Genetics

Genetics (from ancient Greek γενετικός, ghenetikós, “relating to birth,” from γένεσις ghénesis, “genesis, origin”) is the branch of biology that studies genes, heredity and genetic variability in living organisms. The field of study of genetics thus focuses on understanding the mechanisms underlying these phenomena, which have been known since antiquity, along with embryology, but were not explained until the 19th century, thanks to the pioneering work of Gregor Mendel, considered for this reason the father of genetics. For he was the first, although he did not know of the existence of chromosomes and meiosis, to attribute to “traits” inherited independently from parental individuals, the property of determining the phenotype of the individual. In a modern view, the genetic information of organisms is contained within the chemical structure of DNA molecules.

The Mendelian “traits” of the individual correspond to sequences of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) called genes found in the genome. Genes in fact contain the information to produce RNA molecules and proteins that enable the development and regulation of the traits to which they are related. Proteins are produced through transcription of DNA to RNA, which is transported to ribosomes by messenger RNA, which is translated into protein by the ribosomes. This process is known as the central dogma of molecular biology. Some genes are transcribed into RNA but do not become proteins, fulfilling fundamental biological functions.

Although genetics plays an important role in determining an individual’s appearance and behavior, it is his or her interaction with the environment that determines overall appearance. For this reason, identical twins, although having the same genetic makeup, may have different personalities.

From origins to Mendel

Until recent times, the problem of biological inheritance has been predominantly related to that of generation, that is, the ways in which an organism produces another organism similar to itself. In ancient times, according to Aristotle’s conception, it was assumed that it was the action of the soul inherent in the seed that guaranteed the unity of the species and thus generative similarity. According to other theories, due to Hippocrates and Democritus, particles detached from each organ of the body are collected in the seed, which reproduce its characteristics when the seeds of the two parents are fused, forming an organ similar to the parent organ. In the seventeenth century, the preformist conception was advanced that in the egg or sperm is contained in miniature the organism already formed and assumed to be a pre-existing germ created by God at the beginning of the world; parents and children therefore resemble each other because they are derived from germs created according to the same pattern. P.-L. Maupertuis and G. Buffon observed, however, that if the germ is either the egg or the spermatozoon, the child should resemble only the mother or only the father, which is contrary to experience, and taking up the conception of Hippocrates and Democritus they proposed the theory of organic molecules. C. Darwin in the nineteenth century also formulated such a hypothesis, but it was precisely with the development of the theory of evolution that the problem imposed itself in a new way. It emphasized the importance not so much of similarity as of dissimilarity from the parents due to variations affected by selection, a fact long known to breeders and horticulturists who, through appropriate crosses, had obtained new breeds. Such researches were conducted on the assumption that inheritance was continuous, that is, that the characters of the parents mixed in the descendants resulting in an intermediate value. Starting from this conception, F. Galton advocated the theory of ancestral inheritance whereby an individual would derive 1/4 of his characters from his parents, 1/16 from his grandparents, and so on. With statistical research, he also formulated the law of regression that a certain character, e.g., stature, tends to move away from that of the average parent and toward that of the average population.

The theories of Mendel

This theory, in the light of subsequent research, turned out to be unfounded, while the opposite theory, due to G. Mendel and formulated in an 1866 memoir, was confirmed, according to which hereditary traits are transmitted as units that remain constant and distinct in offspring. Mendel made crosses of plants of the same species distinguished by paired and contrasting characters (high-low, smooth-wrinkly) and by following the statistical distribution of these characters in successive generations he was able to identify his famous laws. He further admitted that each observed character corresponded to an element contained in reproductive cells capable of linking with another in a lasting or temporary way in descendants, without alteration. The hypothesis that hereditary characters were produced by material particles contained in reproductive cells, also advanced by A. Weissmann after 1880, was rejected by many as a speculative conception inspired by materialism and mechanism. Mendel’s laws therefore remained unknown and in any case not understood by the few who knew them until the early 20th century, when H. De Vries, C. Correns and E. von Tschermak confirmed their soundness with their work.

The twentieth century

From the early twentieth century onward it was a succession of discoveries and formulations of theories. K. E. Correns and T. Boveri in 1902, W. S. Sutton in 1903 pointed out the close parallelism between the behavior of chromosomes in gametogenesis and fertilization and the trend of Mendelian characters from one generation to the next. They thus came to formulate the “chromosomal theory of inheritance,” according to which the genes carrying inherited characters are located in chromosomes and transmitted with them, through gametes, from one generation to the next. Subsequently, other important discoveries were those made by T. H. Morgan and his students. Morgani discovered sex chromosomes, correctly interpreted sex-linked inheritance and formulated the concept of linkage (association) and crossing-over (exchange) of genes. Other discoveries followed, but perhaps the most resounding came in 1953, when J. D. Watson, F. H. Crick and M. Wilkins elucidated the structure of the nucleic acid molecule. The code by which information is recorded in DNA molecules was discovered by M. W. Nirenberg and S. Ochoa. Thus, the analysis of genetics has truly reached the basis of life phenomena and it is now possible to study how genes function and how they are expressed. § The fields of application of genetics are the most varied, from medical to chemical, pharmaceutical, industrial, agricultural and animal husbandry. Of long-standing tradition and experience is the obtaining in animal husbandry and agriculture of animal (transgenic animals) and plant (GMO, genetically modified organisms, obtained by genetic manipulation) varieties endowed with particular requirements and well adapted to breeding and production in particular environments (for example, the obtaining of short-legged cattle that less easily can escape from pens and that of numerous varieties of wheat each adapted to cultivation in particular places). Even more numerous are the research fields of genetics, three in particular have had very extensive developments: bacterial genetics, population genetics, and gene genetics.

Fields of application and research

Bacterial genetics

Studies the phenomena of transmission of hereditary traits in Bacteria and viruses, so it is more correct to speak of genetics of microorganisms. This field of research has assumed considerable importance because of the practicality with which it is possible to conduct work on living material and because of the very large number of individuals on which it is possible to experiment. These possibilities allow the discovery of genetic events that in other living things cannot be determined in vivo and that, often, are deferred in time so they become extremely rare to observe. It takes only a few agar plates (a kind of gelatin) to cultivate and assay billions of generations of Bacteria in a very short time; thus it has been established that they are capable of recombining their genetic material by three mechanisms: transformation, transduction and conjugation. The latter has been noticed mainly among colibacilli (Escherichia coli). If two cultures labeled, for example, with P+R genes one and PR+ genes the other are mixed, recombinant strains such as PR and P+R+ can be obtained. Today the main features of this phenomenon are clear, and it is known that when two bacteria conjugate, continuity is established between them by means of cytoplasmic bridges. At this point the injection of genetic material from one bacterium to the other takes place. It is customary to denote by F+ the donor bacterium (male) and by F the recipient bacterium (female). The bacterium that received the genetic material carries, for a time, a diploid set-up that subsequent division by cleavage changes back to haploid. Now in bacteria derived by cleavage from a bacterium that had behaved as F there may be recombinant types present, if crossing-over has occurred. Based on the above example, if P and R result in the inability to synthesize two substances “q” and “s,” the P+R strain of bacteria grows only if “s” is present in the laboratory culture, and the PR+ strain grows only if “q” is present. Among recombinant types PR grows only if both “q” and “s” are present, while P+R+ grows well even on normal (called minimal) culture medium in which both substance “q” and “s” are absent. It is thus possible to trace, from the frequencies of occurrence of the recombinant strains, the distance of the two P and R gene loci and locate (map) them on the chromosome of the Bacteria under investigation. Another method used to map the chromosome of Bacteria is one that relies on the time required for the transfer of gene complexes from F+ to F. In fact, the chromosome takes a certain amount of time to accomplish this step, and it is possible, by interrupting conjugation, for example by violent agitation, to determine the time required for the transfer of the various gene complexes. It has been conceded that the chromosome of Bacteria is circular and can open up (by the intervention of enzymes capable of cutting DNA) to initiate transfer now at one point now at another. This hypothesis was made because of the fact that it is not always the same gene complex that is transferred first. The electron microscope later confirmed this model because of the possibility it gave of photographing the various stages of the recombination mechanism under consideration. The enormous importance, from a practical point of view, of bacterial genetics lay until recently only in the possibility of obtaining strains of microorganisms capable of producing ever new types of antibiotics. Research in recent years, shedding new light on the concept of genes and the way they operate, has also opened up new possibilities for the study and treatment (gene therapy) of certain hereditary diseases and cancers.

Population genetics

Studies gene frequencies and how genes are distributed in natural populations; of course, it is also interested in the causes (mutation, selection, etc.) that determine variations in the frequencies themselves. One of the main purposes of population genetics is to elucidate the evolutionary mechanisms and evolutionary prospects of a population, since biological evolution is ultimately linked precisely to changes in gene frequencies. By identifying gene frequency variabilities within a natural population, population genetics studies groups of individuals (populations) that stand between the individual and the species as it believes they constitute the basic unit in which all evolutionary processes occur, or at least all those essential to the transformations of one species into one or more others. Analyses of variations in the gene structure of populations are carried out, today, not only by direct investigations within a population, but also by simulating with mathematical models what takes place in an ideal population, namely, an infinitely large population in which all individuals have an equal probability of mating, as well as even in the absence of mutation and selection. In such a population, equilibrium of genotype frequencies is established within a generation, whatever the initial gene frequencies were. Based on this model, it can be established that genetic equilibrium is achieved, i.e., a new species occurs, when frequencies remain unchanged in subsequent generations.

Another of the most topical problems in population genetics is the study of the causes of variations in gene frequencies among populations. There are two theoretical positions in this regard: the panselectionist and the panneutralist. According to the former, differences in allele frequencies are due to natural selection in that only certain alleles are more or less advantageous in a suitable environment for that population. An example of this is the gene for thalassemia that is frequent in populations in areas where malaria is prevalent and protects heterozygous carriers from the onset of this disease. The second, panneutralist hypothesis considers genes to be selectively neutral and variations in frequency in populations due to random factors. One example is the fact that many electrophoretic variants of proteins show no changes in functional properties, such that it is likely that there are selectively neutral or near-neutral alleles. However, there is no firm evidence to date in favor of either hypothesis. The significance to be attributed to this genetic variability in populations is the main point of discussion among various currents of evolutionary scholars.

Genetic genetics

Studies the behavior, biochemical structure and functions of genes in order to be able to directly intervene (manipulate) the genome of living things. From this opportunity comes the name “gene manipulation” given to this section of genetics. A new science called genetic engineering has even arisen to denote the set of technologies involving the manipulation of genetic material within and across species. This discipline has developed since 1970 when G. Khorana, J. Shapiro and others succeeded, with appropriate manipulation techniques, first to isolate a gene in its pure state, then to link it back to a precise biological function, and finally to “synthesize” it artificially, which is equivalent, within certain limits, to “creating” the elementary basis of life. The development of these techniques, which are based on knowledge of molecular biology, is so potentially disruptive that it led J. Shapiro to withdraw from such scientific work soon after his data were published. Thanks to new methodologies related to genetic manipulation and the ability to rapidly sequence DNA, genetics has undergone tremendous development in both basic knowledge and possible practical applications. With mutation analysis of many genes, it has been possible to study their function in depth and also to identify the genes responsible for many genetically transmissible diseases. Genes from complex organisms can be inserted into simpler organisms, and their role and structural features can be analyzed in great detail. The brewer’s yeast Saccharomyces cerevisiae represents one of the most studied genetic systems in recent years. It is a single-cell eukaryotic organism with a single chromosome set (haploid genome) of 17 chromosomes whose DNA has been completely sequenced in a collective effort of several European and U.S. laboratories (1996). This remarkable achievement makes it possible to identify new genes that regulate the life of this organism. Yeast reproduces by budding of a daughter cell from the mother cell in a simple cell cycle, but it can also have sexual reproduction when two cells with different sexual characteristics mate after a hormonal stimulus. This diploid phase makes possible genetic analysis of mutations in genes that code for essential proteins. Yeast can be transformed (introduction of DNA in the form of a plasmid) with DNA that contains mutated genes or genes from other organisms, such as humans. It is possible in this way to study the functions of human genes in an organism that behaves at the cellular level in a manner very similar to humans. Human genes can be mutagenized in vitro and introduced into yeast to study the effect of these mutations. Through these methods, many human genes have been characterized and structural features important to their function identified. The procedures that are used to identify and isolate mutations are called genetic screening and are generally designed to identify and isolate recessive mutations induced by treatment with mutagenic substances. In organisms with only one chromosome set (haploids), such as Bacteria and yeast, defects caused by these induced mutations can be seen immediately. A mutation in the only available copy of a given gene will indeed have a visible effect on the cell. In diploid (double chromosome set) organisms, the mutation will be visible only if it is present on both chromosome copies (homozygosity). Genes that code for essential proteins are the most important for a genetic analysis. Expression of mutations in essential genes leads to the death of the organism, and therefore, conditional mutants must be used to study the effects of these variations. A mutant protein may be functional at 30 °C, but completely inactive at 37 °C, while the normal protein would grow normally at both temperatures. Strains containing the mutations can be kept alive at the permissive temperature and then for genetic analysis grown at the nonpermissive temperature to study the effect of inactivation of the particular gene. An example of this type of analysis is mutations affecting the cell cycle in yeast. It is possible to follow under an optical microscope the growth and division of yeast that occurs by budding, and the size of the bud is indicative of the various stages of the cycle. At first, temperature-sensitive mutants were identified and later they were analyzed microscopically, in the search for defects in the cycle, at the non-permissive temperature. These mutants did not grow slower than nonmutagenized cells, showing that they did not report a generic metabolic impairment, but they all stopped at a particular position in the cycle. The result showed that the mutated gene product was needed at that particular stage of the cycle. This screening allowed the isolation of CDC (Cell Division Cycle) genes that regulate the cell cycle in many organisms. The phenomenon of gene suppression is used to identify proteins that specifically interact with each other within the cell. The principle of this analysis is based on the fact that a point mutation (replacement of a single amino acid in a protein) can induce structural changes in protein A so that it is no longer able to interact with protein B, which is involved in the same cellular process. However, mutations can occur in protein B that make it able to interact with mutated protein A. Thus, both genes are said to be mutated, but the mutation in B suppresses the A mutation. As we have seen, genetic analysis of a simple organism can give essential information to identify the genes responsible for certain phenotypes, however, it is essential to map precisely along the chromosomes the location of these genes and then isolate them and determine their sequence. The location of a mutation in a given chromosome is the first step in mapping. An example is recessive mutations in the X chromosome of Drosophila, but it can be extrapolated to humans. These X chromosome-related recessive mutations always show a sex-linked segregation pattern in various crosses. When crossed with normal homozygous females, males that carry the mutation produce normal offspring. All male offspring that come from a female homozygous for the mutation (on both chromosomes) will have a mutant phenotype, and all females will be heterozygous (one X from the father and one from the mother) and thus have a normal phenotype. However, the heterozygous females will act as carriers of the mutation and pass it on to 50% of the male offspring. This example shows that any recessive mutation that has a sex-linked segregation pattern can be mapped to the X chromosome.

Perspectives on gene genetics

Mutation types

Human genetic diseases show different patterns of inheritance depending on the type of mutation that causes them. Duchenne muscular dystrophy, a degenerative muscle disease that specifically affects males, is caused by a recessive mutation in the X chromosome and shows a typical sex-linked segregation pattern. Cystic fibrosis depends on a recessive mutation in one chromosome. This type of mutation has a very different segregation pattern. Males and females can be equally affected by the disease. Both parents must be heterozygous carriers of the mutated allele for their children to be at risk of the disease. Each child of heterozygous parents has a 25 percent chance of taking both mutated genes from the parents and thus being affected by the disease, a 50 percent chance of receiving one normal and one mutated allele and being a carrier, and a 25 percent chance of being normal. Huntington’s chorea, a degenerative disease of the nervous system that strikes in adulthood, on the other hand, is caused by a third segregation pattern: autosomal dominant mutations. If both parents are carriers, each child (despite gender) has a 50 percent chance of inheriting the mutated gene and being ill. Mapping an autosomal gene (on a specific chromosome) is certainly more complicated than mapping sex-linked mutations. Once the specific chromosome has been identified, one proceeds with linkage analysis (proximity of other genes or markers) to identify other known markers, on the same chromosome to draw a map. Through analysis of the recombination frequencies between two specific genes or markers, the distance of the markers themselves can be determined, and in fact the further apart two genes are on the same chromosome, the more likely it is that recombination will occur between them. Based on this principle of classical genetics, distance units can be established, and a genetic map unit corresponds to the distance between two positions that have a recombination frequency of 1% and is referred to as a centimorgan in honor of the great Drosophila geneticist, T. H. Morgan. In this organism 1 centimorgan corresponds to about 400 kilobases of DNA. These distances vary from organism to organism because they also depend on the composition of the genome itself.

Recombinant DNA

In experimental organisms commonly used for genetic analysis (Bacteria, yeast, and Drosophila) there are phenotypic markers to map mutations, but in the case of genes associated with communicable genetic diseases this is not possible. However, with the use of recombinant DNA techniques, there are now a large number of molecular markers among which the most widely used are the so-called RFLPs (Restriction Fragment Length Polymorphism). This technique is based on the principle that variations on DNA sequences occur throughout the genome, and since much of the genome does not code for proteins, fairly large sequence variability is possible even in humans. It has been estimated that a sequence change occurs approximately every 200 nucleotides. These changes are referred to as polymorphisms and can be referred to different analysis systems. If substitutions occur in the specific sequences recognized by restriction enzymes, the site in question will no longer be cut and thus a map of restriction sites in a specific region of the genome will contain fragments of different lengths. Based on such variations, it is possible to draw specific maps of the chromosome and associate a particular genetic disorder with a specific restriction fragment. What is the origin of polymorphisms is a very interesting problem in modern genetics. Many mutations form spontaneously along the DNA and can affect regions and sequences that code for proteins. In this case, if their effect is very severe, the mutations will not be passed on to offspring because the one who carries them will die. Others occur in noncoding regions and will have no effect on cellular functions and thus will be passed on without problem to subsequent generations. In humans, polymorphisms appear to be associated with single base changes in DNA. The dinucleotide CpG (Cytosine phosphor Guanine), for example, is a hot spot for single sequence changes, so restriction sites containing that sequence are often polymorphic. These polymorphic sequences are often organized repetitively, from 14 up to 70 base pairs, and are encountered on average along the genome, once every 40 kilobases. Using a molecular biology technique called PCR (polymerizing chain reaction), which is capable of many-fold amplification of DNA, it is possible to determine the exact location of these repeated sequences, in defined regions of DNA, and thus map genes that are adjacent to them. Many families can be identified that are at risk for genetic disease because both parents are heterozygous for a recessive mutation associated with a particular genetic disease. By analyzing the DNA of these families and studying the frequency with which the polymorphic marker (e.g., the CA sequence, repeated 14 times) segregates along with the disease-causing mutant gene, one has a measure of their closeness. The greater the number of families studied, the more precise the mapping of the disease-associated gene. However, because in humans the number of generations that can be analyzed in a family is limited (grandparents, children, grandchildren), the location of a particular disease is very approximate. To overcome these limitations, the use of a new strategy called linkage disequilibrium was introduced. The method is based on the principle that a genetic disease is most likely caused by a mutation that has occurred over many generations. The chromosome of this ancestor will contain markers very close to the mutated gene that will be passed on generation after generation. In contrast markers that are quite distant from the mutated gene will tend to become more and more distant due to recombination. By studying the distribution of specific markers in all diseased individuals in a population, it is possible to identify the genetic locus of the disease, in small regions. In recent years, this methodology has been widely used and, for example, in the Finnish population, where dystrophic dysplasia is common, it has been possible to locate the gene in a 60-kilobase region.

The prospects for gene genetics: gene isolation

Some genetic diseases can be associated with major changes in the structure of the chromosomes themselves, such as deletions of fragments large enough to be visible under a microscope, duplications or translocations of entire regions from one chromosome to another. This makes it possible to limit the region containing the genetic disease locus and use markers specific to that chromosome. In this way, T. Kunkel’s group at Harvard cloned the Duchenne muscular dystrophy gene whose locus is located on the X chromosome. Some patients with that disease showed a rather large deletion right in the X chromosome, and using genetic engineering techniques, the missing fragment was isolated. Then, by comparing the DNA of these patients with that of other patients, who did not show the deletion, it was possible to isolate the gene responsible for the disease. Cloning the muscular dystrophy gene now makes it possible to diagnose it in individuals at familial risk. The history of the study of Duchenne muscular dystrophy provides us with an example of a new methodology in modern genetics called reverse genetics. The deletion of the DNA fragment in the muscular dystrophy gene encodes for a protein later called dystrophin whose function was not known. After isolation of the gene using the techniques described above, it was possible to define the function of dystrophin and to establish the molecular and biochemical causes of the disease. With the sequencing of the entire genome of the yeast Saccharomyces cerevisiae and other organisms and the large amount of data already available for the human genome, reverse genetics represents the methodology to study the functions of unknown genes. Through site-specific recombination techniques, it is possible to inactivate the gene of interest and analyze its effects on the organism.The technique, called gene knockout (gene inactivation), is widely used in yeast and mice and is a very powerful tool for revealing the basic mechanisms of cellular processes. In the mouse, gene knockout enables the study of molecular mechanisms of development and behavior. Mutated or inactivated genes are introduced into embryonic stem cells, which are reintroduced into mouse embryos at the earliest stage of development. The resulting mice are called chimeras and will contain normal cells and cells with the mutated gene. These cells will help form both the germ line (sperm and eggs) and the somatic line. The resulting animals are paired together to determine whether the mutation of interest has been introduced into the germ line. Animals heterozygous for the mutation are paired together to produce a homozygote of the gene of interest. In addition to studying the functions of genes whose phenotype is unknown, this methodology is a very useful genetic tool for studying inherited diseases and developing new protocols for their treatment. After isolating the gene homologous to that in humans for cystic fibrosis, researchers produced mice homozygous for this mutation. These mice exhibit the same symptoms as the human disease and are an excellent model for studying possible therapies, such as gene therapy.

Among the more recent branches of genetics is genomics, which deals with the study of the genome of living organisms from the perspective of its structure, content and evolution. Underlying genomics are the methods of gene cloning and DNA sequencing. Particularly relevant is the Human Genome Project, which, initiated in 1986, led between 2001 and 2003 to the complete mapping of the human genome that opened up new perspectives in genetic research. Today, the boundary between genetics and related disciplines is as thin as ever and destined to become even thinner. In particular, with molecular biology and biochemistry, the areas of research are actually overlapping. In 2020, the scientific community focused the efforts of numerous players (universities, institutions, foundations, pharmaceutical companies) on genetic studies aimed at finding new diagnostic and therapeutic solutions against the Sars-CoV-2 virus responsible for the Covid-19 pandemic.

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