The discoveries of the exon-intron organization of eukaryotic genes and the possibility of alternative splicing have shown that the same nucleotide sequence of the primary transcript can provide the synthesis of several polypeptide chains with different functions or their modified analogs. For example, yeast mitochondria contain the box (or cob) gene, which encodes the cytochrome b respiratory enzyme. It can exist in two forms (Figure 3.42). The "long" gene consisting of 6400 bp has 6 exons with a total length of 1155 bp. and 5 introns. The short form of the gene consists of 3300 bp. and has 2 introns. It is actually a “long” gene devoid of the first three introns. Both forms of the gene are equally well expressed.

After the removal of the first intron of the "long" box gene, on the basis of the combined nucleotide sequence of the first two exons and part of the nucleotides of the second intron, a template is formed for an independent protein - RNA maturase (Fig. 3.43). The function of RNA maturase is to provide the next stage of splicing - the removal of the second intron from the primary transcript and, ultimately, the formation of a template for cytochrome b.

Another example is a change in the splicing pattern of the primary transcript that encodes the structure of antibody molecules in lymphocytes. The membrane form of antibodies has a long "tail" of amino acids at the C-terminus, which ensures the fixation of the protein on the membrane. The secreted form of antibodies does not have such a tail, which is explained by the deletion of the nucleotides encoding this region from the primary transcript during splicing.

In viruses and bacteria, a situation has been described where one gene can simultaneously be part of another gene, or a certain nucleotide sequence of DNA can be a component of two different overlapping genes. For example, on the physical map of the phage ФХ174 genome (Fig. 3.44), it can be seen that the B gene sequence is located within the A gene, and the E gene is part of the D gene sequence. This feature of the phage genome organization was able to explain the existing discrepancy between its relatively small size (it consists of 5386 nucleotides) and the number of amino acid residues in all synthesized proteins, which exceeds the theoretically allowable one for a given genome capacity. The possibility of assembling different peptide chains on mRNA synthesized from overlapping genes (A and B or E and D) is provided by the presence of ribosome binding sites within this mRNA. This allows the translation of another peptide to start from a new reference point.

The nucleotide sequence of gene B is simultaneously part of gene A, and gene E is part of gene D

In the genome of the λ phage, overlapping genes were also found, translated both with a shift in the frame and in the same reading frame. It is also assumed that two different mRNAs can be transcribed from both complementary strands of the same DNA region. This requires the presence of promoter regions that determine the movement of RNA polymerase in different directions along the DNA molecule.

The situations described, indicating the permissibility of reading different information from the same DNA sequence, suggest that overlapping genes are a fairly common element of the organization of the genome of viruses and, possibly, prokaryotes. In eukaryotes, gene discontinuity also enables the synthesis of a variety of peptides based on the same DNA sequence.

With all this in mind, it is necessary to amend the definition of a gene. Obviously, one can no longer speak of a gene as a continuous DNA sequence that uniquely encodes a certain protein. Apparently, at the present time, the most acceptable formula should still be considered "One gene - one polypeptide", although some authors propose to change it: "One polypeptide - one gene". In any case, the term gene should be understood as a functional unit of hereditary material, which by chemical nature is a polynucleotide and determines the possibility of synthesizing a polypeptide chain, tRNA or rRNA.

One gene, one enzyme.

In 1940, J. Beadle and Edward Tatum used a new approach to study how genes provide metabolism in a more convenient object of research - in the microscopic fungus Neurospora crassa. They obtained mutations in which; there was no activity of one or another metabolic enzyme. And this led to the fact that the mutant mushroom was not able to synthesize a certain metabolite itself (for example, the amino acid leucine) and could only live when leucine was added to nutrient medium... The theory "one gene - one enzyme", formulated by J. Bidl and E. Tatum, quickly gained wide acceptance among geneticists, and they themselves were awarded the Nobel Prize.

Methods. the selection of so-called "biochemical mutations" leading to disturbances in the action of enzymes that provide different pathways of metabolism proved to be very fruitful not only for science, but also for practice. First, they led to the emergence of genetics and selection of industrial microorganisms, and then to the microbiological industry, which uses strains of microorganisms that overproduce such strategically important substances as antibiotics, vitamins, amino acids, etc. the notion that "one gene encodes one enzyme." And although this idea is excellent practice brings multimillion-dollar profits and saves millions of lives (antibiotics) - it is not final. One gene is not just one enzyme.

First studies. After in 1902 Garrod pointed out the connection between the genetic defect in alkaptonuria and the inability of the body to break down homogentisic acid, it was important to elucidate the specific mechanism underlying this disorder. Since then it was already known that metabolic reactions are catalyzed by enzymes, it could be assumed that it is the violation of some enzyme that leads to alkaptonuria. This hypothesis was discussed by Driesch (in 1896). It was also expressed by Haldane (1920, see) and Garrod (1923). The important stages in the development of biochemical genetics were the work of Kyuchn and Butenandt on the study of eye color in the mill moth. Ephestia kuhniella and similar studies by Beadle and Ephrussi on Drosophila(1936). In these pioneering works, insect mutants previously studied by genetic methods were selected to elucidate the mechanisms of gene action. However, this approach was unsuccessful. The problem turned out to be too complex, and in order to solve it, it was necessary:

1) choose a simple model organism that is convenient for experimental study;

2) look for the genetic basis of biochemical traits, and not the biochemical basis of genetically determined traits. Both conditions were met by Beadle and Tatum in 1941 (see also Beadle 1945).

The Beadle and Tatum model. The article by these researchers began like this:

“From the point of view of physiological genetics, the development and functioning of an organism can be reduced to a complex system of chemical reactions, which are somehow controlled by genes. It is logical to assume that these genes ... either act as enzymes themselves, or determine their specificity. It is known that physiological geneticists usually try to investigate the physiological and biochemical bases of already known hereditary traits. This approach made it possible to establish that many biochemical reactions are controlled by specific genes. Such studies have shown that enzymes and genes are of the same order of magnitude. However, the possibilities of this approach are limited. The most serious limitation is that in this case, hereditary characters that do not have a lethal effect and, therefore, are associated with reactions that are not very essential for the vital activity of the organism, fall into the field of view of researchers. The second difficulty ... is that the traditional approach to the problem involves the use of externally manifested signs. Many of them represent morphological variations based on systems of biochemical reactions so complex that their analysis is extremely difficult.

Considerations like this led us to following conclusion... Study common problem genetic control of biochemical reactions that determine development and metabolism should be carried out using procedures opposite to the generally accepted one: instead of trying to figure out the chemical basis of known hereditary traits, it is necessary to establish whether genes control known biochemical reactions and how they do it. The neurospore related to ascomycetes possesses properties that make it possible to implement such an approach and, at the same time, serves as a convenient object for genetic research. This is why our program was based on the use of this particular organism. We assumed that X-ray irradiation causes mutations in genes that control certain chemical reactions. Suppose that in order to survive in a given environment, the organism must carry out some chemical reaction, then a mutant, deprived of such an ability, will be unviable under these conditions. However, it can be maintained and studied if grown in an environment to which the vital product of a genetically blocked reaction has been added. ”

4 Action of genes 9

Next, Beadle and Tatum describe the experimental setup (Figure 4.1). The complete medium consisted of agar, inorganic salts, malt extract, yeast extract, and glucose. The minimal medium contained only agar, salts, biotin, and a carbon source. The most studied mutants were those that grew on complete medium and did not grow on minimal. To establish the compound, the synthesis of which is disturbed in each of the mutants, the individual components of the complete medium were added to the minimal agar.

In this way, strains were isolated that were unable to synthesize certain growth factors: pyridoxine, thiamine, and para-aminobenzoic acid. These defects have been shown to be due to mutations at specific loci. The work marked the beginning of numerous studies on neurospore, bacteria and yeast, in which the correspondence of the "genetic blocks" responsible for individual metabolic stages was established, and specific violations enzymes. This approach quickly evolved into a tool that allows researchers to uncover metabolic pathways.

The hypothesis "one gene - one enzyme" received a strong experimental confirmation... As the work of the following decades showed, it turned out to be surprisingly fruitful. Analysis of defective enzymes and their normal variants soon revealed such a class of genetic disorders that led to a change in the function of the enzyme, although the protein itself was still detected and retained its immunological properties. In other cases, the temperature optimum of the enzyme activity changed. Some variants could be explained by a mutation that affects the general regulatory mechanism and, as a result, changes the activity of a whole group of enzymes. Such studies led to the creation of the concept of the regulation of gene activity in bacteria, which included the concept of the operon.

10 4. Action of genes

The first examples of enzymatic disorders in humans. The first hereditary human disease for which it was possible to show an enzymatic disorder was methemoglobinemia with a recessive mode of inheritance (Gibson and Harrison, 1947; Gibson, 1948) (25080). In this case, the damaged enzyme is NADH - dependent methemoglobin reductase. The first attempt at a systematic study of a group of human diseases associated with metabolic defects was made in 1951. In a study of glycogen storage disease, the Corey spouses showed that in eight out of ten cases of the pathological condition, which was diagnosed as Gierke's disease (23220), the structure of liver glycogen was a normal variant, and in two cases it was clearly impaired. It was also obvious that liver glycogen, accumulating in excess, cannot be directly converted into sugar, since patients tend to hypoglycemia. Many enzymes are required in the liver to break down glycogen to form glucose. Two of them, amylo-1,6-glucosidase and glucose-6-phosphatase, were selected for study as possible defective elements of the enzyme system. In liver homogenates with different meanings The pH was measured by the release of phosphate from glucose-6 phosphate. The results are shown in Fig. 4.2. The normal liver showed high activity with an optimum at pH 6-7. Severe liver dysfunction in cirrhosis correlated with a slight decrease in activity. On the other hand, in the case of Gierke's disease with a fatal outcome, the enzyme activity could not be detected at all; the same result was obtained when examining a second similar patient. In two patients with less severe symptoms, there was a significant decrease in activity.

It was concluded that in these cases of Gierke's disease with a fatal outcome, there was a defect in glucose-6-phosphatase. However, in most of the milder cases, the activity of this enzyme was not lower than in liver cirrhosis, and only in two patients it was somewhat lower (Fig. 4.2).

According to the Corey spouses, the abnormal accumulation of glycogen in muscle tissue cannot be associated with a lack of glucose-6-phosphatase, since this enzyme is absent in normal muscles. As possible explanation muscle glycogenosis, they suggested a violation of the activity of amylo-1,6-glucosidase. This prediction was soon confirmed: Forbes discovered such a defect in one of the clinically pronounced cases of glycogen storage disease involving the heart and skeletal muscles. Now we

4. Action of genes 11

known big number enzymatic defects in glycogen storage disease.

Although the degree of manifestation of the various forms of this disease is somewhat different, in the clinical relation between them there is much in common. With one exception, they are all inherited in an autosomal recessive manner. If the enzymatic defects were not disclosed, the pathology of glycogen accumulation would be considered as one disease with characteristic intrafamilial correlations in the severity of the course, the details of the symptoms, and the timing of death. Thus, we have an example when genetic heterogeneity, which could only be assumed on the basis of studying the phenotype (Section 3.3.5), was confirmed by analysis at the biochemical level: the study of enzymatic activity made it possible to identify specific genes.

In subsequent years, the pace of research into enzymatic defects increased, and for the 588 identified recessive autosomal genes that McCusick describes in the sixth edition of his book Mendelian Inheritance in Human (1983), more than 170 cases of specific enzymatic disorders were found. Our successes in this area are directly related to the development of concepts and methods of molecular genetics.

Some stages in the study of enzymatic disorders in humans. We present only the most important milestones in this ongoing process: 1934 Fölling discovered phenylketonuria

1941 Beadle and Tatum formulate the "one gene - one enzyme" hypothesis 1948 Gibson describes the first case of an enzymatic disorder in a disease in humans (recessive methemoglobinemia)

1952 The Corey couple discovered glucose-6-phosphatase deficiency in Gierke's disease

1953 Jervis demonstrates the absence of phenylalanine hydroxylase in phenylketonuria. Bickel reports first attempt to mitigate enzymatic disruption by adopting a diet low in phenylalanine

1955 Smithies develops starch gel electrophoresis

1956 Carson et al. Discovered a glucose-6-phosphate dehydrogenase (G6PD) defect in a case of induced hemolytic anemia

1957 Kalkar et al. Described enzymatic deficiency in galactosemia, showing that humans and bacteria exhibit an identical impairment of enzymatic activity

1961 Root and Weinberg demonstrate enzyme defect in galactosemia in vitro in fibroblast culture

1967 Sigmiller et al. Discovered a hypoxanthine-guanine phosphoribosyltransferase (HPRT) defect in Lesch-Nayhan syndrome

1968 Cleaver describes impairment of excisional repair in xeroderma pigmentosa

1970 Neufeld revealed enzymatic defects in mucopolysaccharidoses, which made it possible to identify pathways for the degradation of mucopolysaccharides

1974 Brown and Goldstein proved that genetically determined overproduction of hydroxymethylglutarylCoA reductase in familial hypercholesterolemia is due to a defect in the membrane-localized low-density lipoprotein receptor that modulates the activity of this enzyme (HMG)

1977 Sligh et al. Demonstrated that mannose-6-phosphate (as a component of lysosomal enzymes) is recognized by fibroblast receptors. A genetic processing defect prevents the binding of lysosomal enzymes, as a result of which their release into the cytoplasm and subsequent secretion into the plasma is impaired (I-cell disease)

12 4. Action of genes

1980 In pseudohypoparathyroidism, a defect in the protein that couples the receptor and cyclase was found.

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The One Gene-One Enzyme Hypothesis is an idea put forward in the early 1940s that each gene controls the synthesis or activity of one enzyme. The concept uniting the fields of genetics and biochemistry was proposed by the American geneticist George Wells Beadle and the American biochemist Edward L. Tatum, who conducted research on Neurospora crassa. Their experiments involved first visualizing the shape to mutation-inducing x-rays and then cultivating it in a minimal growth medium that contains only the essential nutrients necessary for the survival of the wild-type strain. They found that mutant mold strains require the addition of certain amino acids to grow. Using this information, the researchers were able to link mutations in certain genes to disruptions in certain enzymes in metabolic pathways which usually produced the missing amino acids. Today it is known that not all genes encode an enzyme and that some enzymes are composed of several short polypeptides encoded by two or more genes.

This happened in 1941. The "first geneticist" was a fungus with a romantic name - neurospore. Doesn't it sound nice? Moreover, the neurospore is very attractive in appearance. Place the mycelium of the fungus under a strong magnifying glass and admire: thin transparent lace ... You can spend hours looking at the fungus grown in a test tube, admiring the perfect creation of nature. Only American geneticists Beadle and Tatum looked at him as researchers, and not as domestic natural philosophers. Scientists have learned the intricacies of the structure of the fungus to make it work for genetics. And that's what made me happy. Neurospore is a haploid organism. She has only 7 chromosomes, and in ordinary life there are no cells with a double set in the mycelium of the fungus. This means that if a mutant gene appears in a fungus, the consequences of this will appear very soon - after all, the neurospore does not have the second gene, the dominant one!

But that's not all. In neurospores, you can find ... the sexual stage of development. At some point in life, special, "female" cells appear in the mycelium of the fungus. They, like all mycelium cells, are haploid, but unlike them, they are able to merge with any other cell, which thus plays the role of "male". This is how a diploid cell with a double set of chromosomes arises. There are now 14 of them.

At first, the nuclei of such a cell do not fuse, and it divides mitotically several times, forming an islet of diploid cells in the mycelium. By the way, maybe this islet is the "rough version" of nature when creating a multicellular diploid organism of animals and plants?

But in one of the diploid cells, the nuclei merge. In this case, the process of crossing over and reduction division occurs in the nucleus. In a word, the cell makes two meiotic divisions, after which four haploid cells are formed. They are arranged in a shell exactly in a row, like soldiers in a formation. Then each cell mitotically divides once more, and that's where it ends. As a result, 8 cells are formed (they are called ascospores), which are dressed in a shell.

Now let's imagine that a "trouble" happened to one of the genes of the mother's cell - it became mutant. After crossing over, which occurs after the fusion of the nuclei, two hybrid cells will develop, and a mutant gene will enter one of them. Such a cell will also give offspring - four ascospores. The pouch will contain two genetically different types of ascospores. How do you know if there are mutant among them? This is what Beadle and Tatum did. They learned to pick ascospores from the bag and plant them one at a time on a nutrient medium. From each ascospore, after a whole cycle of mitotic divisions, a mycelium grows - its direct descendant. If we compare the properties of mycelium from various ascospores, we can distinguish mutant and normal ones among them.

Here we must say about one more wonderful quality of neurospore.

It is extremely unpretentious and grows well in a nutrient-poor, so-called "minimal" or "hungry" environment (several inorganic salts, glucose, ammonium nitrate and vitamin biotin). From these products, the normal fungus synthesizes all the amino acids, proteins, carbohydrates and vitamins it needs, except for biotin.

But here on one of the genes, scientists "hit" with ultraviolet or X-rays, and it became mutant. If the ability to synthesize any vital amino acid was associated with it, this would be immediately revealed: some ascospores - the descendants of the female cell - will stop growing in a hungry environment. And don't wait for hundreds of generations of the fungus After all, the ascospore does not have a second gene that compensates for the impaired function: its offspring, as we have already said, is haploid, that is, it contains only one set of chromosomes.

It remains to find out which vital function is affected. Beadle and Tatum decided to add different amino acids, vitamins, salts, etc., to the starving environment one by one, and plant whole herds of ascospores there. At last! One of the ascospores germinated on starvation medium with arginine, the other - on medium with tryptophan. This means that the first did not grow because it was not able to create a single molecule of arginine, the second - tryptophan. There is only one reason - on the chromosome of the ascospore, the gene that "controls" the synthesis of tryptophan is affected. In approximately this way, Beadle and Tatum found 380 mutants (!) That carried mutations in 100 separate genes that control vital biochemical reactions.

And here's the curious thing. Several mutants were found for each gene. So, the gene responsible for the synthesis of tryptophan accounted for 30 mutants. Are they all the same? Is everyone's ability to synthesize tryptophan impaired in one place in the gene? To answer this question, scientists crossed all 30 mutants with each other.

In these experiments, the mutants were divided into two groups. The mutants of the first group mutually complemented the mutants of the second group during crossing over. As a result, among ascospores, wild-type * recombinants synthesizing tryptophan were found. This means that two genes must take part in the synthesis of tryptophan: one gene is affected in the mutants of the first group, and the other in the mutants of the second group. But what do these genes control?

* (This is the name of the type that is not changed by mutations, which is most often found in vivo.)

Mutants of both groups grew if serine and indole were added instead of tryptophan, and tryptophan appeared in the medium. This means that all mutants could convert indole and serine into tryptophan. Hence the conclusion: indole and series are tryptophan precursors in the chain of its biosynthesis in a living cell.

This assumption was confirmed when a mutant was found in which this particular function was blocked. It did not produce the enzyme tryptophan synthetase, which is found in the wild neurospore.

The mutants of the first group were also able to synthesize a substance that stimulated the growth of the mutants of the second group. This substance turned out to be anthranilic acid, which apparently acts as a precursor for indole. This means that in the mutants of the first group, the reaction of the conversion of anthranilic acid into indole is disrupted, and the mutants of the second group cannot synthesize anthranilic acid, but are able to convert it into indole.

Based on these data, a method for the synthesis of tryptophan in living cells was discovered: anthranilic acid is converted into indole. Indole combines with serine and, under the influence of the enzyme tryptophan synthetase, is converted into tryptophan. At least three genes are involved in the synthesis of tryptophan, each of them is responsible for the production of enzymes. These genes can be mapped onto the chromosome of neurospores in cross-breeding reactions.

So in 1941, for the first time in the history of natural science, scientists found genes on the chromosome responsible for the synthesis of proteins - enzymes. Beadle and Tatum formulated their findings as follows: "One gene - one enzyme." It is assumed that the genes of a cell control the synthesis of all its enzymes that catalyze metabolic reactions, with each gene controlling only one enzyme.

If you think about it, you can imagine that the scope of this hypothesis is much broader than its name suggests. Indeed. We know that all enzymes are proteins. But in addition to enzymes, the body contains non-enzyme proteins. These are hemoglobin, antibodies and others. Where is the information for their synthesis stored? Also in chromosomal genes. That is why the hypothesis "One gene - one enzyme" now sounds like this: "One gene - one protein", or even: "One gene - one gulipeptide chain."

Until 1941, genetics and biochemistry were separate sciences, and each, by virtue of its capabilities, tried to find the key to the secrets of life: geneticists discovered genes, biochemists, enzymes. The experiments of American scientists Beadle, Tatum and Brenner linked these two units of life together and laid the foundation for the commonwealth of genetics and biochemistry, and at the same time such a progress of knowledge, which had no equal in the entire history of biology. The gene appeared as a specific unit that controls the synthesis of a specific protein. It was high quality new level research.

Experiments with neurospore inspired scientists, but still required an answer to the questions: what is a gene? What substance is it built from? How does it regulate protein synthesis?

Genetics solved these puzzles of nature only after it began to search in the kingdom of bacteria. But before starting the story about the new heroes of genetic experiments, we must finally get to know them better.

Genetics- Science is by no means young, research in it has been carried out for several centuries, from Mendel in 1865 to the present day. The term "gene" to denote a unit of hereditary characteristic was first proposed by Johannsen in 1911, and in the 1940s it was refined by the concept of "one gene - one enzyme", which was proposed by Tatum and Beadle.

This position was determined in experiments on fruit flies, but it applies equally to humans; ultimately, the life of all beings is determined by their DNA. The DNA molecule in humans is larger than in all other organisms, and it is more complex, but the essence of its functions is the same for all living things.

Concept “ one gene - one enzyme", Which arose on the basis of the ideas of Tatum and Beadle, can be formulated as follows:
1. All biological processes are under genetic control.
2. All biochemical processes occur in the form of stepwise reactions.
3. Each biochemical reaction is ultimately under the control of different distinct genes.
4. A mutation in a certain gene leads to a change in the ability of the cell to carry out a certain chemical reaction.

Since then, the concept of "one gene - one enzyme" has expanded somewhat, and now sounds like " one gene - one protein". In addition, recent research suggests that some genes work in conjunction with others to produce unique proteins, that is, some genes can encode more than one protein.

Human genome contains about 3 billion base pairs; it is believed to contain between 50,000 and 100,000. After deciphering the genome, it turned out that there are only about 30,000 genes. The interaction of these genes is much more complicated than expected. Genes are encoded in strands of DNA, which, in combination with certain nuclear proteins, form chromosomes.

Genes- not just pieces of DNA: they are formed by coding sequences - exons, interspersed with non-coding sequences - nitrons. Exons, as an expressed part of DNA, constitute only a small part of the most important molecule of the organism; most of it is not expressed, is formed by nitrons and is often called "silent" DNA.

Approximate size and structure human genome are presented in the figure below. The functional length of the human chromosome is expressed in centimeters. Santimorganida (CM) - the distance over which the probability of crossing over during meiosis is 1%. Gene linkage analysis showed that the human genome is about 3000 cM long.

Average chromosome contains approximately 1,500 genes encoded in 130 million base pairs. The figure below shows schematically the physical and functional dimensions of the genome: the first is calculated in nucleotide pairs, and the second in cm. Most of the human genome is silent DNA and is not expressed.

On the DNA matrix as a result of the transcription process, RNA is synthesized, and then protein. Therefore, the DNA sequence completely determines the sequence of the functional proteins of the cell. All proteins are synthesized as follows:
DNA => RNA => protein

The genetic apparatus of humans and other mammals is more complex than that of other living organisms, since parts of some genes in mammals can combine with parts of others genes as a result of which a completely new protein is synthesized or a separate cellular function is controlled.

Therefore, in humans, it is possible to increase the number of expressed genes without actually increasing the volume of expressed DNA or the absolute number of genes.
In general, about 70% of all genetic material is not expressed.