishes, and their number was approximately the same as on individual dishes. In a parallel experiment, the culture of bacteria was divided into independent subcultures, which were cultured for the same amount of time without the presence of phage. After that, each subculture was grown separately on media in the presence of phage, and it was found that the number of resistant bacteria was very different on different plates (Figure 14.2). Based on the cultivation of independent subpopulations of bacteria in the presence of selection pressure (the phage), Delbrück and Luria confirmed that the emergence of bacteria resistant to bacteriophage T1 is the result of a random mutation that "won" under selection conditions. Therefore, the hypothesis that the emergence of resistant bacteria is an active response to environmental conditions, or that mutations arise "by order" of the environment is false.

Evolution is not a process that must necessarily be connected by a huge period, but we can follow it in real time. An excellent example is the population of peppered moths (Biston betularia) in Great Britain in the 19^thand 20^th Centuries and the phenomenon known as industrial melanism. Before the year 1800, this moth existed in its natural light-coloured form. The melanic form (with dark colouring), which is caused by a mutation in one gene, occurred only very rarely. The industrial revolution brought with it the pollution of the environment by large factories, which resulted in the bark of the trees becoming darkened with soot. Since this moth spends most of the day resting on the bark of trees, the natural form with light-colouring were easily visible on the darker bark of trees, and individuals were more often eaten by predators. In contrast, moths with melanic colouring were less visible and were less likely to be eaten. Although their frequency in the population was initially low, under such conditions the melanic form of the moths began to prevail in the population. Since the 1950s, with more emphasis placed on environmental pollution control, conditions have greatly improved in many industrial areas. The bark of the trees have become paler again and the natural forms of the moth are beginning to dominate again with melanic forms becoming rarer in these areas.

Figure 14.2. Schematic principle of fluctuation test. A bacterial culture is cultivated without the presence of bacteriophage. Then, a single subculture is separated and again cultivated for several generations without phage. This subculture is then plated onto Petri dishes and cultivated in the presence of phage, resulting in a similar number of resistant colonies on each plate. Next, several subcultures are cultivated separately in the absence of phage. When plates onto Petri dishes with phage, the number of resistant bacterial colonies is very different between different subcultures.

One of the basic evolutionary factors is selection. Due to the influence of selection, there is a difference in survival and successful reproduction in harsh environmental conditions. Abiotic factors (climate, altitude, salinity etc.) but also biotic factors (availability of food, predators, parasites, pathogens) can act as selection factors. The intensity of selection in evolutionary biology is determined by the so-called selection coefficient, which we express in the range 0-1. If the selection is maximal, i.e., each bearer of the given character is excluded from reproduction under the given conditions, the selection coefficient has a value of 1.

We also recognise different types of selection with variation in how selection affects the preservation of the frequency of individual phenotypes, and thus genotypes. One type of selection is selection in favour of heterozygotes, an example of which is the maintenance of the mutated Hbs allele for β-haemoglobin, whose carriers in the homozygous state suffer from sickle cell anaemia (see also Chapter 5 – Mutations: how they arise and what to do about them). In the heterozygous state, the Hbs allele provides carriers with an advantage in the form of resistance to malaria. It is therefore important to note that this allele can be considered advantageous only under severe selection conditions, which in this case is a geographical area with a high prevalence of the Anopheles mosquito, the carrier of malaria. If a human intervenes in the selection process with this activity, we call it artificial selection. A typical example of artificial selection is breeding.

There are also different views on the level at which selection operates. The classic "Darwinian" view says that selection acts at the population level, but it can also act at the level of individuals or groups of individuals. The British ethologist and evolutionary biologist Richard Dawkins brought a different perspective on this question. Dawkins claims that selection acts at the level of genes and he published his ideas in 1976 in the well-known work "The Selfish Gene". In 1973, John Smith and George Price developed the theory of evolutionarily stable strategy, which asserts that individuals with a trait that conditions the highest possible fitness (reproductive fitness, the ability to reproduce in a given environment) will not prevail in the population, but such a behavioural phenotype will be established under the given conditions, which cannot be replaced by a better one by natural selection. Environmental conditions and relationships between individual members of the population thus help to keep the fitness of each individual in check.

A prerequisite for the action of selection is the existence of variability between organisms. Therefore, mutations are another important factor in evolution. We divide mutations in connection to selection as follows:

negative mutations – they disadvantage the carrier of the mutation and reduce its fitness. As a result, the frequency of the given mutation in the population will gradually decrease. There are many examples of negative mutations that cause human diseases (e.g., cystic fibrosis, phenylketonuria).
positive mutations – carriers of such a mutation are favoured. Such positive mutations result in a higher fitness than other individuals in the population, so the frequency of such an allele will gradually increase. At a certain time, the so-called fixation arises, which means that each individual will have a given mutation (e.g. butterflies that have a longer proboscis will more easily get nectar from a flower, and after time, all butterflies of a given species will have a long proboscis).
neutral mutations – selection is not directly affected as these mutations do not change the coding information in DNA. We can also consider as neutral mutations those that are reflected in the phenotype, but different phenotypes in this case do not affect fitness. However, populations do not have an infinite size, they cannot increase indefinitely, although individuals produce a large number of gametes, only a certain part of them is used and gives rise to new individuals. Thus, the frequency of alleles that carry given mutations can change due to a random process known as genetic drift (Figure 14.3). We could describe the difference between selection and genetic drift by saying that selection is a controlled process while genetic drift is random, but both can lead to the disappearance of a certain genotype.

With the advent of molecular biology methods, it has become clear that neutral mutations and genetic drift are not marginal factors in evolution. In 1968, the Japanese scientist Motoo Kimura came up with the theory of neutral evolution, which says that neutral mutations are responsible for most evolutionary changes at the molecular level, and changes within species and between species occur mainly through genetic drift. This theory is also supported by the fact that mutations in sites that do not affect protein functions or non-coding regions are the most common.

Figure 14. 3. Schematic representation of genetic drift. An example is a population in which individuals form gametes of 2 different genotypes (red allele and green allele). During the creation of offspring, the selection of gametes is random, therefore, in the next generation, the representation of alleles in the population may be changed. After a time, a certain allele may even disappear (green) and the other allele will be fixed in the population (red).

Recent human evolution

In the year 2000, the well-known palaeontologist Stephen J. Gould declared that no change has taken place in humans for 40,000-50,000 years, and the evolution of humans is only slight or has already ended. On the other hand, it is obvious that variability still exists at the biological level and the environmental conditions in which people live are different, so selection can still affect us. An example of recent human evolution is a mutation in the LCT1 gene, which ensures the activity of the lactase enzyme until adulthood. People without this mutation often suffer from lactose intolerance. The mutation spread sometime between 4,500 and 7,500 years ago, around the time when farming probably appeared in the northern Balkans as a means of subsistence. Carriers of the mutated LCT1 allele were favoured because they could consume more dairy products. However, some recent studies indicate that this positive selection may have occurred later and originally spread from Russian shepherds. There are many places in human DNA where similar changes can be observed by bioinformatic analyses, for example, the EPAS1 and EGLN1 genes, which are related to the adaptation to high altitudes of Tibetans, or mutations in genes related to pigmentation and reactions of the immune system.

Evolution can progress relatively quickly and similar features of various organisms can develop independently of each other

We cannot consider evolution as a purely random process. The occurrence of mutations is random, but the selection in favour of a certain variant is always determined by something. An example is the work of the prominent American evolutionary biologist Jonathan Losos, who devoted most of his scientific career to the study of the ecology and evolution of lizards of the genus Anoles. About 400 species of these lizards live in South and Central America, while about 200 live on islands in the Caribbean Sea. Such islands represent huge "test tubes" for terrestrial animals, where it is possible to independently observe how a given species develop under harsh conditions. The islands in the Caribbean Sea offer different ecological environments, different types of predators, and competitors, and are often hit by tropical storms. In the 1980s, Losos and his colleague conducted an experiment in which they brought lizards to islands where no lizards had previously lived and observed what changes they would undergo. They observed the signs of the environment that the lizards inhabit (tops of trees, grass or the ground, etc.), they also observed the joints of the legs, length of the digits and the colour of the chin lobe of the lizards. After approximately 10 years, they evaluated how the lizards performed in this new environment. They found that all the lizards chose a certain type of environment for their new life, while lizards living in the same conditions on different islands independently developed similar behavioural traits. For example, lizards living on the ground have longer limbs that allow them to escape from predators faster, while lizards living in treetops have shorter limbs to be more agile when moving. In addition, the lizards living in the treetops have longer last digits (toes) so that they can better hold on to the branches. Lizards on islands where hurricanes are more frequent also have longer toe joints. As for the chin lobe, which they use as a warning against enemies, but also to attract attention during mating, the lizards living in darker environments had a paler chin lobe, so that the environment would have a stronger contrast. On the other hand, lizards living in sunny places had their chin lobes more prominently coloured. The experiments by Losos showed that evolution can be relatively fast if the selection pressure is very strong. Moreover, if the selection conditions are the same, organisms can independently acquire the same characteristics (we call this phenomenon convergent evolution).

Did you know that...

...although the biological evolution of man as a species is very small, human society is subject to the so-called phenomenon of cultural evolution? Under cultural evolution, we could imagine that the characteristics of culture, such as language, ideas, knowledge, skills, or technology, all influence our behaviour and success, and we pass them on to the next generations. Patrick Savage and his collaborators published a study in which they traced the evolution of Japanese and English folk songs. Using a sample of 10,062 melodies, they demonstrated that songs undergo gradual modification. Like DNA mutations, changes in tones are more common if they do not significantly affect the overall melody of the song. In contrast to mutations at the DNA level, where mutations of the type of insertions and deletions are rare, in the case of the evolution of melodies, insertion or deletion of tones is more frequent than substitution. Using this example, they demonstrated that even creative art is the subject of an evolutionary process that carries an analogy with the evolution of genes, language, or other elements of culture.

Want to read more?

Some Assembly Required: Decoding four billion years of life, from ancient fossils to DNA by Neil Shubin

Your Inner Fish: A journey in the 3.5 billion year history of the human body by Neil Shubin

Sapiens: A brief history of humankind by Yuval Noah Harari

Chapter 15: DNA as evidence – forensic genetics

Forensic genetics is a scientific discipline where we use the knowledge of genetics and DNA analysis to identify persons of interest and as evidence in court proceedings. It is not only the determination of the perpetrator of the crime, but also the identification of persons, for example in mass accidents, the identification of skeletal remains, or the determination of paternity, or other kinship.

DNA holds the answer

The main role in forensic genetics is played by the DNA molecule, the structure of which is described in more detail in Chapter 3 - Meet DNA, the bearer of genetic information. At this point, we must remember that DNA has to be packaged very precisely to fit into the cell nucleus. Several important proteins (e.g. histones) participate in the process of wrapping DNA and compaction, and DNA together with proteins forms a structure known as a chromosome. A human somatic cell has 46 chromosomes (23 pairs). Those marked with numbers are called autosomes and besides them, we have 2 sex chromosomes (gonosomes), X and Y. Females have two X chromosomes, and males have an X and a Y. We have two copies of each chromosome, one inherited from our mother, the other from the father. These chromosomes are the same in that they carry the same genes (e.g. the gene for the blood antigen that determines the blood group), but the form of the gene (allele) may not be the same – we could inherit the allele for blood group B from the mother and the allele for blood group A from the father. Before the introduction of DNA analysis in forensic genetics, the identification of blood groups played an important role in criminal investigations (Figure 15.1).

Figure 15.1 Inheritance of blood groups. Schematic representation of possible genotypes in the offspring of parents with IBi and IAi genotypes.

Thus, our DNA is a combination of the DNA of our ancestors, while the process of meiosis - during which sex cells are formed and recombination between homologous (same) chromosomes occurs - significantly contributes to the increase in genetic variability. Since the Y chromosome is found only in men, it is inherited through the paternal lineage. In addition to the DNA inside the nucleus of the cells, DNA is also found in the mitochondria, which are stored in the cytoplasm of the cell. After fertilisation, the zygote acquires the cytoplasm of the mother's egg cell, and therefore the mitochondrial DNA is inherited from the mother. With the help of mitochondrial DNA analysis, we can trace the maternal lineage. What is interesting here is that mitochondrial DNA is found in multiple copies, so even if the nuclear DNA present in the forensic trace were degraded, mitochondrial DNA may still be detectable.

Human genetic information consists of 3x10⁹ base pairs. We refer to the order of individual nucleotides as the DNA sequence and the method by which we can determine this order is called sequencing (more in Chapter 4 - How do you work with DNA?). The first, almost complete sequence of human DNA was revealed in 2003, thanks to the Human genome project (HUGO). HUGO found, among other things, that the DNA of two unrelated people differs only in 0.1-0.3% of positions out of a total of three billion possible positions. In the case of identical twins, the DNA sequence is the same. Genes are the regions of DNA that code for some functional product (see Chapter 3 - Meet DNA, the bearer of genetic information). Genes make up only about 1.5% of the total genetic information of a person (it is assumed that a person has 20,000-22,000 protein-coding genes). The majority of genetic information consists of the so-called non-genic or non-coding DNA. Some of these non-coding regions have a regulatory function and can influence the transcription of genes, but in general, not much is known about the function of several parts of non-coding DNA.

If we want to compare the DNA of two people and distinguish them based on such an analysis, we have to focus on positions that are different between people. We refer to such positions as DNA polymorphisms. An important type of polymorphism is sequence polymorphisms, often referred to as single nucleotide polymorphisms - SNPs (Figure 15.2). This means that if, for example, an individual has an adenine at a certain location, someone else will have a guanine at that exact position in their DNA.

Figure 15.2 Representation of DNA sequence polymorphism

One of the methods by which we can determine differences in DNA between people is by restriction fragment length polymorphism analysis - RFLP. In this method, we use enzymes (restriction endonucleases) isolated from bacteria, which can specifically cleave (cut) DNA based on the sequence found in a certain place (Figure 15.3). For example, the EcoRI enzyme cleaves DNA only if it recognises consecutive GAATTC nucleotide sequence. If this sequence is the place of a DNA polymorphism, for example a cytosine instead of thymine in a given position, then the DNA can not be cleaved by the EcoRI enzyme at that place. This will then give a different pattern when run on a gel electrophoresis.

Figure 15.3 DNA sequence polymorphisms in forensic genetics. A. Restriction fragment length polymorphism (RFLP) representation. B. Schematic visualisation of RFLP polymorphism by agarose gel electrophoresis. M – molecular weight marker, represents DNA fragments of known length, 1 – example of visualisation of a polymorphic site that does not undergo restriction enzyme cleavage, 2 – example of visualisation of a polymorphic site that undergoes restriction enzyme cleavage.

However, if we cut our entire DNA with such an enzyme, it would cut it in many places, which would greatly complicate the analysis of the results. Therefore, we have to define a section of DNA in which we know that the desired polymorphism occurs, and then analyse just that piece. We use the polymerase chain reaction (PCR) method to limit and multiply a certain defined section of DNA. This method was invented and described in 1983 by Kary Mullis and is explained in more detail in Chapter 4 - How do you work with DNA. Any cells can be used as a material for DNA isolation (e.g. lymphocytes, hair roots, sperm, skin cells...), and for profiling people, a swab of the buccal mucosa (inner side of the cheek) is most often used.

DNA profiling

DNA analysis for human profiling (also called DNA fingerprinting) was first published by Alec Jeffreys from the University of Leicester (Great Britain) in 1985. DNA fingerprinting played an important role in solving the criminal case of the murders of two young women in the English county of Leicestershire. During the investigation, Richard Buckland, who was only 17 years old at the time, was accused based on the matching of the blood group and the relatively rare property of excreting blood antigens into body fluids. Under the pressure of the investigation, he finally confessed to one of the crimes. After some time, however, the investigators learned about the possibility of DNA analysis through the research of Alec Jeffreys. After comparing the RFLP profile from the crime scene samples to that of Richard Buckland, it was found that they did not match. So, the police reopened the case and summoned approximately 5,000 men from the surrounding cities to take samples. Those whose samples matched the crime scene samples by blood type were subjected to RFLP analysis. However, to the great disappointment of the investigators, they did not find a match. After some time, a woman came to the police claiming that she had heard Colin Pitchfork, a baker at a local inn, persuade another man to go and hand over the sample to the police instead of him. Based on this testimony, the police arrested Colin Pitchfork, conducted a DNA analysis, and found that his RFLP profile matched the DNA from the crime scene samples. It was a great success because, for the first time in history, DNA analysis contributed to the clarification of such a serious crime and the conviction of the real murderer.

An even more accurate (and thus more informative) method of DNA analysis in the identification of people are DNA length polymorphisms (Figure 15.4). In STR (short tandem repeats) length polymorphisms, two individuals differ in the number of repeats of short DNA segments. The advantage of this approach is that STR polymorphisms are much more diverse than SNP polymorphisms, so they allow many different people to be distinguished from each other. Thus, there are two possible combinations from the RFLP analysis (either the DNA is cleaved or not), while with STR polymorphisms there are many more possible combinations, depending on the variability of the polymorphism in the population. For example, a certain individual may have five repetitions of one STR polymorphism on one chromosome and seven on the other, and another individual may have eight repetitions of the given polymorphism on one chromosome and four repetitions on the other chromosome (Figure 15.4). STR polymorphisms are usually analysed by amplifying a given section using PCR (similar to RFLP), but their detection is most often done by capillary electrophoresis. We also refer to this technique as fragmentation analysis and it is much more accurate when distinguishing DNA fragments with small differences in length.

Figure 15.4 DNA length polymorphisms. A. Representation of length polymorphism. B. Example of STR polymorphism possibilities of various individuals.

Currently, 13 (in some cases up to 20) different STR polymorphisms are investigated in standardised forensic laboratories for human identification, while the probability of a match between two unrelated people in such an analysis is up to 1:10¹², that is, close to zero. In 1998, the international CODIS database was established by the American FBI agency, in which the DNA profiles of all individuals investigated in any court proceedings are collected. The CODIS software is also used by a large number of countries across the world for their own law enforcement, but also to identify missing and unidentified individuals.

Historical DNA profiling

As already mentioned, in some cases nuclear DNA can be degraded and it is challenging to use it for polymorphism analysis. One way to solve this problem is to use mitochondrial DNA for analysis. Mitochondrial DNA analysis played an important role in identifying the remains of the Romanov dynasty. The Romanovs were a Russian imperial dynasty whose members, headed by Tsar Nicholas II. were murdered by the Bolsheviks in Yekaterinburg in 1918. Their bodies were thrown into a mass grave that was discovered in the late 1970s. However, the remains of two bodies, Tsarevich Alexei and one of his sisters, probably Anastasia, were not found among the remains. This created a space for speculators who claimed to be the so-called tsarinas. It wasn't until several decades later that another grave was discovered nearby, where additional skeletal remains were found that suggested they might be members of the family. Based on mitochondrial DNA analysis, it was confirmed that they are descendants of the Romanov dynasty. The DNA of Prince Philip, Duke of Edinburgh (husband of Queen Elizabeth II, British Royal family), who was a relative of the Romanov family on his mother's lineage, was used for the analysis (Figure 15.5).

Figure 15.5 Family tree of the Romanov dynasty. Square symbol – man, circle symbol – woman. The dark squares of the circle represent family members who are descendants of Princess Alice through the maternal lineage.

Polymorphisms have been described on the Y chromosome, the association of which has been linked with the geographical origin of a person. Based on the presence of a certain haplotype (grouping of polymorphism markers) on the Y chromosome, we can identify whether a given man is African or Central American, for example. In the investigation of a crime, when the DNA profile of some trace at the crime scene is not matched in the first phase with the DNA profiles in the database, any approximation of the possible appearance of the person is of great benefit to the investigation process. The trend today is to sequence the entire genome (the entire genetic information of an organism) using second-generation sequencing methods, so-called forensic genomics. However, by analysing several SNP polymorphisms, even without additional information, it is possible to estimate some phenotypic features of a person, with the most accurate results achieved when determining pigmentation - the colour of eyes, hair, and skin. In addition to DNA analysis, RNA analysis is also used in some cases. Although the cells of a given individual contain DNA with the same sequence, they differ in what RNA molecules are present and in differing amounts. Thus, we can use RNA analysis to identify the type of tissue and sometimes also to determine the time when the trace was left at the crime scene. The disadvantage of RNA molecules is that they are less stable than DNA molecules and degrade faster.

The DNA molecule undergoes several modifications, the most famous of which is methylation (adding a methyl group) to cytosine. Such modification on certain known areas of DNA can be related to age and lifestyle (see Chapter 7 - From Epigenetics to human diseases) and consequently advances in epigenetic analysis have suggested that DNA methylation markers provide more information to forensic analysis. The ability to gain detail on the age of the unknown sample and their lifestyle, such as smoking or drinking habits, can better guide police investigations when the suspect is not known.

Did you know that...

...it is now possible to distinguish identical twins using DNA analysis. Identical twins arise from a single fertilised egg and when the egg splits into two twin embryos, they acquire their own genetic mutations and epigenetic pattern. Such monozygotic twins have a near-identical genetic sequence but display variation in their epigenome. There have only been a few criminal cases worldwide that have involved monozygotic twins. In Boston in 2004, two women were abducted and assaulted. A suspect was identified based on the genetic profile from evidence gathered, but it matched the suspect, Dwayne McNair, as well as his identical twin brother. At the time, the techniques were not available to distinguish the two brothers and it was not until 2014 that DNA technology had progressed to the point at which it could be used to definitively rule out Dwayne McNair’s twin brother. In 2018 McNair was convicted and sentenced to 16 years in prison.

Want to read more?

Website: Forensic Science

DNA fingerprinting in forensics: past, present, future (2013) L. Roewer Investigative Genetics 4:22

Encoded evidence: DNA in forensic analysis (2004) M.A. Jobling & P. Gill Nature Reviews Genetics 5:739-751

Website: How forensic DNA evidence can lead to wrongful convictions

Website: How do DNA ancestry tests really work?

Chapter 16: Genetics in sport

Everyone who takes up a sport would like to win a competition at least once. To be the best in their discipline. How can they achieve this goal? It may be necessary to start with the correct choice of sports discipline, since it is possible to describe a naturally suitable body type for each discipline. Many athletes won at the Olympics and World Championships precisely because their physique was different from others, and this helped them to perform better in sports. Jamaican sprinters have long legs and narrow hips, which are an advantage when running and jumping. Marathon runners from Kenya and Ethiopia have very slim calves and ankles. Athletes who rotate in the air during performance, such as figure skaters and gymnasts, are shorter. In water polo and handball, longer forearms allow for a more effective swing when throwing. Longer forearms also allow kayakers and canoeists to perform powerful, repetitive strokes, while weightlifters need shorter forearms. A short torso and relatively long legs are important in basketball and volleyball, while a long torso and short legs ensure swimmers move faster in the water. The morphology and physiology of the body, which is largely genetically determined, is an important factor in choosing a sport.

Is it all about practice or does biology help?

Elite sports performance is the result of many hours of practice, and it is thought that we need to train 10,000 hours to be successful in sports. It also assumes that just that much time is needed for the natural differences between individuals to disappear and the difference in training to manifest itself. Constant repetition is sufficient to improve an individual's performance, but the question remains as to why some athletes achieve better results than others. So does the 10,000-hour rule apply equally to everyone? The answer may lie in the structure of our muscles and what affects their performance. Muscles are composed of slow type I muscle fibres and fast type II muscle fibres. Type I slow-twitch muscle fibres have a smaller diameter, contain many mitochondria, and are more resistant to fatigue. In these fibres, the aerobic (oxidative) energy system dominates. Fast-twitch (type II) muscle fibres contract about twice as fast as slow-twitch fibred during explosive movements, but they tire very quickly. In these muscle fibres, the anaerobic (glycolytic) energy system dominates. When exposed to strength training, the fast-twitch fibred grow more than slow-twitch ones, thus the more fast-twitch fibres a muscle contains, the greater its ability to grow. Most people have muscles that are composed of slightly more than half slow-twitch muscle fibres. However, for athletes, the ratio of these fibres is different and corresponds to the sports discipline they are engaged in. Sprinters need to quickly produce explosive power, so their calf muscles contain up to 75% fast-twitch muscle fibres. Athletes who run 800 m have 50% slow and 50% fast fibres in these muscles. Endurance runners have more slow-twitch fibres, which fatigue very slowly - in marathon runners they make up to 80% of the muscle fibres.

Muscle composition affects endurance, strength, and speed. Endurance ability means performing a certain physical activity with a given intensity for a longer period, without reducing performance. From the point of view of endurance abilities, the dominant energy system is the aerobic energy system. Aerobic endurance requires the ability of the cardiovascular system to deliver oxygen to the working muscles and the ability of the muscles to use this oxygen. The most common quantification of endurance is the maximum rate of oxygen uptake (VO₂ max), which represents the amount of oxygen that the lungs can extract from inhaled air and deliver, via the bloodstream, to the working muscles per unit of time. The higher the VO₂ max, the more oxygen that gets to the muscles. Aerobic capacity represents the total amount of energy released in an oxidative way, without disturbing the metabolic homeostasis associated with an increase in the level of lactate in the blood.

Muscle strength is the interaction between the force and speed of muscle contraction. The amount of force developed depends on the structure of the muscle (type, proportion, and diameter of muscle fibres), and the number of contracting muscle fibres and is also influenced by other factors, such as energy status, integrity of muscles, and their innervation. Muscular strength is crucial in athletics such as sprinting, jumping, and weightlifting. But do athletes acquire their unique combination of muscle fibres through training, or is it genetically determined? Much evidence suggests the latter possibility. No training study has shown that it is possible to change slow-twitch fibers into fast-twitch fibers. Aerobic training can thus make fast fibres more persistent and strength training slow fibres stronger, but they never completely "switch".

Trainability, or an athlete's response to exercise training, also depends on genetic factors. Already in the 1970s, scientists were dealing with the influence of genes on sports performance, with the first studies mainly focused on identical twins who lived separately for a long time in different environments. This made it possible to investigate the influence of heredity and distinguish external environmental factors. In 1998, the British doctor Hugh Montgomery began to deal with sports predispositions at the genetic level. He mainly focused on gene polymorphism mutations that could affect sports performance. We define DNA polymorphisms as the existence of DNA sequence variants that are not associated with any observable phenotypic change and can be found anywhere in the human genome. Thus, polymorphism means two alternative forms of a chromosomal region (allele) that differ either in the nucleotide sequence or in the number of repeats of a short DNA sequence (see Chapter 15 - DNA as evidence).

Single nucleotide polymorphisms (SNP) correspond to a difference in one nucleotide pair. From the point of view of the position in a certain part of the genetic information, we can divide SNPs into coding and non-coding. Coding point mutations are found in the coding regions of genes. At the same time, they can be synonymous (silent), which does not change the amino acid, or non-synonymous, which changes the codon, which changes the amino acid in the protein or shortens the protein. Non-coding point mutations are found in non-coding regions of genes or outside genes (you can read more about mutations in Chapter 5 - Mutations: how they arise and what to do with them). Since these regions contain various regulatory elements necessary for the correct transcription and translation, they can affect the function of the gene.

An insertion-deletion polymorphism (indel) is a type of genetic variation in which a specific nucleotide sequence is present (insertion) or absent (deletion). If they are not a multiple of three nucleotides, they lead to frameshift mutations in the coding region of the gene, resulting in the formation of a completely different set of amino acids, or the occurrence of a premature stop codon. These mutations can fundamentally affect the structure and function of the given protein. The indel variants, which are multiples of three, do not affect the other amino acids, but the resulting protein sequence is altered, with either amino acid changes occurring or some missing.

Genetic factors can affect some components of athletic performance, such as strength, endurance, flexibility, or neuromuscular coordination. Indeed, there are specific areas of DNA that can differ between individuals, and it is through these polymorphisms that it is possible to explain why some individuals have a different reaction to sports training than others. In certain sports, the presence of specific polymorphisms can also contribute to a higher level of performance. Currently, more than 200 candidate sports genes are known, of which more than 20 are directly related to the elite level of the athlete. These genes affect physiological factors such as blood circulation, blood pressure control, lung and heart capacity, muscle fibre composition and hypertrophy, muscle metabolism, mitochondrial protein synthesis, or adaptation to training load. The most studied genes include ACE, ACTN3, and PPARA.

The ACE gene polymorphism was described in 1998 as the first-ever genetic marker of sports predisposition. This gene encodes angiotensin-converting enzyme (ACE), which is part of the renin-angiotensin system (RAS). The RAS is a hormonal system that regulates blood pressure, fluid and salt homeostasis in the human body. The ACE enzyme changes angiotensin I to angiotensin II, which increases blood pressure due to its vasoconstrictive effect. The ACE gene is located on chromosome 17 and the polymorphism of this gene consists in the insertion (allele I) or deletion (allele D) of a 287 bp DNA segment in the non-coding part of the gene. Thus, there are three genotypes in the population: II, ID, and DD. The presence of allele I leads to a lower level of the ACE enzyme in serum and tissue, which is associated with a higher proportion of type I slow-twitch muscle fibres, higher efficiency of aerobic performance, better resistance to fatigue, higher oxygenation of peripheral fibres during activity and a more pronounced aerobic response to training. This allele represents an advantage for endurance athletes. Among the given disciplines are endurance running, triathlon, cross-country skiing, swimming over medium (200-400 m) and long distances (over 400 m), and mountain climbing. The D allele, on the other hand, is responsible for a higher level of the serum enzyme and is associated with speed and strength performance. The DD genotype is associated with a higher percentage of type II fast-twitch muscle fibres, which are essential for maximal strength output over a short period. A high representation of the D allele was recorded in athletes of speed-power disciplines with a predominance of anaerobic performance. The given disciplines include sprinting, long and high jump, weightlifting, swimming, and short distance running (up to 200 m).

The structural protein α-actinin 3 (ACTN3) functions as part of the Z-line of muscle cells and has an important role in the anchoring of actin filaments (Figure 16.1). It is encoded by the ACTN3 gene, which is located on chromosome 11 in the human genome. The polymorphism of this gene consists of the substitution of cytosine for thymine at position 577, which leads to the exchange of the codon for the amino acid arginine (R) with a stop codon (X). In this case, the R allele represents the normal functional version of the gene, while the X allele contains the sequence change that prevents the production of functional α-actinin 3. A quarter of people of East Asian descent have the XX genotype, compared to only 18% in the European population and only 1% in the African population.

Figure 16.1 Structure of skeletal muscle. A muscle is composed of muscle fibers. A myofibril is a protein complex that represents the functional unit of a muscle fibre.

In sports genetics, the ACTN3 gene is also known as the “speed gene” since speed-power athletes need fast type II muscle fibres rich in glycolytic enzymes for their performance. Several studies have confirmed that elite sprinters had a high frequency of the RR genotype and an absence of the XX genotype. Allele R thus represents an advantage for speed-power sports such as weightlifting, sprints, and short-distance swimming. Loss of α-actinin 3 (genotype XX) leads to a decrease in the amount of muscle mass, muscle strength, and diameter of fast muscle fibres and increases the ratio of slow muscle fibres associated with endurance.

PPARα (peroxisome proliferator-activated receptor alpha) is a transcription factor that regulates lipid, glucose, and energy balance in the cell. It is encoded by the PPARα gene located on chromosome 22. In tissues involved in the use of fatty acids, such as the liver, skeletal muscles, and heart, the production of PPARα is increased, and the level of this receptor is also higher in slow muscle fibres. As endurance training stresses fatty acid metabolism, PPARα is an important factor in the adaptive response to training load. In this case, the SNP polymorphism replaces guanine with cytosine in the non-coding region of the gene. A high frequency of the C allele has been observed mainly in strength-oriented athletes, while both the G allele and the GG genotype represent an advantage for endurance athletes. In addition, the analysis of muscle fibre composition revealed a higher proportion of slow muscle fibers in the GG genotype compared to the CC genotype. The G allele is therefore considered an advantage in endurance performance.

Resistance to injury and the ability to recover from injury is another critical factor for optimal sports performance, as some injuries can lead to recurring problems. Collagens are the basic structural component of connective and supporting tissues, making up to 35% of all proteins in the body. They occur in connective tissue such as ligaments and tendons, but also in skin, bones, cartilage, or blood vessels. The task of collagens is to ensure the strength of the traction tissue, the elasticity of the muscles, the transfer of muscle forces to the bones, and much more. Currently, 28 different types of collagens have been described, which differ in shape and function, although about 90% of collagens belong to types I, II, and III. Polymorphisms of genes that code for collagen chains are currently being investigated concerning the risk of injury in sports. An example of a polymorphism that can affect resistance to injury is the COL1A1 gene polymorphism.

Type I collagen is found in connective tissues such as cartilage, ligaments, and tendons. It is composed of two α1 polypeptide chains encoded by the COL1A1 gene and one α2 polypeptide chain encoded by the COL2A1 gene (Figure 16.2). This gene is located on chromosome 17, has a length of 17,554 bp, and several different polymorphisms have been identified within the gene, which are related to the development of human diseases. For example, the disease osteogenesis imperfecta, known as brittle bones disease, is caused by a mutation in the genes responsible to produce type I collagen (COL1A1 and COL2A1). The mutation leads to a total change in the structure and quantity of collagen.

Figure 16.2 Structure of collagen. Type I collagen is a heterotrimer composed of two α1 chains and one α2 chain, which are folded together into a right-handed helix.

Osteoporosis is an age-related disease characterised by low bone mineral density, which leads to increased fragility and a high risk of fractures. Currently, the association between the development of osteoporosis and an SNP polymorphism located at position 1245 is being studied. In this polymorphism, a substitution of guanine for thymine occurs, with the presence of the T allele representing a higher risk of both osteoporotic and non-osteoporotic fractures and reduced bone mineral density. The TT genotype is associated with a higher likelihood of progressive degeneration of intervertebral discs and an increased likelihood of anterior cruciate ligament (ACL) tears in skiers. In other studies, the TT genotype was associated with a reduced incidence of ACL rupture, Achilles tendon rupture, and shoulder dislocation. It is known that tendon and ligament injuries that are related to sports performance are accompanied by various risk factors that are different for each sport. Since the individual studies were focused on different sports disciplines, the mechanism of injury was different, which could lead to conflicting observations. However, larger studies have shown that this polymorphism may be associated with the risk of ACL injury and the TT genotype may have a protective role.

Therefore, if someone wants to become an elite athlete, it is an advantage to know their sporting genetic predispositions. Accordingly, a person can more easily decide whether to choose speed-power sports or endurance disciplines, whether they will be the best in individual sports, or whether they have a better chance of success in collective disciplines. However, genes are only a small part of success. Without daily training, no one becomes a winner.

[Or1]In online text this has different font type or size

Did you know that...

...a mutation in the ACTN3 gene appeared more than 50,000 years ago when modern humans migrated from Africa to the colder climate of central and northern Europe. Indeed, natural selection favoured the XX genotype of this gene in a colder environment. People lacking α-actinin 3 were found to maintain body temperature better during immersion in cold water due to a change in skeletal muscle thermogenesis. Better body resistance to cold in individuals with the XX genotype was not accompanied by increased energy expenditure, but by an energetically efficient increase in the tone of type I slow-twitch muscle fibres that generate heat.

Want to read more?

Genetics and sports performance: the present and future in the identification of talent for sports based on DNA testing (2022) D. Varillas-Delgado et al. European Journal of AppliedNPhysiology 122:1811-1830

Genetic influence on athletic performance (2014) L.M. Guth & S.M. Roth Current Opinion in Pediatrics 25(6):653-658

Website (The Physiological Society) Sports genetics: Physical performance potential is dependent on genome

Chapter 17: Genetically modified organisms

Genetically modified organisms (GMOs) are organisms whose DNA has been intentionally altered by humans in a way that is not possible through nature (usually by means of genetic engineering). GMOs find applications mainly as part of modern biotechnologies and in scientific research.

Ancient biotechnology

During the development of human society, the creation of various types of technology played a fundamental role. These could initially be primitive (e.g., simple hunting tools), but were gradually improved and became irreplaceable in most key activities, including food production and maintenance of safe environment. In the period of the Neolithic revolution, the so-called biotechnologies – activities of engineering, using living organisms to create different types of products – emerged. The oldest biotechnologies included the domestication and cultivation of agriculturally important plants (around 10,000 BC in the so-called Fertile Crescent) and later, similarly significant domestication of animals (around 8,000 BC). These basic biotechnologies have enabled humans to purposefully use living organisms to obtain food. In the next period, the process of fermentation was discovered, which made possible the production of fermented drinks (beer, wine), but also to bake bread (in approximately 7,000 BC in China). Eventually, newer technologies were developed, such as breeding of economically important crops and animals (5,000 BC) or the use of natural antibiotics (500 BC in China) and insecticides (100 BC in China).

Modern technologies in agriculture, industry, and medicine

As the technological progress of human society accelerated over time, the original biotechnologies were improved or replaced by their more accessible and cheaper variants. Mechanisation and automation became more intensive in agriculture (the wheelbarrow was invented in China around the year 230) and the medical industry also developed rapidly. Especially, the advances in medicine were closely linked to biology and biotechnologies. Breakthrough discoveries in this field include the establishment of vaccines and vaccination (in 1798, Edward Jenner was the first to use the smallpox vaccine) along with the later discovery of antibiotics (in 1928, Alexander Fleming discovered penicillin). As the mankind constantly expanded and the demands for the quantity and quality of food, medicine and other commodities continued to rise, more effective biotechnologies were developed, making use of crucial discoveries from the field of genetics. In the second half of the 20^th century it was definitively confirmed that DNA is the carrier of genetic information, its function and the mechanism by which it is realised were described, the genetic code was deciphered (see chapter 3, meet DNA), and in the 1970s the techniques of recombinant DNA and genetic engineering emerged (Chapter 10). Knowledge and scientific progress in this area, combined with the improvement of modern technologies in all branches of human activity subsequently led to the creation of new forms of biotechnologies. One of the most significant, and at the same time the most controversial, of them is Genetically Modified Organisms (GMOs).

Targeted change of the organism's genetic information

GMOs are organisms whose DNA has been modified in a way that is not possible in nature, and genetic engineering methods are usually used for this purpose. In addition to these methods, however, humanity has had another very effective approach to DNA modification of various types of organisms – breeding. By breeding we mean the intentional selection of individuals (mostly plants or animals, in specific cases also microorganisms) with similar characteristics (e.g., the size of plant fruits or the amount of muscle processed into meat in cattle), which can be crossed with each other. From the offspring, individuals can be selected which have the most suitable characteristics, and the process repeated many times. It is obvious that in this way people purposefully (because they carry out selection) change the genetic information (most of the characteristics of each organism are determined by genes) of the given organism even without laboratory equipment. Using this approach, it was possible to breed different races of dogs (the ancestor of all dogs was similar to a wild wolf), cattle or other animals (Figure 17.1).

Figure 17.1 The dog breeding process. The arrows indicate the differently coloured offspring of the selected parent, with the darkest individuals selected for the next crossing (indicated by an orange line). After several generations, a dog with black spots has been bred.

If we look at the well-known Belgian blue cattle (Fgure 17.2) with today's technical knowledge, we will find that the enormous amount of muscle produced by these animals is due to a mutation in gene encoding the myostatin protein.This protein inhibits the building of muscle and ensures that the body's muscular system is appropriate for its overall constitution. In the case of Belgian blue cattle, this gene is mutated so that myostatin is not functional and consequently the cattle produce an extreme amount of muscle. When the breeding process of this type of cattle began, the mutation appeared randomly, but thanks to its physiological consequences, the individuals with the mutation were selected and further bred. After some time, the originally wild species has become, at least at a first glance, a completely different organism. Obviously, its special properties are caused by changes in genetic information, even though the breeders probably did not understand that at the beginning. Many other organisms have gone through a similar process in the past, including corn – a typical example of a plant species bred over thousands of years (breeding began around 5000 BC). The original ancestor of corn – teosinte – is a plant with a similar fruit structure, but significant differences in the number of seeds, their shape and size. All these characteristics (determined by genetic information) have been gradually modified by humans to create the corn we know today.

Figure 17.2 Belgian Blue Cow. The cow is a breed that is used to produce lots of beef for food.

Therefore, it is clear that humans have influenced the genetic makeup of economically important domesticated plants and animals since the distant past. But why don't we consider these organisms GMOs? The answer is hidden in the second part of the definition, which says that an organism is considered a GMO only if the changes in its genetic information could not have occurred naturally. In the case of breeding, of course the changes did not occur in a natural way since the crosses were carried out by breeders according to their breeding intention. On the other hand, we must ask ourselves the question: "Could the two organisms that we selected for breeding, at least theoretically, meet freely in nature and have the offspring that we obtained?" To this question, in the case of bred organisms, we must answer: "Yes, it is theoretically possible, even though extremely unlikely.” As a result, we do not consider bred species of plants and animals to be GMOs, despite the fact that their genetic information has been altered by humans.

The development of GMOs

If we take all organisms whose genetic information has been changed by human activity and among them select only those, whose changes could not even theoretically occur naturally, we will formally speak of GMOs – organisms created by methods of recombinant DNA technology and genetic engineering. This approach, compared to the hard-to-predict and slow process of breeding, allows us to make a precisely planned intervention in the genetic material of the organism, with a relatively easy-to-predict result. In reality, the creation of GMO starts with the isolation of DNA from one organism and identification of a gene that is useful to us (economically or scientifically). Afterwards, we multiply this gene, separate it from the rest of the DNA and connect it with the carrier DNA (e.g., part of a virus or plasmid), which will facilitate its incorporation into the genetic material of another organism. In the laboratory, it is possible to insert such recombinant DNA (it is a combination of the carrier DNA and the gene that we want to transfer) into the cell of the host organism, where it merges with the original genome (Figure 17.3).

Figure 17.3 Modification of genetic information by genetic engineering. The traditional breeding process (left) is where the most optimal characteristics are chosen after each round of crossing/breeding. This in in comparison to the genetic engineering mechanism (right) where the DNA from a different organism, a bacteria, is inserted into the apple.

In 1973, the first GMO bacterium was created, which included a gene for resistance to the antibiotic kanamycin (originating from another type of bacterium). In the following years, it was possible to modify the DNA of a mouse in a similar way (1974) or to insert an antibiotic resistance gene into the genome of a tobacco plant (1983). As soon as it became clear that changing the genetic information of various organisms is possible using the same basic set of tools, biotechnologists began to come up with ideas for modifying individual economically important organisms. What was the purpose of these modifications? The same thing that was central to every new biotechnology - to get food, medicine, and other commodities in larger quantities, faster and easier than ever before.

Commercially available GMOs

As soon as it was possible to purposefully modify the DNA of economically important organisms, a whole branch of modern biotechnology emerged, which tried to adjust these organisms and their use mainly (but not only) for the food and pharmaceutical industry. The very first GMO commercially available in the form of food was Flavr Savr tomato (launched in 1994), which included a gene that slowed its rotting. The product of this gene prevented the natural formation of the enzyme polygalacturonase, which degrades the cell walls on the surface of the tomato, reducing the firmness of the fruit and enabling a faster onset of rotting. For this reason, tomatoes might begin to rot even before they reach the store shelves. Growers often solve this by picking unripe fruits and later treating them with ethylene, which signals the cells to begin ripening. When using this trick, however, they do not have time to create all the necessary substances in the fruits and thus might end up "tasteless". Flavr Savr tomatoes did not suffer from this, because even though they were picked after they were already ripe, the fruits were still fresh for sale or production. Like all GMOs, these tomatoes had to go through a strict inspection by the Food and Drug Administration (USA) before being put on the American market, which excluded them from posing a risk to consumers or the environment. The first GMO animal used for human consumption was the AquAdvantage salmon (introduced in 2015), which is genetically modified in such a way that a gene regulating growth hormone activity allows this salmon to feed and grow not only in the spring and summer (like regular salmon), but through the entire year. Thanks to this modification, the salmon grows to much larger sizes and generates larger amount of meat.

GMO plants of the first and second generation

Even though many different types of GMO food have been created since the 1990s, GMO variants of agriculturally important crops – rice, corn, soy, and others – still have the greatest impact on public life worldwide. The original strategy for modifying these crops was to insert genes into them that would allow these plants to resist herbicides that kill most other plants. Afterwards, it was possible to intensively spray the fields with herbicides and get rid of all the weeds, while the cultivated plants survived without damage. This procedure created the entire first generation of GMO crops, most of which are resistant to the herbicide Roundup (commonly called "Roundup Ready"). The list of Roundup Ready plants today includes soybeans, corn, beets, wheat, and others. This type of modification allows growers to obtain a larger amount of harvest on the same fields, thus bringing them the main advantage. Compared to the first generation of GMO crops, the second-generation GMO plants were designed so that their modification brings benefits to consumers. A typical example of such plant is the so-called golden rice, a genetically modified rice with three introduced genes allowing the synthesis of β-carotene in the seeds. β-carotene, a precursor of vitamin A, is an antioxidant and is important for the proper development of vision in childhood. Since in some of the Asian (e.g., India, Vietnam, Laos) and African countries, vitamin A is poorly represented in the available food, many (especially poor) people suffer from its deficiency. As a result, in 2005, up to 190 million children were likely affected by vision disorders or, in extreme cases, blindness. Golden rice was subsequently planted in these countries to help solve the problem. Other similar projects included the preparation of tomatoes producing resveratrol and other plants with various antioxidants overproduced in their fruits.

GMO producers of pharmaceutical substances

In addition to GMOs, which we can use directly in the form of food, there are many genetically modified organisms producing substances needed in medicine, agriculture, or industry. In this case, the genetically modified organisms themselves do not encounter the consumer, but the substances produced by these organisms are important. These are often microorganisms (usually Escherichia coli bacteria) cultivated in large fermenters and producing antibiotics, human hormones, vitamins, and other important substances. This way we can obtain large quantities of humulin – human insulin used in the treatment of diabetes, erythropoietin – a hormone supporting blood formation used in the treatment of anaemia and blood cell differentiation disorders, or somatotropin – a human growth hormone used in the treatment of growth and development disorders. In case of infectious diseases, practically all types of antibiotics (e.g., penicillin, streptomycin, tetracycline) are synthesised using GMO producers, and various branches of industry (especially the food industry) use substances produced by GMOs to create the necessary materials and products (e.g., recombinant chymosin is used for treatment of milk during cheese production).

GMOs in science and research

Besides the employment of GMOs in biotechnology, genetic modification of various types of organisms is a common part of scientific work. If a scientist is interested in the function of an unknown gene, one possibility is to remove this gene from the organism's genome (using molecular biology methods) and then observe the properties of this organism. If any of the observed characteristics changes, it is possible that the studied gene is involved in the development of this characteristic (if the colouration of the organism was to change, it is likely that the gene of interest is responsible for the standard colouration, etc.). If the scientist has some basic information about the studied gene, they can also use more complicated forms of genetic modification. For example, if the gene encodes a protein whose localisation inside the cell is unknown, this gene can be combined with a gene encoding a green fluorescent protein (originally isolated from the sea jellyfish Aequorea victoria). As a result, the protein product of the studied gene will be labelled with a fluorescent label that can be visualizsed using a fluorescent microscope. Using this approach, it is possible to monitor the movement and location of various proteins in a wide variety of cells (Figure 17.4).

Figure 17.4 Proteins labelled with green fluorescent protein. The image on the left shows a protein located freely in the cytoplasm (almost the entire cell is green), the image on the right shows a protein located in the cytoplasmic membrane (the cell is bordered by a fluorescent green line). Both pictures were kindly provided by A. Cillingova.

When investigating the properties of a certain protein, another option is to introduce mutations designed by scientists into the gene that encodes this protein. If the activity, localisation, or function of the resulting protein changes, we can conclude that the mutation has affected one of its important parts (not all parts of a protein are equally important, so some mutations have practically no consequences, while others can lead to a complete loss of function, described in detail in chapter 5 – mutations how they arise and what to do with them). In some cases, the genetic modification of a certain organism is not even used as a goal of scientific research, but rather as a tool for examining the genes of different organisms. Thus, scientists can introduce certain genes from one species into another and observe a change in its properties. Depending on how the properties change, it is possible to recognise some of the functions of the studied gene and its role in the organism in which it is naturally found. Due to these types of observations, scientists can understand the functions of individual genes and their mutual relationships.

Risks and misinformation

It is obvious that GMO technology comes with several risks and potential dangers. In particular, the planting of GMO plants of the first generation led to increased use of various types of toxic sprays in the past (mostly Roundup), which got into the soil and damaged the surrounding ecosystem. It is also possible that the introduction of some GMOs into the wild could compromise natural biodiversity, as these organisms could interbreed freely with wild individuals of the same species, and the offspring of these organisms could displace wild species from their natural environment. Another risk is that if one organism produces proteins that are typical of a completely different type of organism, it could cause unexpected allergic reactions during consumption (if used as food). However, excessive concerns about allergic reactions are generally misplaced for a simple reason – all GMO foods are very strictly tested by agencies responsible for food safety at the state level, as well as at the level of the European Union (in case of most European states). Theoretically, it is also possible that the use of various pest resistance genes might lead to the creation of conditions in which we would select (since only those would survive) extremely resistant types of pathogens. This concern exists, but experimental data do not indicate such a situation anywhere in the world. It is therefore reasonable to use GMO technologies with caution, while continuously controlling the state of biodiversity, pathogens, and other important factors in the environment. Unfortunately, in addition to these (to various degrees) real risks, GMO technology is met with a lot of misinformation, hoaxes and conspiracy theories suggesting that genetically modified organisms can modify human DNA (which they cannot), are less healthy as a food, or contain fewer beneficial substances than their unmodified variants (which does not make sense, since they are designed to produce extra substances in addition to everything that the standard organism normally produces), or they cause diseases (which they do not). It is therefore important for each of us to draw information about GMOs (and other topics as well) from reliable sources and avoid being misled by inaccurate or distorted pieces of information (to find out how to deal with conspiracies and hoaxes in genetics, read chapter 18 - The most common hoaxes in genetics).

Did you know that...

...the methods of genetic engineering have already been used to edit human genes? Despite the loud criticism from the majority of professional public, in 2018, in his laboratory, the Chinese doctor He Jiankui and his team genetically modified two human embryos. After these embryos were implanted, twins Lulu and Nana were born – the first people with a gene modified by genetic engineering techniques. Specifically, it was a modification of the CCR5 gene, which encodes a receptor involved in HIV infection. The modification should ensure increased resistance of the modified cells to infection, making it easier for the twins to live in a part of the world where HIV is a threat. However, almost the entire scientific community, ethical commissions of scientific institutes, and the lay public objected to this procedure. The reason for that is simple: no matter how good the original intention may have been, such an intervention in the human genome can have unpredictable consequences for the girls. This is because we still do not perfectly know all the processes in which the CCR5 receptor is involved, nor can we be completely sure whether some unexpected event did not take place in the editing process that could cause Lulu and Nana health problems in the future. The case of these two sisters is a reminder to all of us that the methods and technologies of genetic engineering must be handled with utmost caution and in accordance with international ethical standards. In conclusion, we will only add that at the time of writing this text, both girls are healthy and happy.

Want to read more?

Genetically modified organisms for the environment: Stories of success and failure and what we have learned from them, Cases, I., Lorenzo, V., International Microbiology (2015).

Chapter 18: The Most Common Hoaxes in Genetics - Myths & Facts

Nowadays there is a lot of information available on the internet, and for the non-expert it is often difficult to find out which of them are true. Hoaxes or “fake news” are deceptive news items that appear to be true at first glance. There are several reasons why people tend to believe hoaxes. One is the nature of human thinking, which is subject to what is known as confirmation bias (the tendency to interpret and prioritise information that confirms our pre-existing beliefs) and conjunction fallacy (when two things happen at the same time, we consider it implausible that it could be a coincidence). Another reason why people believe hoaxes, is the lack of critical thinking and the inability to distinguish between correlation and causation (Figure 18.1). While correlation expresses that two phenomena have a certain relationship and happen at the same time, causality expresses that there is a causal relationship between these phenomena, that is, one phenomenon is the cause of the other. For example, if a person is clapping and it begins to snow at the very same time, these phenomena are correlated but not causally related. Similarly, the administration of an experimental drug to a patient and their recovery suggests a correlation between these phenomena. There could also be a causal relationship, i.e., that the administration of the potential drug caused the patient to be cured, but this needs to be proven by further investigation. However, the patient could also have been cured without the administration of the drug. It must be added that the causality of approved drugs has been confirmed by many years of research and numerous laboratory tests. In the rest of this chapter we deal with interesting and current hoaxes related to genetics.

Figure 18.1 Correlation and causality: the relationship between ice cream sales and shark attacks over the course of a year. The graph shows a correlation between the amount of ice cream sold and the number of shark attacks on humans. However, there is no causality between these phenomena, i.e., one phenomenon is not the cause of the other. Rather, both phenomena are causally related to the temperature of the environment in each month.

Hoax #1: Long-term smoking is beneficial to health

The positive effects of smoking on human health were promoted in the 1930s and 1940s, when physicians were in advertisements that recommended certain brands of cigarettes would be less irritating to the throat. However, physicians were often influenced by tobacco companies, which sent them many cigarettes in return for these recommendations. In advertising, similar claims were made by doctors until the 1960s, even though doctors themselves had already confirmed the negative effects of long-term smoking on human health. Today we know that long-term smoking can trigger the development of various cancers (e.g. lung cancer), heart disease, infertility and weakening of the immune system. In the United States of America, as many as one in five deaths is due to smoking. Based on this information, we can say that long-term smoking is not beneficial to health in any way.

Hoax #2: Man evolved from the ape

The origin of this hoax dates back to 1965 when F. C. Howell used the picture "The Road to Homo Sapiens" in the book "The Early Man" (Figure 18.2). The picture showed different species of primates in a row, starting with a chimpanzee-like ape, suggesting that humans evolved from apes. However, the image was used in the book to refute the claim that humans evolved from the present-day ape species. However, this image spread without further context and led people to believe that man was an evolutionary descendant of apes. The truth, however, is that humans and today's chimpanzees share a common ancestor. About 5.4 million years ago, a branch of the evolutionary tree split into one that led to chimpanzees and another that led to humans. It is also true that humans did not evolve from species such as Homo habilis or Homo erectus, but these species and Homo sapiens share a common ancestor.

Figure 18.2 The Road to Homo Sapiens. This is now an iconic image showing the evolutionary route to man from our hypothetical fossil ancestors in a lineage. The original picture was drawn by Rudolph Zallinger in the book "The Early Man". However, this popoular version was designed by José-Manuel Benitos and modified by M. Garde.

Hoax #3: Within 200 years, all "blondes" will disappear

The first reports that people with blond hair were gradually disappearing appeared as early as the 19^th century. However, this news did not spread intensively until the 1960s, when several magazines and newspapers reported that blond people would disappear from various parts of the world within 50 to 140 years. At the beginning of the 21^st century, this hoax became very popular when an article appeared on the BBC News website in 2002 that blond people would disappear completely within 200 years. A similar report was published four years later in the Sunday Times, also citing a study by the World Health Organization (WHO). However, a member of the WHO, Rebecca Harding, flatly denied that the organisation had ever conducted such a study. After some time, it turned out that the mis-information about the extinction of blond people in 200 years first appeared in the popular German magazine Allegra, which quoted a WHO anthropologist. But the organisation, in turn, stated that such a person had never worked for the WHO, and might never even existed. People with blond hair are more likely to be born and live in Nordic areas, with the highest frequency in Europe being in Finland, Sweden, Norway and Iceland. This fact could also contribute to the spread of this hoax, as the last blond person is said to be born in Finland in the year 2202.

A person's hair colour is determined by many genes. Among the best studied genes is MC1R (melanocortin 1 receptor), the product of which is located on the cytoplasmic membrane of melanocytes, which produce the pigment melanin. When an individual carries at least one dominant allele of the MC1R gene, melanocytes produce the pigment eumelanin, which is responsible for the formation of dark, i.e., black or brown, hair. However, if an individual carries only recessive alleles of this gene, pheomelanin is formed instead of eumelanin, which is responsible for the formation of red hair. People with blond hair, like redheads, have both recessive alleles of the MC1R gene, but their melanocytes produce almost no eumelanin or pheomelanin, due in part to the influence of other genes. For blonds to disappear completely the recessive alleles of the MC1R as well as other genes would have to disappear from the population. However, this is not possible within 200 years. Furthermore, as blond people are not disadvantaged in any way and produce offspring, it is highly unlikely that they will disappear completely from the population. Even if there were no blond people in one part of the population, recessive alleles for this hair colour would be present in heterozygotes of dark-haired people, so that blond people would again be born in approximately one quarter of cases in the offspring of two such heterozygotes (Figure 18.3).

Figure 18.3 Schematic representation of a cross between two parents (heterozygotes) for dark hair. When crossing two heterozygotes for gene A (Aa x Aa), e.g., the MC1R gene for hair colour, one quarter of the offspring will be recessive homozygotes. This cross explains how the offspring of two dark-haired people (Aa) can also produce blond children (aa).

In order for the recessive allele of the MC1R gene to disappear completely, it would have to carry a significant disadvantage. This could happen in the case of this allele if the climatic conditions on Earth were to deteriorate to the point where it was a permanent tropical summer. Blond people often have fair skin, which is not as well adapted to heat and intense sunlight as the skin of dark-haired people. But even in this case, blond people would probably be able to adapt to the worsened conditions (they would seek shade, use sunscreen) and could therefore continue to reproduce, allowing the recessive allele to spread further in the population (including through heterozygotes as already mentioned). In summary, it is very unlikely that blonde people will completely disappear in the near future.

Hoax #4: Vaccines cause autism

Probably the best known vaccine myth is the claim that vaccines cause autism. This stems primarily from an article by Wakefield and his team published in the prestigious medical journal The Lancet in 1998. Children with autism are characterised by repetitive behaviour, sensitivity to sound, and an inability to interpret emotions. These children also seek solitude, avoid eye contact, and do not respond when their name is called. The main cause of this disease are genetic factors (gene variants or mutations), but environmental factors (e.g., advanced parental age, epigenetic changes, pollution) are also thought to have an influence. However, Wakefield and his team believed that the MMR (a triple vaccine against three viral diseases Measles, Mumps and Rubella) vaccine could also cause autism and claimed to have demonstrated a causal link between the measles vaccine, which is part of the MMR vaccine, and autism in eight children. In addition, they presented the mechanism by which this vaccine causes autism, where they claimed that the measles virus from the live vaccine, when it enters the body, causes inflammation of the gut. Damaged intestinal walls then release harmful proteins into the blood, which then enter the brain where they cause changes that lead to autism. This whole process sounds more credible precisely because it was published in a prestigious scientific journal. The problem, however, is that this is the only study that supposedly "proves" the link between vaccines and the development of autism. On the contrary, since the publication of this paper, dozens of independent studies have been conducted on hundreds of thousands of children that refute any negative effect of vaccines in relation to the development of autism. Eventually, even the study by Wakefield and his team was retracted, i.e., labelled as untrue.

Vaccination opponents still believe that the MMR vaccine causes autism because it is given to children at a similar age when the first symptoms of autism appear. However, this assumption is flawed and only suggests that these phenomena are correlated, not causally related. Moreover, there are dozens of studies that refute that vaccines cause autism. Consequently, all the above facts confirm that vaccines do not cause autism.

Hoax #5: Vitamin C is a panacea against cancer

The positive effects of vitamin C in the treatment of cancer were first observed by Linus Pauling and Ewan Cameron. They published two studies in 1976 and 1978 claiming that giving high doses of vitamin C to patients in the terminal stages of cancer had a beneficial effect on their survival. However, these studies were criticised because no control subjects were included in the experiments. A few years later, two more studies were published that refuted Pauling and Cameron's findings, claiming that such high doses of vitamin C in tablet form would be absorbed by the intestine and only low doses of this vitamin would reach the blood and tissues. However, in 2008, a study was published in which authors observed a reduction in the weight of mouse tumours (brain, pancreatic and ovarian) after injecting high doses of vitamin C directly into these tumours. The study authors even suggested a mechanism for how this reduction occurred. They believed that vitamin C interacts with the unique chemistry of tumour cells, resulting in increased production of hydrogen peroxide. This is toxic to the tumour cells and subsequently the tumour itself is eliminated. This mechanism was confirmed with a second experiment by injecting catalase, an enzyme that breaks down hydrogen peroxide into water and oxygen, into the tumours in addition to vitamin C. In this case, they did not see any reduction in the weight of the tumours, suggesting that the production of hydrogen peroxide is critical to the elimination of the tumours.

So, the question arises, how is it possible that injecting high doses of vitamin C has not yet been used to treat human tumours? The answer is simple: there is no evidence that vitamin C is also effective in reducing the weight of human tumours. To date, neither the effects of high doses of vitamin C on patients' tumours nor the long-term side effects of this potential treatment have been studied. Furthermore, it is not known whether vitamin C can interact with other substances used in cancer treatment.

Based on these facts, it can be argued that vitamin C cannot yet be considered a cure for cancer. Moreover, there are up to 200 different types of cancer, and it is highly unlikely that vitamin C could be a panacea for all these diseases in the future.

Did you know that...

...fake news is not new? In the sixth century AD, Procopius of Caesarea (~500-554 AD) was the principal historian of Byzantium (now Istanbul). After the death of Emperor Justinian, Procopius used fake news to discredit the old Emperor. As he was dead, there could be no questioning or investigation. In these times, as there was no means to validate and verify the authenticity of the information, any challenge to authority was classed as treason! In the 20^th century there was an increase in the presence of misinformation in newspapers but with the rise of the internet and then social media, the level of fake news has multiplied! A study by the Central Statistics Office estimates that 62% of all information on the internet is unreliable, with around 20% of people believing in the fake news.

Want to read more?

Pennycook and Rand (2021) The Psychology of Fake News. Trends in Cognitive Sciences 25(5):388-402

Chapter 19: Genetics in science fiction and pop culture

Genetics, genetic engineering, and mutations appear repeatedly in fictional form in various media – literature, movies, music, computer games, board games and more. Even though, in many cases, an interesting and credible scientific idea is not the centre of attention, the stereotypical portrayal of these phenomena (often in a negative context) contributes to the formation of the public's basic image of them. Since this type of "science fiction" is commonly used in popular culture solely to justify the presence of a completely unrealistic element (e.g., a superhero or a monster), the image that the public can get is often distorted and detached from reality.

Science fiction and its importance for society

From the distant past to present day, science fiction and its honest depiction in popular culture has fulfilled several important roles. One of them was to provide an open space for speculation about technologies and discoveries that could be interesting and useful to people. In the 19^th Century, the pioneer of this discipline was Jules Verne (it was during this period that the science fiction genre began to develop), who described several important technological discoveries in his works, some which were later turned into reality. The most important ones include a spaceship/rocket (novel "From the Earth to the Moon"; 1865), a submarine (novel "20,000 leagues under the sea"; 1871) or a helicopter (novel "Robur the Conqueror"; 1886). Another important role of science fiction is to hold up a mirror to our society at any given time and ask the question: "What would become of us if we had this type of technology at our disposal?", with many classical works focused on the psychological and social aspect of technological progress. In the 1950s and 1960s, Isaac Asimov (e.g., novels “I,Robot”; 1950 and "Foundation"; 1951), Robert A. Heinlein (e.g., novel "A Stranger in a Strange Land"; 1961) and Arthur C. Clarke (e.g., novel "2001: A Space Odyssey"; 1968) were the key figures bringing this point of view into the sci-fi genre. As a result, besides elaborate science fiction, more complicated topics related to the essence of humanity, definition of life, creation and control of artificial intelligence, emerging field of robotics, and many others reached literature and film. The third important role of science fiction is that it enables the creation of a (fictional) space for stories that could not take place in a real earthly environment. This tool allows authors and consumers of popular culture to fully explore their own imagination, develop abstract thinking and consider a wide range of hypothetical situations and problems, as well as the solutions to them. The film industry in particular was able to use the potential of science fiction to bring viewers a sense of wonder from unknown corners of the universe, planet Earth, one's own body, the world of dreams, the biosphere and many other environments. Finally, science fiction also brings the possibility of setting the science aside and use it simply as a background to bring the audience adventures, action, or horror tension in a new disguise. Probably the most famous representatives of this stream of sci-fi is the "Star Wars" saga (1977), "Alien" series (1979), and "Back to the Future" movies (1985).

The development of genetics and science fiction

One of the key topics that science fiction has repeatedly opened since its beginnings in the 19^th Century is the creation and modification of new forms of life. At a time when the basic rules of heredity and their molecular essence were not yet known, fiction of this type was completely abstract, or based on medical knowledge or a modified form of religious belief. Throughout the 19^th and 20^th Centuries, however, genetics began to develop intensively, and it also began to penetrate the popular culture. The seminal work of Gregor Johann Mendel, in which he described his original idea of heredity, was published in 1866 (for more details about Mendel and his work, see chapter 1 - In the beginning there was Mendel). Unfortunately, his idea fell into oblivion for several decades and had to be rediscovered in 1900 by a trio of scientists Carl Correns, Hugo de Vries, and Erich von Tschermak. In the following decades, genetics came more and more to the forefront of scientist’s interest, and after the molecular structure of DNA was described in 1953 by Watson, Crick, Franklin and Wilkins, a massive development of molecular genetics started (for more details on the basic structure of DNA, see chapter 3 - Meet DNA: the bearer of genetic information). However, the history of science fiction starts even deeper into the past (Figure 19.1).

Figure 19.1 Timeline – the development of genetics and science fiction.

The novel "Frankenstein" (1818) by the English author Mary Shelley is generally considered to be the very first work of the science fiction genre (however stories with certain elements of science fiction were already published in the distant past). Even before the description of the structure of DNA, the topic of artificial preparation of human embryos or humanized animals appeared in the works of Herbert G. Wells (novel "The Island of Dr. Moreau"; 1896) and Aldolus Huxley (novel "Brave New World"; 1932), but the real boom of genetics in science fiction came along with the so-called "golden era of sci-fi" in the 1950s. Genetics then quickly entered the mainstream, becoming the basis for comic book creation, horror films and entered the fantasy genre. Nowadays, we come across the topic of genetic modification, mutations or cloning quite commonly in practically any form of popular culture.

The paradox of genetics in science fiction

As mentioned above, genetics and its various forms are a common part of science fiction today. However, if we take a closer look at the current state of various genres in which genetics is often depicted (science fiction, fantasy, horror), we can notice a strange paradox – genetics is very often mentioned and presented as the trigger of various unusual situations (the emergence of a mutant monster, the discovery of supernatural abilities of one of the characters), but at the same time there are very few instances when the authors present some sort of interesting scientific explanation. In practice, this means that more and more often, especially in filmmaking, we encounter genetic engineering in the position of an unspecified cause of some type of disaster, or a shorthanded explanation of the presence of special abilities in the main character. Since most of the time the viewer is not given any details, they have no choice but to accept that "there is a technology that caused the situation". However, in this science-lacking sci-fi environment, it is still possible (albeit relatively rare) to discover truly interesting and original scientific and technological ideas.

Genetics in science fiction – examples

Some of the most famous examples of genetics being portrayed in popular culture are the stories of superheroes. A successful representative of this type of fiction is the "X-Men" series. In these stories, the "mutation in the gene X" leads to a wide variety of superpowers (laser vision, telepathic abilities, the ability to control magnetism, weather, etc.) in individual characters. However, the fiction does not explain how a change in a single protein or RNA molecule can lead to such complex and completely unreal consequences. Here, the mutation is used as a simple justification for any idea the story creators have, and its basis is never further inspected. The concept of human cloning is depicted the same way in the second episode of the "Star Wars" saga - "Attack of the Clones" (2002). In this movie, the technology of cloning serves solely as a tool to explain the presence of a huge anonymous army in the story. Furthermore, a completely unrealistic image of genetic recombination can be seen in the film "The Fly" (1986), in which the main protagonist gradually transforms into a monster with both human and fly characteristics. On the other hand, the fiction describing the inheritance of "Hen Ichaer" – the gene responsible for the powerful magical abilities in the popular series "The Witcher" (1992) is simple, but very appropriately chosen. In this fiction, magical talent is caused by a specific allele of a gene, and the way in which this talent is passed down from one generation to another exactly corresponds to the basic rules of Mendel's conception of inheritance. Of course, the fiction does not explain how the gene enables its bearer to practice magic, but the rules of inheritance themselves are scientifically accurate.

Another example of well-thought-out science fiction, containing elements of genetics, evolutionary biology, but mainly developing critical thinking and a general understanding of the methodology of science, can be found in the fan fiction work "Harry Potter and the methods of rationality" (2015) by Eliezer Yudkowski. In this work, we meet Harry Potter, who, unlike in the original novel and film series, is not a bullied orphan, but a self-confident boy with perfect rational thinking. He is thus able to effectively study magic and the essence of reality, in which he suddenly found himself at the age of eleven. Finally, one of the most convincing science fictions of all time is represented by the original film "Jurassic Park" (1993), showing the artificial creation of dinosaurs after palaeontologists discover a piece of amber with a mosquito embedded in it, many millions of years old. Since this mosquito was once a parasite on real dinosaurs, scientists working at the park service can obtain the DNA of the dinosaurs, and by filling in the missing parts with the DNA of a specific species of frog, they can bring the dinosaurs back to life. In addition to this basic fiction, we also learn that the dinosaurs cannot leave the park because they cannot produce the amino acid lysine, which they must get in their diet, or they would die (lysine is indeed an essential amino acid). We also find out that dinosaurs cannot reproduce naturally, since all the individuals present in the park are females (if you watch the film carefully, you will come across another – not quite realistic – element of science fiction, which explains why the dinosaurs eventually reproduced on their own). Thus, in addition to an exciting adventure story, "Jurassic Park" also provides us with a relatively sophisticated look into the world of science.

What can a mutation really do?

When talking about genetics, one of the tropes most frequently used in science fiction is mutation. Mutations cause a change in the properties of various creatures (often they are people, but it is not a rule), which subsequently play important roles in stories. If we were to ask ourselves the question: "What superpowers can a genetic mutation really bring to a person?", the answers would be much simpler than those usually presented on the silver screen. It is obvious that since the final product of the expression of each gene is a functional RNA or a protein, mutations primarily affect the properties of these molecules. It follows that the mutation can never cause "laser sight" or "magnetic skin". However, mutations can really bring new, previously unseen abilities to people – for example, lactose intolerance is a completely standard feature of adult people, since milk is primarily the food of mammalian newborns. In the past, however, a mutation appeared in the human population, thanks to which the human body produces the enzyme lactase (it enables the digestion of milk sugar) even in adulthood, and thus we can consume milk throughout our entire lives. As this mutation spread through the human population, milk can now be consumed by many adults, although few would call this ability a "superpower" (for more information on lactase and other examples of mutations that contribute to ongoing human evolution, see chapter 14 – the principle of evolution).

A similar phenomenon can also be observed in the human body's ability to tolerate higher amounts of alcohol in drinks. In Asian populations we can very often come across a variant gene encoding the alcohol dehydrogenase enzyme, which allows the decomposition of ethanol into toxic acetaldehyde several times faster than the wild type allele. Thanks to this enzyme variant, it seems that Asian people get drunk much more easily. Conversely, mutations that slow down the production of acetaldehyde make it easier for people to tolerate higher amounts of alcohol. In a similar way, mutations in some genes allow the inhabitants of the Himalayan regions to survive at extremely high altitudes, even though there is significantly less oxygen in the air in those places. On the other hand, just as a mutation (or a combination of several mutations) can help our body to adapt to a particular type of environment, it can also cause certain RNAs or proteins inside the body to not function correctly. As a result of these mutations, many types of metabolic diseases (e.g., phenylketonuria), and food intolerances, but also other serious genetically determined diseases occur. Mutations can indeed bring new abilities to a person, but rather than the ability to bend steel beams with one's hands, we should imagine the ability to metabolize milk or alcohol without difficulty.

Stereotypes in the portrayal of genetics in popular culture

Science fiction represents a wide space for various depictions of genetics and technologies derived from it, however, we often encounter several recurring stereotypes. One such stereotype is the creation of a dangerous monster by genetic engineering, or other type of immediate threat that humanity faces because of the reckless use of this type of technology. Another similar stereotype is a dystopian future in which humanity threatens its own existence and basic human rights and freedoms due to the uncontrolled use of genetic engineering techniques. The unfortunate feature of these stereotypes is that they usually portray genetic engineering in a negative light and, despite the clear benefits of its use, instil a sense of fear and threat in the consumers. A very common visual stereotype is the "scientist observing the model of double-stranded DNA". This type of visual display of DNA analysis is common in movies and comes from the widespread (and correct) idea of the spatial organisation of DNA. However, real DNA analyses do not usually use this type of secondary structure imaging and (unless the scientist in question is investigating this very property) it does not provide them with any useful information. Real analysis of DNA usually employs methods based on electrophoretic separation, where individual fragments form specific bands, or a more modern approach of sequencing, which determines the sequence of individual nucleotides in the analysed sample and writes them down in the form of text (if you are interested in how to analyse DNA properly, turn to chapter 4 – How to work with DNA). Since the inaccurate depiction of technologies based on recombinant DNA and overused stereotypes related to gene engineering in popular culture can significantly distort the image of these techniques in the eyes of the common consumer of science fiction, it is important for all of us to carefully distinguish the facts from fiction in all media.

Did you know that...

...another of the original ideas of science fiction literature – combining the cells of different types of organisms into a single embryo – has become a reality in 2021? This idea was discussed in 1986 by H.G. Wells in the novel "The Island of Doctor Moreau", in which the mad doctor Moreau uses the methods of medical science and surgery to create creatures consisting of parts of the bodies of various animals (fox-bear, mare-rhino) or even combinations of animals and people (wolf-man, leopard-man, wolf-woman). A scientific group led by Chinese experimenter Tao Tan has not yet done anything similar, but they managed to create long-tailed macaque embryos into which they implanted human stem cells. These cells were subsequently able to divide and contribute to the formation of multiple germ cell lines, typical of the developing embryo. Of course, such embryos did not develop into viable individuals, so we do not have to fear the arrival of ape-people, but the very fact that human and monkey cells can coexist in one embryo is a significant and extremely ethically controversial discovery.

Want to read more?

Brave new world, Huxley, A., Chatto & Windus (1932).

Jurassic park, Crichton, M., Alfred A. Knopf (1990).

See links throughout this chapter for more information on the books and movies referred to!

Chapter 20: How to become a model...in biology

We live in a complicated world. The role of scientists, citing Francis Bacon (1561 – 1626), is to be “merchants of light and to promote relief of human’s estate”. To this end, scientists collect data, formulate possible explanations (hypotheses) of the corresponding phenomena and then subject them to rigorous experimental testing. Hypotheses that fail the experimental test are replaced by those that are more viable. As a result, they propose models that not only explain the available observed data, but also predict previously unknown properties of the given phenomenon. The crucial role in understanding basic principles governing life was (and still is) played by so-called model organisms. Each model organism exhibits unique characteristics, but at the same time illustrates the unity of life, which works on the same principles, regardless of whether it is a bacterium, fungus, plant, or animal. This chapter provides :

a description of a "good model organism";
rules for choosing a model organism suitable for the study of a relevant biological phenomenon;
examples of conventional as well as “exotic” model organisms with their advantages and limitations.

While it is only a superficial overview of the topic, it should persuade the reader that model organisms are not only instrumental in understanding the general principles of the complicated living world we live in but can help us in gaining insights about ourselves as a distinct species.

Biological phenomena should be studied in the simplest organisms that exhibit them

One strategy for studying complicated biological phenomena is to reduce them to simpler questions. In biology, this approach means that when we want to understand how a complex organism, like humans, function, it is practical to first table the problem in a more simpler organism that displays the same phenomena.

The principle of minimum complexity - that is, the study of complex biological phenomena in simpler organisms - can be applied thanks to the fact that living organisms share a common ancestor (Figure 20.1). Thanks to Charles Darwin (1809 – 1882) and his followers, we know that in the long-term evolutionary perspective, more complex forms of organisms arise from simpler ones through a combination of the generation of genetic variability (e.g., through mutations or recombination) and the selection of those genetic variants that are best adapted to the given environment as measured by their ability to produce offspring (more information can be found in Chapter 14 – the principle of Evolution). The so-called phylogenetic trees illustrate that all living forms are related to each other (Figure 20.1). Some are more related - located on the same branches of the tree – than others less - located in more distant parts of the tree. During this evolutionary diversification there was (1) preservation of fundamental biological principles that apply to all living forms and (2) specialisation in individual branches, which led to the emergence of a defined taxonomic group or a specific biological species. This unity of life, illustrated by a phylogenetic tree, makes it possible to apply Delbrück's principle who with his collaborators revealed the molecular basis of heredity using simple bacterial viruses (see chapter 14 for more details). In other words, we can formulate models about the functioning of the relevant biological phenomenon based on the study of simpler - model - organisms.

Figure 20.1. Phylogenetic tree of selected representatives of the groups Bacteria, Archaea and Eukaryota. The tree illustrates that all living organisms share a common ancestor, from which the combination of genetic variability and natural selection led to diversification into individual taxonomic groups. The closer these groups are within the crown of the tree, the more evolutionarily related they are. Following the Delbrück’s principle, the choice of model organism depends on what biological phenomenon is the purpose of the study.

Model organisms enable the construction of relevant models about biological phenomena

The use of simpler organisms to create models explaining the functioning of living nature has a long tradition, dating back to ancient Greece. However, it was only fully developed during the Enlightenment. For example, Antoine Lavoisier (1743 – 1794) used experiments with guinea pigs to investigate the nature of respiration (Figure 20.2). Lavosier went on to study how the body metabolises food and surely if he had not ended up as a victim of the French Revolution on the guillotine, his contribution to the understanding of human physiology would have been even more profound.

Figure 20.2. The study of guinea pig respiration led to a model of respiration as a slow form of combustion. A guinea pig is placed in an ice calorimeter – a container which is placed into a second insulated container filled with ice - into which air is supplied in a controlled manner. The amount of exhaled carbon dioxide from the guinea pig was analysed. The amount of heat was determined based on the volume of water produced as a result of melting ice. Lavoisier proved that the heat produced is the same as the amount of oxygen consumed by breathing. From this he concluded that breathing is a slow form of combustion. Image of the ice calorimeter is redrawn from Kleber (1961) The Fire of Life.

The use of animals for the research of biological phenomena providing information valid for humans gained momentum in the 19^th Century. Joseph von Mering (1849 – 1908) and Oskar Minkowski (1858 – 1931), for example, showed in dogs that surgical removal of the pancreas lead to a dramatic increase in the concentration of sugar in the urine and other symptoms typical of diabetes mellitus. Frederick Banting (1891 – 1941) used the pancreas of dogs (and then cattle) to isolate insulin. Also using dogs, Ivan Petrovich Pavlov (1849 – 1936) discovered the phenomenon of conditioned reflexes and thus laid the foundation of modern neurophysiology. The basics of innate immunity was understood not thanks to mammals, but to gastropods. At the turn of the century, Ilya Mechnikoff (1845 – 1916) observed what was happening in the wound area of the gastropod Charonia tritonis. He observed cells (later labelled macrophages) at the wound site that had the ability to eat (phagocytose) bacteria that had begun to multiply in the wound.

We can move even further away from humans, jumping to another branch of the phylogenetic tree, and still find an organism that, unexpectedly, tells us a lot about us. Today, the classic experiments of Gregor Johann Mendel (1822 – 1884) on the pea (Pisum sativum) revealed the foundations of heredity (see chapter 1 – in the beginning there was Mendel). Are not plants simple enough for you? As we shall see in the upcoming paragraphs, there is basically no limit for simplicity of the model organism and some breakthrough discoveries about fundamental biological processes were performed on organisms as simple as bacterial viruses. But before we dwell into some specific examples, let us list basic requirements that must be met by a suitable model organism.

A model organism must meet several requirements

As this is a book about genetics, we can start the discussion about the properties of a “good” model organism using Mendel's hybridization experiments as an example. It illustrates that when choosing an organism for the study of a given phenomenon, it is necessary to be very careful and at the same time to be lucky. The fact that Mendel's results did not find a response in the scientific community at the time of their publication was due, among other things, to the fact that he tried to prove that his rules also apply to another plant. His choice of plant to prove his theory was the hawkweed (Hieracium), which although studied by botanists in the 19^thCentury, had one feature that disqualified it as a model organism for the study of the foundations of heredity: this genus of plants reproduces apomictically. This means that an egg can also form an embryo without fusing with the male gamete. Of course, Mendel could not obtain the same results for the hawkweed as for the pea, which reproduces sexually and thus his genius had to wait for recognition for 20 years when his results were “re-discovered”.

How can we avoid a similar mistake that happened to Mendel? Here is a list a few properties that a good model organism should meet (Table 20.1).

Table 1. Properties of a good model organism.

Property	Explanation
Size	For the statistical evaluation of experiments, it is necessary to have data on a large number of individuals.
Availability and easy maintenance in the laboratory	The financial and technical means of maintaining the organism in the laboratory should be as low as possible.
Short reproduction cycle	It is ideal to do as many experiments as possible in the shortest possible time, e.g. enabling their easy repeatability
Possibility of use for the study of a wide spectrum of phenomena	It is advantageous if the organism is not useful for study of a single phenomenon but can be used to address several problems.
Easy observability and possibility of quantification of the studied property	"Hidden" properties require more sophisticated and therefore more financially/technically demanding methods.
Techniques enabling targeted (e.g. genetic) manipulations	Advantageous for genetic analysis of studied properties.
Genome size	Too large a genome complicates genetic manipulation and interpretation of results.
Generalisation of the results	Advantageous in the case of phenomena with a more general occurrence; it may not apply if we are interested in a property specific to a given type of organism.

Now, we know what it takes to be a good model organism, we can introduce a few examples.

The basic principles of regulation of cellular activities apply equally to bacterial cells as to human cells

Our cells are exposed to a constantly changing environment and therefore must be able to evaluate these changes correctly and respond adequately. For example, when an interesting source of nutrients occurs in their vicinity, they must be able to recognise it, get it into the cytoplasm and process it efficiently. They do so with the help of enzymes, coded by genes, which the cell "turns on" after detecting the nutrient and "turns off" in its absence (gene expression is described in Chapter 3). How is this control carried out? How does a cell know which gene to turn off/on and when to do it?

Important answers to these questions were provided by research on cells, namely on bacterial cells. Jacques Monod (1910 – 1976), a French biologist, conducted an experiment in which the bacteria Escherichia coli were cultured in a medium that either contained a mixture of sugars glucose and maltose, or a mixture of glucose and lactose (Figure 20.3). Bacteria in the glucose+maltose medium had a smooth growth curve, while in the case of the glucose+lactose mixture, the growth stopped for a period halfway through the experiment. Only after restarting did the number of cells reach same number of cells as the bacteria growing in a glucose+maltose mixture. Monod correctly assumed that this pausing occurred as after all the glucose was consumed, the cells then adapted to a new carbon source (lactose). Genetic analysis of this phenomenon led to a model of the regulatory circuit responsible for this adaptation (Figure 20.3). It turned out that the principle of gene activity regulation identified in E. coli is also used in certain variations by more complex (e.g. human) cells. This led Monod to make a metaphorical generalisation: "What is true for E. coli is also true for an elephant."

Figure 20.3 Basic principles of gene activity regulation were described in Escherichia coli. (A) Growth of E. coli is smooth when in the presence of glucose + maltose. However, when E. coli cells are cultured in a medium containing glucose + lactose as a carbon source, the bacteria first use up all the glucose and only after several tens of minutes of adaptation begin to use lactose and continue to grow. (B) Simplified diagram of the circuit that regulates the activity of genes that encode proteins involved in lactose catabolism, respectively.

When we want to understand the functioning of a eukaryotic cell, the model organism must be an eukaryote

How to choose from the enormous number of species of eukaryotic organisms? Figure 20.1 illustrates the great diversity of eukaryotes, most of which are single-celled organisms that for the purposes of this text (though incorrectly in terms of modern taxonomy) can be called protozoa. When we are interested in the functioning of human cells, the first step is to look for a type of organism that meets the requirements for a model organism but that is also closely related to humans. On our simplified version of the phylogenetic tree, we see that this requirement could be met by a representative of the group of fungi. Not the macroscopic mushrooms that we collect in forests or put on our pizzas, but microscopic species that can exist in a single-celled state. Fungi called yeast fulfil this requirement.

Yeast are eukaryotic microorganisms that can ferment various carbon sources, for example the sugar from freshly crushed grapes (so-called grape must). In this case, it is a yeast of the species Saccharomyces cerevisiae, which has accompanied mankind for the past several thousand years as an assistant in the production of wine, beer, and baked goods. Louis Pasteur (1822 – 1895) proved that S. cerevisiae is an agent that converts sugar into alcohol, and since then this yeast has not only been the workhorse of classical biotechnology but has become an honorary member of the imaginary Pantheon of model organisms.

Although the cells of S. cerevisiae are relatively small (3-5 mm compared to the approx. 100 mm diameter of a typical human body cell), their structure is very similar to mammalian cells (Figure 20.4). A yeast cell is specific in some respects, for example, it has a cell wall, it divides by budding (that's why S. cerevisiae is sometimes referred to as budding yeast). However, yeast cells also contain organelles characteristic of eukaryotic organisms, its genome is separated from the rest of the cell by a membrane that encloses the cell nucleus, and its cell cycle is regulated in an analogous manner to mammalian cells. This was proved by the American biologist Leland Hartwell, who in the 1970s prepared a collection of cell division cycle (cdc) mutant strains of S. cerevisiae that were unable to pass through one of the phases of the cell cycle. A few years later, the British geneticist Paul Nurse identified cdc mutants in the cell cycle in another yeast species Schizosaccharomyces pombe (or simply fission yeasts) and at that time attempted a very brave experiment to answer the question: Would it be possible to replace a faulty yeast gene with a human gene, which would restore the yeast cells the ability to divide? Together with his student Melanie Lee, they discovered that there really is a human gene that can fully function in yeast cells. This result underscored the evolutionary relatedness of such seemingly dissimilar organisms as fungi and mammals and confirmed that the study of biological phenomena in simple organisms enables the formulation of models valid for human biology as well.

Figure 20.4 Yeast is an excellent organism for studying phenomena related to the eukaryotic cell. (A) The yeast Saccharomyces cerevisiae divides by budding. (B) A diagram of S. cerevisiae showing the cells have a cell wall, but otherwise the organelles are very similar to a mammalian cell. (C) The cell cycle of S. cerevisiae goes through the same stages as mammalian cells. G1 is where the cells increase in size, S stage is where DNA is replicated. In G2, the cell is preparing to divide and division occurs in M phase. Genetic control of the S. cerevisiae cell cycle can be analysed using mutants unable to pass through one of its phases (shown by the red “STOP”).

It turns out that the regulation of the cell cycle of yeast and mammalian cells is fundamentally similar, and its disturbances have the same consequences: either loss of the ability of cells to divide, or division even in conditions where it should be stopped. This is, for example, the case of tumour cells that are not able to correctly evaluate the signals that indicate to the cell that it should stop the cell cycle. Such a signal can be, for example, DNA damage, which a normal cell evaluates by stopping the cell cycle, repairing the damage, and only then restarting the cell division. A tumour cell that has lost this ability due to a mutation in the genes controlling the cell cycle will continue to divide even if the DNA damage is not repaired, increasing the likelihood of further mutations that promote uncontrolled division. You have read more about this in Chapter 9 – when cells go crazy.

The impressive list of crucial discoveries stemming from yeast research covers a wide range of phenomena including protein secretion, biogenesis of organelles, autophagy, bioenergetics, genome stability, chromosome segregation, and many, many more. As in case of the mechanisms of cell cycle regulation, the results shed more light on the cellular processes occurring in normal human cells including specialised cell types such as pancreatic beta-cells or neurons as well as in cells exhibiting pathologies causing various human diseases.

In addition to these fundamental discoveries, research on yeasts contributed to the development of new technologies, which subsequently found application in the research of more complex organisms. For example, the first eukaryotic organism in which the complete order (sequence) of nucleotides in all chromosomes was determined in 1996 was the yeast S. cerevisiae, which opened the way for the sequencing of the human genome at the beginning of this millennium.

The biggest celebrity among genetic model organisms is the fruit fly Drosophila melanogaster

When Thomas Morgan (1866 – 1945) first saw the white-eyed male fly Drosophila melanogaster in his laboratory at Columbia University in New York, he had no idea that he was at the beginning of a discovery that would bring him the Nobel Prize 20 years later. Indeed, an experiment in which he crossed white-eyed mutant flies with standard red-eyed partners that led to the discovery that the genes are located on chromosomes (Figure 20.5).

Figure 20.5 The experiment that led to the discovery of the localisation of genes on chromosomes. A white-eyed male and a red-eyed female produce exclusively red-eyed offspring. When their daughters are crossed with a red-eyed male, all the daughters will be red-eyed, but half of the sons will have white eyes. Try to explain this result when you know that the mutant allele of the gene controlling eye colour is located on the X sex chromosome.

Thanks to Morgan and his students at Columbia University in New York, Drosophila became the "main figure" for the study of genetic control of biological processes in animals. Its short life cycle (up to 14 days), easy breeding and wide repertoire of techniques that allow for sophisticated genome manipulations are some of the attributes that have made Drosophila probably the most widely used model organism.

After Morgan laid the foundation for the chromosome theory of inheritance, several hundred mutants were prepared that showed various developmental disorders either in the larval stage or in adult flies. Examples of such mutants are Ultrabithorax, which produces one extra winged limb; Antennapedia, which has an extra pair of legs instead of antennae; or eyeless (ey), lacking eyes. Remarkably, when the genes whose alteration leads to developmental disorders in mutants were identified, similar genes were found to occur in the human genome. And not only that, the products of these genes are also involved in the correct course of ontogenesis. And just as Paul Nurse showed in yeast, mammalian and Drosophila genes were found to be interchangeable. That is, when the human gene was inserted into the genome of the Drosophila mutant, its proper ontogeny was restored. Conversely, Drosophila genes worked well in mammals. The fact that the ey gene is involved in the formation of the eye was proven by the fact that when it was artificially activated in different parts of the body of Drosophila (for example, on the legs), the formation of eyes took place in them. When such (so-called ectopic) expression was done with the mouse gene Pax6, which is similar to ey, the result was the same (eye formation in "inappropriate" body parts). The results of these fascinating experiments confirmed that the ontogeny processes of Drosophila and mammals are under similar genetic control, so the results obtained in these small dipterans are also relevant for animals like us.

The small but mighty nematode is an excellent model

At the end of the 1960s, the South African biologist Sydney Brenner (1927 – 2019) decided to leave the field of molecular biology, with an explanation that everything essential in it had already been discovered. He decided to find an animal that would help him answer important questions in developmental biology: what determines cell differentiation during ontogenesis.

From all the candidates, a small (approx. 1 mm) self-fertilising worm Caenorhabditis elegans, met all the requirements for a model organism (Table 20.1), including simple breeding (in Petri dishes with agar and bacteria that are food for the worms). C. elegans has a short life cycle (~21 days at 20^oC), starting with embryogenesis inside the egg. The nematode hatches as a 1^st stage larvae (L1) and proceeds through 4 periods of growth (L1-4), each separated by a molt – where the cuticle (skin) of the worm is shed, much like a snake (Figure 20.6). Upon reaching adulthood, each worm will produce around 300 genetically identical progeny over a 4 day period. After reproduction, the worms start to age, in a similar manner as humans. An interesting phenomenon of the life cycle of C. elegans is that, in the case of starvation during the 2^nd larval stage (L2), the worm can exit the trajectory leading to an adult individual and reach the stage of so-called dauer larva, where the worm will undergo a significant slowdown in metabolism and various other physiological and anatomical changes (e.g., closing of the mouth opening). This allows its survival for several months instead of 2 weeks. Genetic analyses of this developmental "bypass" identified several genes that control entry (and exit) from the dauer larva stage. It has been shown that changes in these genes in adults can lead to a 3-fold extension of their life. Interestingly, these genes code for proteins that participate in the regulation of metabolism in human cells and belong to the pathways that are regulated by the hormone insulin. The study of such an exotic developmental stage as the dauer larva in C. elegans has significantly contributed to our understanding of the molecular mechanisms of aging, even in mammals like us.

Figure 20.6 The life cycle of Caenorhabditis elegans makes it possible not only to study different stages of development in a short time. (A) The worms start life as an egg and then go through 4 larval stages to reach adulthood within 3 days. If conditions are poor, the worm can divert to an alternative state, dauer, for a period of time before returning to the usual life cycle when it is more favourable. (B) After the final phase of L4 to adulthood, the worms start to produce their 300 genetically identical offspring. Then, for the last 2 weeks of life, the worms will start to show classic signs of aging, until they die. Images provided by S. Hughes.

In addition to the advantages mentioned above, C. elegans provides a few more important benefits: First, we know the exact pedigree of every cell that makes the worm. Together with colleagues, Robert Horvitz and John Sulston, mapped the fate of each cell and created a cellular family tree, which is commonly called a lineage. The fact that each individual worm has the same cell pedigree (we call this eutely) makes it possible to identify genes involved in cell fate determination in C. elegans bodies. The importance of the study of C. elegans for human medicine was shown, for example, in the study of genetic control of cell death. Of the 1090 cells that are created during ontogenesis, exactly 131 die in a process known as apoptosis, so that the adult hermaphrodite worm has 959 cells. By identifying mutants in which this type of programmed cell death does not occur, it was possible to reveal the genes whose products are involved in the implementation of apoptosis. Some genes activate apoptosis (inactivation of such a gene led to an excess number of cells in an adult), while others inhibit it (such a mutant would have fewer cells). One of the genes discovered in this way was ced-9(the abbreviation ced is derived from cell death), which encodes an inhibitor of apoptosis. Human cells have been shown to contain a gene encoding a similar protein called Bcl-2. In the case of some oncological diseases (e.g., B-cell lymphomas), this protein is produced in high concentration, which leads to the suppression of apoptosis even under conditions that normal cells evaluate as dangerous. Due to the overproduction of the apoptosis inhibitor Bcl-2, tumour cells lose this ability and continue to divide instead of dying. Similar to the case of ced mutants, genes were also found in C. elegans that regulate the formation of specialised cell types, including 302 neurons. The defined (and low) number of neurons makes C. elegans an excellent object for the study of neurogenesis (formation of the nervous system), because all connections are mapped in detail and it is thus possible to study their role in the relevant biological process (e.g., movement, excitability, responses to odour signals).

The second benefit of C. elegans is that it has had its genome completely sequenced so that we now know what every gene is and where it is in the genome. In fact, this humble 1mm long worm was the first multicellular eukaryotic organism to have its genome sequenced. Containing 100 million bases of DNA, the genome has around 20,500 protein-coding genes arranged across 6 chromosomes (5 autosomes and a sex chromosome).

Taken together, C. elegans is an extremely valuable model organism with many thousands of researchers working with it on various topics. Research with C. elegans continues to be an important source for new discoveries, as shown by the many Nobel Prizes awarded to people working with worms!

For studying the molecular basis of memory one needs an organism that has large neurons and easily measurable indicator of memory formation

This requirement is met by the sea slug Aplysia californica, which was chosen by the American neurobiologist of Austrian origin Eric Kandel as a model organism for the study of biochemical events that accompany the creation of a memory (Figure 20.7).

Figure 20.7 Aplysia californica makes it possible to study processes associated with memory formation at the level of the whole organism and individual synapses. (A) Top view of a sea slug. Mechanical irritation leads to reflex withdrawal of the gills (green colour) and siphon (brown colour) resulting in the discharge of coloured fluid. However, if there is no predator attack after irritation, repeated irritations no longer lead to these reflexes. Biochemical events at the level of individual synapses that accompany the formation of a memory can be studied on dissected large neurons outside the animal's body (so-called in vitro conditions). (B) A synapse is the connection between two neurons that allows a chemical signal to be passed between them. The presynaptic neurone releases a chemical signal (the neurotransmitter, shown in blue) into the synaptic cleft. The neurotransmitter diffuses across the gap and will bind to receptors on the post-synaptic neurone..

Aplysia reacts to mechanical irritation by retracting its gills and the siphon, an organ through which it sucks water into the body. The retraction is a way for the sea slug to protect vital organs from a possible attack by a predator. When the irritation is repeated without a real predator attack, Aplysia stops reacting to it (it remembers that the irritation is not associated with danger). How is this memory stored at the neuronal level? Kandel studied what happens at the level of the connection (synapse) between the neuron and the muscle cell that is responsible for retracting the siphon during memory retention. He used the fact that this reflex can also be studied outside the animal's body and that the studied cells are relatively large, which facilitates experimental work with them. He described a series of biochemical reactions that are triggered in response to repeated stimuli and that "mark" the corresponding synapse, thereby changing its properties and the way it communicates with the target muscle cell. It turned out that these reactions are responsible for the creation of memory traces in other animals, including humans. It was therefore not surprising that Kandel won the Nobel Prize in Physiology or Medicine in 2000.

Amphibians make it possible to observe embryogenesis outside the mother's body

The fact that many amphibians undergo fertilisation of eggs and subsequent development of the embryo outside the mother's body makes them an excellent model for developmental biology. It should also be added that many species of amphibian (e.g., the clawed frog Xenopus laevis) produce a large number eggs that are quite big in size, which makes it possible to obtain a substantial amount of experimental material.

After fertilisation, the cells divide for several generations synchronously, so it is possible to study what happens during the individual phases of the cell cycle. After the multiplication of the mass of cells, a spherical structure of the blastula is formed, which is comprised of undifferentiated cells covered by a single layer of cells. Subsequently, the creation of the so-called gastrula, which results in three germ layers (ectoderm, endoderm and mesoderm), from which individual tissues later develop (Figure 20.8).

Figure 20.8 Amphibians make it possible to study embryogenesis outside the mother's body. (A) In the first stages of embryogenesis, the gastrula is formed, when cells from the so-called the organiser migrate inside the multicellular mass and form three germ layers. The ectoderm is in blue, endoderm in green and mesoderm is shown in yellow. (B) By transplanting the organiser (in orange) of one embryo into another embryo Spemann and Mangold demonstrated the existence of a substance that induces this stage of differentiation. A few years after their experiment, it became clear that this inducer is proteinaceous in nature and participates in gastrulation in other organisms.

Gastrulation begins in a specific location of the blastula, which is referred to as the "organiser". Cells from the organiser region migrate into the blastula and eventually form the three-layered gastrula. But how do the cells in the organiser area "know" to start migrating? This interested a German embryologist, Hans Spemann (1869 – 1941), and his student Hilde Mangold (1898 – 1924). They hypothesised that a substance is formed in the organiser area that provides the cells with the instruction to start migrating. To test their hypothesis, Mangold transplanted an organiser from an embryo that had just begun gastrulation into the blastula of another embryo. She observed that in the second embryo, gastrulation began in two places: in the area of the original and the area of the implanted organiser. The result was two mirror-orientated connected gills (Figure 20.8). The identification of the substance that is the inducer for starting gastrulation took a few more years, but it turned out that it is basically the same in both amphibians and mammals. The Nobel Prize for Spemann in 1935 underlined the importance of this discovery for human medicine.

John B. Gurdon also used the advantages of working with amphibians in his experiments, where he proved that the nucleus of differentiated body cells can be "reprogrammed" to the state in which it was in the fertilised egg stage. In his experiments, Gurdon removed the nucleus from the differentiated body cell of the frog and introduced it into an unfertilised egg, the nucleus of which he had previously destroyed. Subsequently, he stimulated the egg cell with the "new" nucleus to divide, and the result was that embryogenesis started, leading to a frog that was a genetic clone of the nucleus donor (Figure 20.9). This experiment indicated that it should be possible to clone even more complex animals. Considering that the whole procedure is not technically trivial, it took several years before Gurdon's experiment was also carried out in mammals. The result was Dolly the sheep, whose nucleus came from the udder cell of an adult sheep. The fact that the nucleus of a differentiated body cell can be reprogrammed opened the way for an experiment whose goal was to find out what the molecular mechanism of this "dedifferentiation" is. Japanese biologist Shinya Yamanaka identified these reprogramming factors in human cells. He discovered that the activation of these factors in body cells leads to a change in their properties so that, similar to embryonic cells, they become so-called pluripotent. This means that such reprogrammed cells (induced pluripotent cells), like embryonic cells, are capable of giving rise to many types differentiated cell. This opened the door to the so-called therapeutic cloning, in which, for example, it is possible to take a population of body cells from the patient's body, reprogram them to be pluripotent, correct genetic disorders in them and, after re-differentiation, bring them back to the patient and thus help him heal. Gurdon's experiments on amphibians thus led to a model that Yamanaka used to make a discovery with previously unimaginable therapeutic potential. Both therefore deservedly shared the Nobel Prize for Physiology or Medicine in 2012.

Figure 20.9 The possibility of reprogramming the nucleus of a body (somatic) cell was first demonstrated in amphibians. The nucleus of a body cell of a tree frog was introduced into the egg (purple) without a nucleus (enucleated egg), which, after stimulation of division, gave rise to a frog with the same genetic information (the so-called clone).

There are many organisms that may seem exotic, yet they provided essential information about fundamental biological principles

So far, we briefly introduced organisms that can be considered as “conventional”. This adjective is substantiated by the fact that their use in scientific investigations resulted approximately forty Nobel prizes. However, the list of model organisms does not end here. In fact, there are dozens of species that, for one reason or another, have been exploited in understanding various biological phenomena. Examples of such organisms are provided in Figure 20.10.

Figure 20.10 Examples of organisms and biological phenomena that they helped to uncover.

For understanding some phenomena, we need to use organisms that are evolutionarily more closely related humans

Although the study of simple model organisms has provided a lot of detailed valid knowledge, in some cases we need to study one of the placental mammal species in order to obtain an adequate explanation of some phenomenon related to human biology. Perhaps the most popular model organisms from this group are the rodents such as mouse, rat, and guinea pig. All of them meet the basic requirements for a model organism, and at the same time their genetic makeup and physiological parameters are very similar to humans. Also, for this reason rodents are used for studying behaviour (primarily rats), or modelling of human diseases (mainly mice). Today we have at our disposal many strains of rodents that have been modified in such a way that they show typical symptoms of oncological, cardiovascular or neurological diseases. Most of these strains are the product of long-term inbreeding, thus they have a very low degree of genetic variability, which simplifies the interpretation of the obtained results and identification of the genes involved in a particular trait. Laboratory rodents are routinely used in preclinical tests of potential drugs. In addition, there are special strains of these animals that have some useful modification. For example, the mouse strain called SCID (severe combined immunodeficient mice) has a suppressed immune system, which enables human tissue transplants for use in, for example, oncology research. You can read more about SCID in Chapter 10 - Gene therapy.

In addition to their use in medicine, rodents are suitable for developing new technologies. An excellent example is the technique called optogenetics, which enables targeted and regulated control of a selected biological process. It is significant that the beginnings of optogenetics can be found in research on the phototactic behaviour of algae. Their cells have a protein present on their plasma membrane that can capture photons and subsequently make the membrane temporarily permeable to ions. This actually changes the electrical conditions on the cell membrane, i.e., exactly what happens when a nerve signal is transmitted along the membrane of a neuron. Creative biologists tested what would happen if this photosensitive ion channel was introduced into the membranes of selected mouse neurons. In theory, it would then be possible to control the activity of these neurons by turning on (or turning off) the light. Experiments proved that this assumption was correct, and today optogenetics is a standard strategy used not only to control the activity of neurons, but also of other cell types.

Rodents are still almost 200 million years away from our common ancestor. This means that not all results obtained on mice can be applied to humans. In our phylogenetic tree, we must move to the branches that are directly attached to the one leading to Homo sapiens. Primates have many times provided biologists with answers to questions that other model organisms could not help to solve. Of these, our closest evolutionary relative is the chimpanzee (Pan troglodytes). While we separated from a common ancestor about 6 million years ago, and at the nucleotide sequence level, our genomes are nearly 99% similar. The chimpanzee brain is approximately 3 times smaller than the human brain, and although there is a large difference in the number of neurons in the cerebral cortex, both brains have a very similar architecture. Above all, however, the cognitive abilities of chimpanzees are at least in some ways similar to those of young children, so studying the neurobiology of these primates can tell us a lot about human neurobiology.

Among all the examples, one of the most interesting is that in the brain of a chimpanzee performing some activity, a specific group of neurons in the corresponding part of the brain is activated. Italian neurobiologists led by Giacomo Rizzolati discovered that the same group of neurons is activated in a chimpanzee observing the performer. It turned out that such so-called mirror neurons also exist in the human brain. The discovery of mirror neurons indicates the neurobiological basis of empathy, empathising with another's world of ideas, emotions, way of thinking and attitudes. Empathy is a very important tool for social cohesion, and the fact that we share its foundation with our primate relatives underscores how deeply it is embedded in our biology.

In some cases, the only possible organism formulating adequate models about human biology are... humans

Finally, we are left with the ultimate model organism, Homo sapiens. Indeed, we can learn about some aspects of human biology only by studying humans. Leonardo da Vinci (1452 – 1519), drew a model of the so-called Vitruvian Man (Figure 20.11), which is based on the Ten Books on Architecture, written in the 1^st Century by Marcus Vitruvius Pollio (approx. 80 BC – 15 BC). The Vitruvian Man illustrates the perfect proportions of the human body, drawn by Leonardo around 1490. It was not the first, but certainly the most beautiful and elaborate.

”The length of the outspread arms is equal to the height of the man. From the hairline to the bottom of the chin is one-tenth of the height of the man. From below the chin to the top of the head is one-eighth of the height of the man. From above the chest to the top of the head is one-sixth of the height of the man. From above the chest to the hairline is one-seventh of the height of a man. From the chest to the head is a quarter of the height of the man. The maximum width of the shoulders contains a quarter of the man. From the elbow to the tip of the hand is a quarter of the height of a man; the distance from the elbow to the armpit is one-eighth of the height of the man; the length of the hand is one-tenth of the man. The virile member is at the half height of the man. The foot is one-seventh of the man. From below the foot to below the knee is a quarter of the man. From below the knee to the root of the member is a quarter of the man. The distances from the chin to the nose and the hairline and the eyebrows are equal to the ears and one-third of the face.“

Figure 20.11 Vitruvian man.

While not the first model of a human, it is surely the most beautiful and elaborate. The Vitruvian Man is a model that is an idealistic representation of reality. Similar to models of DNA, the molecular basis of heredity, intergenerational gene transfer, the cell cycle, the development of a monocellular body, aging, programmed cell death, photosynthesis, or social interactions, it attempts to explain known data and provide inspiration for useful predictions for the future. The adequacy of these models depends on the level of our knowledge. Thanks to model organisms, this level is increasing every day, but it is still not enough to make these predictions accurate. Therefore, we must take the undoubted achievements of contemporary biology with humility; what we do with our knowledge no longer depends on model organisms but is fully in our hands.

Did you know that...

...Tardigrades, also known as water bears, were first described in 1773 (Figure 20.12). These microscopic animals are surprisingly cute when observed under the microscope and are quite interesting to study. Tardigrades have been shown to survive at very cold temperatures (down to -273^oC) and at very hot temperatures of 150^oC – that is hotter than boiling water!! It is possible that these cute little water bears have been on Earth for over 600 millions years – before the dinosaurs. Know, scientists are using the amazing ability of tardigrades to survive space radiation as a model organism in astrobiological studies. Read more about tardigrades as a model organism here.

Figure 20.12 The tardigrade Hypsibius exemplars

Want to read more?

Your genome: A webpage listing the main characteristics of the most commonly used model organisms. In addition, the websitehas a lot of interesting information about the genomes of various organisms and in relation to human medicine.

iBiology: On this webpage there are several video lectures by biologists who use model organisms to study interesting biological phenomena.

Catalogue of life: An online catalogue of species of living organisms maintained by a group of taxonomists from around the world.

Nobel Prize: The official website of the Nobel Foundation, which contains all the information related to the Nobel Prizes and recordings of the lectures of the laureates.

Ankeny RA, Leonelli S. (2020). Model organisms. Elem Philos Biol https://doi.org/10.1017/9781108593014.

Goldstein B, King N. (2016). The future of cell biology: Emerging model organisms. Trends Cell Biol 26:818–824.

Katz PS. (2016) “Model organisms” in the light of evolution. Curr Biology 26:R649–R650.

Kleiber M. (1961) The fire of life. John Wiley & Sons Inc. New York.

Kováč L. (2000). Fundamental principles of cognitive biology. Evol Cogn 6:51–69.

Mora et al. (2011). How many species are there on Earth and in the ocean? PLOS Biol 9:e1001127.

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