Molecular biology as a science lecture by Ph.D. tazabaeva k.a

Molecular biology

a science that sets as its task the knowledge of the nature of life phenomena by studying biological objects and systems at a level approaching the molecular level, and in some cases reaching this limit. The ultimate goal in this case is to clarify how and to what extent the characteristic manifestations of life, such as heredity, reproduction of one's own kind, protein biosynthesis, excitability, growth and development, storage and transmission of information, energy transformations, mobility, etc. , are due to the structure, properties and interaction of molecules of biologically important substances, primarily the two main classes of high molecular weight biopolymers (See Biopolymers) - proteins and nucleic acids. A distinctive feature of M. b. - the study of the phenomena of life on inanimate objects or those that are characterized by the most primitive manifestations of life. These are biological formations from the cellular level and below: subcellular organelles, such as isolated cell nuclei, mitochondria, ribosomes, chromosomes, cell membranes; further on - systems that stand on the border of animate and inanimate nature - viruses, including bacteriophages, and ending with the molecules of the most important components of living matter - nucleic acids (See Nucleic acids) and proteins (See Proteins).

M. b. - a new field of natural science, closely related to long-established areas of research, which are covered by biochemistry (See Biochemistry), biophysics (See Biophysics) and bioorganic chemistry (See Bioorganic Chemistry). The distinction here is possible only on the basis of taking into account the methods used and the fundamental nature of the approaches used.

The foundation on which M. developed. was laid by such sciences as genetics, biochemistry, physiology of elementary processes, etc. According to the origins of its development, M. b. inextricably linked with molecular genetics (See Molecular Genetics) , which continues to make up an important part of M. banking, although it has already formed to a large extent into an independent discipline. M.'s isolation. from biochemistry is dictated by the following considerations. The tasks of biochemistry are mainly limited to ascertaining the participation of certain chemical substances in certain biological functions and processes and elucidating the nature of their transformations; the leading value belongs to information about the reactivity and about the main features of the chemical structure, expressed by the usual chemical formula. Thus, in essence, attention is focused on transformations involving principal-valent chemical bonds. Meanwhile, as was emphasized by L. Pauling , in biological systems and manifestations of vital activity, the main importance should be given not to principal-valent bonds acting within the same molecule, but to various types of bonds that determine intermolecular interactions (electrostatic, van der Waals, hydrogen bonds, etc.).

The end result of a biochemical study can be represented in the form of a system of chemical equations, usually completely exhausted by their representation on a plane, that is, in two dimensions. A distinctive feature of M. b. is its three-dimensionality. The essence of M. b. M. Perutz sees it in interpreting biological functions in terms of molecular structure. We can say that if before, when studying biological objects, it was necessary to answer the question “what”, that is, what substances are present, and the question “where” - in which tissues and organs, then M. b. makes it his task to get answers to the question “how”, having learned the essence of the role and participation of the entire structure of the molecule, and to the questions “why” and “what for”, having found out, on the one hand, the connections between the properties of the molecule (again, primarily proteins and nucleic acids) and the functions it performs and, on the other hand, the role of such individual functions in the overall complex of manifestations of vital activity.

The mutual arrangement of atoms and their groupings in the general structure of the macromolecule, their spatial relationships acquire a decisive role. This applies to both individual, individual components, and the overall configuration of the molecule as a whole. It is as a result of the emergence of a strictly determined volumetric structure that biopolymer molecules acquire those properties, due to which they are able to serve as the material basis of biological functions. This principle of approach to the study of the living is the most characteristic, typical feature of M. b.

Historical reference. The great importance of studying biological problems at the molecular level was foreseen by I. P. Pavlov , who spoke about the last step in the science of life - the physiology of the living molecule. The very term "M. b." was first used in English. scientists W. Astbury in application to research related to elucidating the relationship between the molecular structure and the physical and biological properties of fibrillar (fibrous) proteins, such as collagen, blood fibrin, or contractile muscle proteins. Widely use the term "M. b." steel since the early 1950s. 20th century

M.'s emergence. as a mature science, it is customary to refer to 1953, when J. Watson and F. Crick in Cambridge (Great Britain) discovered the three-dimensional structure of deoxyribonucleic acid (DNA). This made it possible to speak about how the details of this structure determine the biological functions of DNA as a material carrier of hereditary information. In principle, this role of DNA became known somewhat earlier (1944) as a result of the work of the American geneticist O. T. Avery and coworkers (see Molecular Genetics), but it was not known to what extent this function depends on the molecular structure of DNA. This became possible only after the laboratories of W. L. Bragg, J. Bernal, and others developed new principles of X-ray diffraction analysis, which ensured the use of this method for a detailed knowledge of the spatial structure of protein macromolecules and nucleic acids.

Levels of molecular organization. In 1957, J. Kendrew established the three-dimensional structure of Myoglobin a , and in subsequent years, this was done by M. Perutz in relation to Hemoglobin a. Ideas about different levels of spatial organization of macromolecules were formulated. The primary structure is a sequence of individual units (monomers) in the chain of the resulting polymer molecule. For proteins, the monomers are amino acids. , for nucleic acids - Nucleotides. A linear, filamentous molecule of a biopolymer, as a result of the occurrence of hydrogen bonds, has the ability to fit in space in a certain way, for example, in the case of proteins, as shown by L. Pauling, it can take the form of a spiral. This is referred to as a secondary structure. Tertiary structure is said to be when a molecule that has a secondary structure further folds in one way or another, filling three-dimensional space. Finally, molecules that have a three-dimensional structure can enter into interaction, regularly located in space relative to each other and forming what is designated as a quaternary structure; its individual components are commonly referred to as subunits.

The most obvious example of how a molecular three-dimensional structure determines the biological functions of a molecule is DNA. It has the structure of a double helix: two threads running in a mutually opposite direction (antiparallel) are twisted one around the other, forming a double helix with a mutually complementary arrangement of bases, i.e. so that against a certain base of one chain there is always such a the base that best provides the formation of hydrogen bonds: adepine (A) pairs with thymine (T), guanine (G) with cytosine (C). Such a structure creates optimal conditions for the most important biological functions of DNA: the quantitative multiplication of hereditary information in the process of cell division, while maintaining the qualitative immutability of this flow of genetic information. When a cell divides, the strands of the DNA double helix, which serves as a template, or template, unwind, and on each of them, under the action of enzymes, a complementary new strand is synthesized. As a result of this, two completely identical daughter molecules are obtained from one parent DNA molecule (see Cell, Mitosis).

Similarly, in the case of hemoglobin, it turned out that its biological function - the ability to reversibly attach oxygen in the lungs and then give it to tissues - is closely related to the features of the three-dimensional structure of hemoglobin and its changes in the process of implementing its physiological role. When binding and dissociating O 2, spatial changes in the conformation of the hemoglobin molecule occur, leading to a change in the affinity of the iron atoms contained in it for oxygen. Changes in the size of the hemoglobin molecule, reminiscent of changes in the volume of the chest during breathing, made it possible to call hemoglobin "molecular lungs".

One of the most important features of living objects is their ability to finely regulate all manifestations of vital activity. M.'s major contribution. scientific discoveries should be considered the discovery of a new, previously unknown regulatory mechanism, referred to as the allosteric effect. It lies in the ability of substances of low molecular weight - the so-called. ligands - to modify the specific biological functions of macromolecules, primarily catalytically acting proteins - enzymes, hemoglobin, receptor proteins involved in the construction of biological membranes (See Biological membranes), in synaptic transmission (see Synapses), etc.

Three biotic streams. In the light of M.'s ideas. the totality of the phenomena of life can be considered as the result of a combination of three flows: the flow of matter, which finds its expression in the phenomena of metabolism, i.e., assimilation and dissimilation; the flow of energy, which is the driving force for all manifestations of life; and the flow of information, penetrating not only the whole variety of processes of development and existence of each organism, but also a continuous series of successive generations. It is the idea of ​​the flow of information, introduced into the doctrine of the living world by the development of biomaterials, that leaves its own specific, unique imprint on it.

The most important achievements of molecular biology. Swiftness, scope and depth of M.'s influence. progress in understanding the fundamental problems of the study of living nature is rightly compared, for example, with the influence of quantum theory on the development of atomic physics. Two intrinsically related conditions determined this revolutionary impact. On the one hand, a decisive role was played by the discovery of the possibility of studying the most important manifestations of vital activity under the simplest conditions, approaching the type of chemical and physical experiments. On the other hand, as a consequence of this circumstance, there was a rapid involvement of a significant number of representatives of the exact sciences - physicists, chemists, crystallographers, and then mathematicians - in the development of biological problems. In their totality, these circumstances determined the unusually rapid pace of development of M. b., the number and significance of its successes, achieved in just two decades. Here is a far from complete list of these achievements: disclosure of the structure and mechanism of the biological function of DNA, all types of RNA and ribosomes (See Ribosomes) , disclosure of the genetic code (See genetic code) ; discovery of reverse transcription (See transcription) , i.e. DNA synthesis on an RNA template; study of the mechanisms of functioning of respiratory pigments; discovery of a three-dimensional structure and its functional role in the action of enzymes (See Enzymes) , the principle of matrix synthesis and mechanisms of protein biosynthesis; disclosure of the structure of viruses (See Viruses) and the mechanisms of their replication, the primary and, in part, the spatial structure of antibodies; isolation of individual genes , chemical and then biological (enzymatic) gene synthesis, including human, outside the cell (in vitro); transfer of genes from one organism to another, including into human cells; the rapidly progressing deciphering of the chemical structure of an increasing number of individual proteins, mainly enzymes, as well as nucleic acids; discovery of the phenomena of "self-assembly" of some biological objects of ever-increasing complexity, starting from nucleic acid molecules and moving on to multicomponent enzymes, viruses, ribosomes, etc.; elucidation of allosteric and other basic principles of regulation of biological functions and processes.

Reductionism and integration. M. b. is the final stage of that direction in the study of living objects, which is designated as "reductionism", i.e., the desire to reduce complex life functions to phenomena occurring at the molecular level and therefore accessible to study by the methods of physics and chemistry. Achieved M. b. successes testify to the effectiveness of this approach. At the same time, it must be taken into account that in natural conditions in a cell, tissue, organ, and the whole organism, we are dealing with systems of increasing complexity. Such systems are formed from lower-level components through their regular integration into wholes, acquiring a structural and functional organization and possessing new properties. Therefore, as the knowledge of patterns available for disclosure at the molecular and adjacent levels is detailed, before M. b. the task of understanding the mechanisms of integration as a line of further development in the study of the phenomena of life arises. The starting point here is the study of the forces of intermolecular interactions - hydrogen bonds, van der Waals, electrostatic forces, etc. By their combination and spatial arrangement, they form what can be designated as "integrative information". It should be considered as one of the main parts of the already mentioned flow of information. In M.'s area. examples of integration can be the phenomena of self-assembly of complex formations from a mixture of their constituent parts. This includes, for example, the formation of multicomponent proteins from their subunits, the formation of viruses from their constituent parts - proteins and nucleic acids, the restoration of the original structure of ribosomes after the separation of their protein and nucleic components, etc. The study of these phenomena is directly related to the knowledge of the main phenomena " recognition” of biopolymer molecules. The point is to find out what combinations of amino acids - in protein molecules or nucleotides - in nucleic acids interact with each other during the processes of association of individual molecules with the formation of complexes of a strictly specific, predetermined composition and structure. These include the processes of formation of complex proteins from their subunits; further, selective interaction between nucleic acid molecules, for example, transport and matrix (in this case, the discovery of the genetic code has significantly expanded our information); finally, this is the formation of many types of structures (for example, ribosomes, viruses, chromosomes), in which both proteins and nucleic acids participate. The disclosure of the corresponding laws, the knowledge of the “language” underlying these interactions, is one of the most important areas of mathematical linguistics, which is still awaiting development. This area is considered as belonging to the number of fundamental problems for the entire biosphere.

Problems of molecular biology. Along with the specified important tasks M. would. (knowledge of the laws of "recognition", self-assembly and integration) the actual direction of scientific search for the near future is the development of methods that allow deciphering the structure, and then the three-dimensional, spatial organization of high-molecular nucleic acids. This has now been achieved with respect to the general plan of the three-dimensional structure of DNA (double helix), but without exact knowledge of its primary structure. Rapid progress in the development of analytical methods allows us to confidently expect the achievement of these goals over the coming years. Here, of course, the main contributions come from representatives of related sciences, primarily physics and chemistry. All the most important methods, the use of which ensured the emergence and success of M. b., were proposed and developed by physicists (ultracentrifugation, X-ray diffraction analysis, electron microscopy, nuclear magnetic resonance, etc.). Almost all new physical experimental approaches (for example, the use of computers, synchrotron, or bremsstrahlung, radiation, laser technology, and others) open up new possibilities for an in-depth study of the problems of M. b. Among the most important tasks of a practical nature, the answer to which is expected from M. b., in the first place is the problem of the molecular basis of malignant growth, then - ways to prevent, and perhaps overcome hereditary diseases - "molecular diseases" (See Molecular diseases ). Of great importance will be the elucidation of the molecular basis of biological catalysis, ie, the action of enzymes. Among the most important modern directions of M. b. should include the desire to decipher the molecular mechanisms of action of hormones (See. Hormones) , toxic and medicinal substances, as well as to find out the details of the molecular structure and functioning of such cellular structures as biological membranes involved in the regulation of the processes of penetration and transport of substances. More distant goals M. b. - knowledge of the nature of nervous processes, memory mechanisms (See Memory), etc. One of the important emerging sections of M. b. - so-called. genetic engineering, which sets as its task the purposeful operation of the genetic apparatus (Genome) of living organisms, starting with microbes and lower (single-celled) and ending with humans (in the latter case, primarily for the purpose of radical treatment of hereditary diseases (See. Hereditary diseases) and the correction of genetic defects ). More extensive interventions in the human genetic basis can only be discussed in a more or less distant future, since in this case serious obstacles, both technical and fundamental, arise. Concerning microbes, plants, and it is possible, and page - x. For animals, such prospects are very encouraging (for example, obtaining varieties of cultivated plants that have an apparatus for fixing nitrogen from the air and do not need fertilizers). They are based on the successes already achieved: the isolation and synthesis of genes, the transfer of genes from one organism to another, the use of mass cell cultures as producers of economic or medically important substances.

Organization of research in molecular biology. M.'s rapid development. led to the emergence of a large number of specialized research centers. Their number is growing rapidly. The largest: in the UK - the Laboratory of Molecular Biology in Cambridge, the Royal Institute in London; in France - institutes of molecular biology in Paris, Marseille, Strasbourg, the Pasteur Institute; in the USA - departments M. b. at universities and institutes in Boston (Harvard University, Massachusetts Institute of Technology), San Francisco (Berkeley), Los Angeles (California Institute of Technology), New York (Rockefeller University), health institutes in Bethesda, etc.; in Germany - Max Planck institutes, universities in Göttingen and Munich; in Sweden, the Karolinska Institute in Stockholm; in the GDR - the Central Institute for Molecular Biology in Berlin, institutes in Jena and Halle; in Hungary - Biological Center in Szeged. In the USSR the first specialized institute M. would be. was created in Moscow in 1957 in the system of the Academy of Sciences of the USSR (see. ); then the following were formed: the Institute of Bioorganic Chemistry of the Academy of Sciences of the USSR in Moscow, the Institute of Protein in Pushchino, the Biological Department at the Institute of Atomic Energy (Moscow), and the departments of M. b. at the institutes of the Siberian Branch of the Academy of Sciences in Novosibirsk, the Interdepartmental Laboratory of Bioorganic Chemistry of the Moscow State University, the Sector (later the Institute) of Molecular Biology and Genetics of the Academy of Sciences of the Ukrainian SSR in Kyiv; significant work on M. b. is conducted at the Institute of Macromolecular Compounds in Leningrad, in a number of departments and laboratories of the Academy of Sciences of the USSR and other departments.

Along with individual research centers, organizations of a wider scale arose. In Western Europe, the European Organization for M. arose. (EMBO), in which more than 10 countries participate. In the USSR, in 1966, at the Institute of Molecular Biology, a Scientific Council on M. B. was established, which is the coordinating and organizing center in this field of knowledge. He published an extensive series of monographs on the most important sections of M. b., “winter schools” on M. b. are regularly organized, conferences and symposiums are held on topical problems of M. b. In the future, scientific advice on M. would. were created at the Academy of Medical Sciences of the USSR and many republican Academies of Sciences. The journal Molecular Biology has been published since 1966 (6 issues per year).

For rather short term in the USSR the considerable group of researchers in the field of M. has grown; these are scientists of the older generation who have partially switched their interests from other fields; for the most part, they are numerous young researchers. From among the leading scientists who took an active part in the formation and development of M. b. in the USSR, one can name such as A. A. Baev, A. N. Belozersky, A. E. Braunshtein, Yu. A. Ovchinnikov, A. S. Spirin, M. M. Shemyakin, V. A. Engelgardt. M.'s new achievements. and molecular genetics will be promoted by the resolution of the Central Committee of the CPSU and the Council of Ministers of the USSR (May 1974) "On measures to accelerate the development of molecular biology and molecular genetics and the use of their achievements in the national economy."

Lit.: Wagner R., Mitchell G., Genetics and metabolism, trans. from English, M., 1958; Szent-Gyorgy and A., Bioenergetics, trans. from English, M., 1960; Anfinsen K., Molecular basis of evolution, trans. from English, M., 1962; Stanley W., Valens E., Viruses and the nature of life, trans. from English, M., 1963; Molecular genetics, trans. With. English, part 1, M., 1964; Volkenstein M.V., Molecules and life. Introduction to molecular biophysics, M., 1965; Gaurowitz F., Chemistry and functions of proteins, trans. from English, M., 1965; Bresler S. E., Introduction to molecular biology, 3rd ed., M. - L., 1973; Ingram V., Biosynthesis of macromolecules, trans. from English, M., 1966; Engelhardt V. A., Molecular biology, in the book: Development of biology in the USSR, M., 1967; Introduction to molecular biology, trans. from English, M., 1967; Watson, J., Molecular Biology of the Gene, trans. from English, M., 1967; Finean J., Biological ultrastructures, trans. from English, M., 1970; Bendoll, J., Muscles, Molecules, and Movement, trans. from English, M., 1970; Ichas M., Biological code, trans. from English, M., 1971; Molecular biology of viruses, M., 1971; Molecular bases of protein biosynthesis, M., 1971; Bernhard S., Structure and function of enzymes, trans. from English, M., 1971; Spirin A. S., Gavrilova L. P., Ribosome, 2nd ed., M., 1971; Frenkel-Konrat H., Chemistry and biology of viruses, trans. from English, M., 1972; Smith C., Hanewalt F., Molecular Photobiology. Processes of inactivation and recovery, trans. from English, M., 1972; Harris G., Fundamentals of human biochemical genetics, trans. from English, M., 1973.

V. A. Engelhardt.

Great Soviet Encyclopedia. - M.: Soviet Encyclopedia. 1969-1978 .

I will briefly recall the so-called central dogma of molecular biology, originally formulated by Francis Crick. In general, it states that genetic information is transferred from nucleic acids to proteins during implementation, but not vice versa. More specifically, it is possible to transfer DNA → DNA ( replication), DNA → RNA ( transcription) and RNA → protein ( broadcast). There are also much less common ways that are characteristic of some viruses: RNA → DNA ( reverse transcription) and RNA → RNA ( RNA replication). Let me also remind you that proteins consist of amino acid residues, the sequence of which is encoded in the genetic code of the organism: three nucleotides (they are called codon, or triplet) code for one amino acid, and the same amino acid can be coded for by several codons.

In the second half of the 20th century, technologies were developed recombinant DNA(that is, DNA manipulation methods that allow changing the sequence and composition of nucleotides in a molecule in various ways). It is on their basis that all molecular biological methods are being developed to this day, although they have become much more complex, both ideologically and technologically. It is molecular biology that has caused such a rapid growth in the amount of biological information over the past half century.

I will talk about the methods of manipulating and studying DNA and RNA, and touch on proteins quite a bit, since in general the methods associated with them are closer to biochemistry than to molecular biology (although the line between them has recently become very blurry).

Cutting and stitching

Enzymes are proteins that speed up chemical reactions. They are very efficient: the acceleration can be several orders of magnitude! For example, an enzyme catalase, splitting hydrogen peroxide, speeds up the reaction by about 12 orders of magnitude, that is, a trillion times! At the same time, an inorganic catalyst - finely dispersed platinum - accelerates the same reaction by only six orders of magnitude, or a million times. However, this comes at the cost of very strict working conditions for most of them.

Restriction endonucleases

Figure 2. Restriction sites. Above Sma I, during the work of which "blunt" ends are formed. Bottom- restriction enzyme target sequence Eco RI, during which "sticky" ends are formed.

One of the first and most important steps in molecular biology was the ability to cut DNA molecules, and in strictly defined places. This method was invented during the study in the 1950s-1970s of such a phenomenon: some types of bacteria, when foreign DNA was added to the environment, destroyed it, while their own DNA remained intact. It turned out that they use enzymes for this, later called restriction nucleases or restrictases. There are many types of restrictases: by 2007, more than 3,000 were known. An important property of each such enzyme is its ability to cut a strictly defined - target- DNA nucleotide sequence (Fig. 2). Restriction enzymes do not affect the cell's own DNA, because the nucleotides in the target sequences are modified so that the restriction enzyme cannot work with them. (True, sometimes, on the contrary, they can cut only modified sequences - to combat those who modify DNA, protecting themselves from the above restriction enzymes.) Due to the fact that the target sequences are of different lengths, their frequency of occurrence in DNA molecules varies: the longer the required fragment, the less likely it is to appear. Accordingly, the DNA fragments formed during treatment with various restrictases will have different lengths.

New endonucleases continue to be discovered to this day. Many of them have not yet been cloned, that is, the genes that encode them are not known, and some purified fraction of proteins with the necessary catalytic activity is used as an “enzyme”. The Novosibirsk company SibEnzyme has been successfully competing for a long time with New England Biolabs, a world-renowned leader in the supply of restrictases (that is, it offered the same or more different restrictases, some of which are very exotic). - Ed.

For the isolation of the first restriction enzyme, the study of its properties and the first use for chromosome mapping, Werner Arber ( Werner Arber), Dan Nathans ( Dan Nathans) and Hamilton Smith ( Hamilton Smith) in 1978 received the Nobel Prize in Physiology or Medicine.

DNA ligases

To create new DNA molecules, of course, in addition to cutting, it is also necessary to be able to link two strands together. This is done with the help of enzymes called DNA ligases that cross-link the sugar-phosphate backbone of two strands of DNA. Because the chemical structure of DNA does not differ between organisms, you can crosslink DNA from any source, and the cell will not be able to distinguish the resulting molecule from its own DNA.

Separating DNA Molecules: Gel Electrophoresis

Often you have to deal with a mixture of DNA molecules of different lengths. For example, when processing DNA chemically isolated from the body with restriction enzymes, a mixture of DNA fragments will be obtained, and their lengths will differ.

Since any DNA molecule in an aqueous solution is negatively charged, it becomes possible to separate a mixture of DNA fragments of various sizes along their length using electrophoresis, . The DNA is placed on a gel (usually agarose for relatively long and very different molecules, or polyacrylamide for high resolution electrophoresis) which is placed in a constant electric field. Because of this, DNA molecules will move towards the positive electrode ( anode), and their speeds will depend on the length of the molecule: the longer it is, the more the gel prevents it from moving and, accordingly, the lower the speed. After electrophoresis, mixtures of fragments of different lengths in the gel form bands corresponding to fragments of the same length. Using markers (mixtures of DNA fragments of known lengths), one can determine the length of molecules in a sample (Fig. 3).

There are two ways to visualize the results of phoresis. The first, most frequently used recently, is the addition of substances to the gel that fluoresce in the presence of DNA (rather toxic ethidium bromide has traditionally been used; recently, safer substances have come into use). Ethidium bromide glows orange when irradiated with ultraviolet light, and upon binding to DNA, the luminescence intensity increases by several orders of magnitude (Fig. 4). Another method is to use radioactive isotopes, which must first be included in the analyzed DNA. In this case, a photographic plate is placed on top of the gel, which is illuminated above the DNA bands due to radioactive radiation (this imaging method is called autoradiography).

Figure 4. Agarose gel electrophoresis using ethidium bromide to visualize results in ultraviolet ( left). The second track from the left is a marker with known fragment lengths. On right- Installation for electrophoresis in gel.

In addition to the "ordinary" electrophoresis in a gel plate, in some cases they use capillary electrophoresis, which is carried out in a very thin tube filled with a gel (usually polyacrylamide). The resolution of such electrophoresis is much higher: it can be used to separate DNA molecules that differ in length just one nucleotide. Read about one of the important applications of this method below in the description of the Sanger DNA sequencing method.

Identification of a specific DNA sequence in a mixture. Southern blotting

Using electrophoresis, you can find out the size of DNA molecules in solution, but it will not tell you anything about the sequence of nucleotides in them. By using DNA hybridization it is possible to understand which of the bands contains a fragment with strictly defined sequence. DNA hybridization is based on the formation of hydrogen bonds between two strands of DNA, leading to their connection,.

First you need to synthesize DNA probe, which is complementary to the sequence we are looking for. It is usually a single-stranded DNA molecule 10–1000 nucleotides long. Due to complementarity, the probe will bind to the desired sequence, and due to the fluorescent label or radioisotopes embedded in the probe, the results can be seen.

For this, a procedure called Southern blotting or Southern carryover, named after the scientist who invented it ( Edwin Southern). Initially, a mixture of DNA fragments is separated by electrophoresis. A sheet of nitrocellulose or nylon is placed on top of the gel, and the separated DNA fragments are transferred to it by blotting: the gel lies on a sponge in a bath with an alkali solution that seeps through the gel and nitrocellulose due to the capillary effect of paper towels folded on top. During leakage, the alkali causes DNA denaturation, and already single-stranded fragments are transferred to the surface of the nitrocellulose plate and fixed there. The nitrocellulose sheet is carefully removed from the gel and treated with a radioactively labeled DNA probe specific to the required DNA sequence. The nitrocellulose sheet is thoroughly washed so that only those sample molecules that hybridize with DNA on nitrocellulose remain on it. After autoradiography, the DNA with which the probe has hybridized will be visible as bands on the photographic plate (Fig. 5).

The adaptation of this technique to determine specific RNA sequences is called, in contrast to Southern blotting, northern blotting (northern blotting: southern in English means "southern", and northern- "northern"). In this case, electrophoresis is carried out in a gel with mRNA molecules, and a single-stranded DNA or RNA molecule is chosen as a probe.

DNA cloning

We already know how to cut the genome into pieces (and stitch them together with arbitrary DNA molecules), separate the resulting fragments by length, and select the necessary one using hybridization. Now it's time to find out how, by combining these methods, we can clone a section of the genome (for example, a specific gene). In the genome, any gene occupies an extremely small length (compared to the entire DNA of the cell). DNA cloning literally means the creation of a large number of copies of a certain fragment of it. It is precisely through this amplification we get the opportunity to isolate a piece of DNA and get it in sufficient quantity for study.

How to divide DNA fragments by length and identify the desired one was described above. Now we need to understand how we can copy the fragment we need. There are two main methods: the use of rapidly dividing organisms (usually bacteria Escherichia coli- Escherichia coli - or yeast Saccharomyces serevisiae) or do a similar process, but in vitro by using polymerase chain reaction.

replication in bacteria

Since bacteria (like any other cell, except for the precursors of germ cells) double their DNA with each cell division, this can be used to multiply the number we need DNA. In order to introduce our DNA fragment into a bacterium, it is necessary to "sew" it into a special vector, which is usually used as a bacterial plasmid(small - relative to the bacterial chromosome - a circular DNA molecule that replicates separately from the chromosome). Wild-type bacteria often have similar structures: they are often transferred "horizontally" between different strains or even species of bacteria. Most often, they contain genes for resistance to antibiotics (it was because of this property that they were discovered) or bacteriophages, as well as genes that allow the cell to use a more diverse substrate. (Sometimes they are "selfish" and do not carry any functions.) It is these plasmids that are usually used in molecular genetic studies. Plasmids necessarily contain an origin of replication (the sequence from which replication of the molecule begins), the target sequence of the restriction enzyme, and a gene that allows the selection of those cells that have this plasmid (usually, these are genes for resistance to some antibiotic). In some cases (for example, when studying very large DNA fragments), not a plasmid is used, but an artificial bacterial chromosome.

The necessary DNA fragment is inserted into the plasmid with the help of restriction enzymes and ligases, after which it is added to the bacterial culture under special conditions that provide transformation- the process of active capture of DNA from the external environment by the bacterium (Fig. 6). After that, bacteria are selected, the transformation of which was successful, by adding an antibiotic corresponding to the gene in the plasmid: only cells that carry the resistance gene (and, consequently, the plasmid) remain alive. Further, after the growth of the cell culture, plasmids are isolated from it, and “our” DNA fragment is isolated from them using restrictases (or I use the entire plasmid). If the gene was inserted into a plasmid in order to obtain its protein product, it is necessary to provide the culture with conditions for growth, and then simply isolate the required protein.

At this point, the question should immediately arise: how could all this be used before the genomes were deciphered, and reading the DNA sequence was still expensive and not common? Suppose, with the help of restriction and cloning of the resulting fragments, we get DNA library, that is, a set of bacteria carrying various plasmids containing in total the entire genome (or a noticeable part of it). But how can we understand which of the fragments contains the necessary gene? For this, the hybridization method was used. First, it was necessary to isolate the protein of the desired gene. After that, its fragment is sequenced, the genetic code is reversed, and the nucleotide sequence is obtained (of course, many different options had to be tried due to the degeneracy of the genetic code). In accordance with it, a short DNA molecule was chemically synthesized, which was used as a probe for hybridization.

But in some cases, this method failed - for example, this happened with blood clotting factor VIII. This protein is involved in blood clotting, and malfunctions in its functionality are the cause of one of the most common genetic diseases - hemophilia A. Previously, for treatment, this protein had to be isolated from a large number of organisms, because it could not be cloned for production by bacteria. This was due to the fact that its length is about 180,000 base pairs, and it contains many introns (non-coding fragments between coding ones) - it is not surprising that this gene did not get entirely into any plasmid.

On the mechanisms of blood coagulation, see " How does blood clotting work? ». - Ed.

Polymerase chain reaction (PCR)

The method is based on multiple selective copying of a certain DNA segment with the help of enzymes under artificial conditions. In this case, only the DNA segment that satisfies the specified conditions is copied, and only if it is present in the sample under study. Unlike DNA replication in the cells of living organisms, relatively short DNA segments are amplified using PCR (usually no more than 3000 base pairs, however, there are methods that allow you to "raise" up to 20 thousand base pairs - the so-called Long Range PCR).

In fact, PCR is an artificial multiple replication of a DNA fragment (Fig. 7). DNA polymerases are designed in such a way that they cannot synthesize new DNA simply by having a template and monomers available. To do this, you also need a seed ( primer) from which they start the synthesis. A primer is a short, single-stranded nucleic acid fragment that is complementary to a DNA template. During replication in the cell, such primers are synthesized by a special enzyme primase and are RNA molecules that are later replaced by DNA. However, artificially synthesized DNA molecules are used in PCR, since in this case the step of removing RNA and synthesizing DNA in their place is not needed. In PCR, primers limit the amplified region on both sides.

Figure 7. DNA replication- the most important process for living organisms, the basis of many molecular biological methods. Since each of the DNA chains contains a sequence of nucleotides that is complementary to the other chain (their information content is the same), when DNA is duplicated, the chains diverge, and then each chain serves as a template on which a new DNA chain complementary to it is built. As a result, two DNA duplexes are formed, each of which is an exact (without taking into account synthesis errors) copy of the original molecule.

So, it's time to explain how PCR works. Initially, the reaction mixture contains: a DNA template, primers, DNA polymerase, free nucleosides (future "letters" in the newly synthesized DNA), as well as some other substances that improve the functioning of the polymerase (they are added to special buffers used in the reaction).

To synthesize DNA complementary to the template, it is necessary that one of the primers form hydrogen bonds with it (as they say, "anneal" on it). But the matrix already forms them with the second chain! So, first you need to melt the DNA - that is, to destroy the hydrogen bonds. This is done by simple heating (up to ≈95 ° C) - a stage called denaturation. But now the primers cannot burn on the matrix due to the high temperature! Then the temperature is lowered (50–65 °C), the primers are annealed, after which the temperature is slightly raised (up to the optimum operation of the polymerase, usually about 72 °C). And then the polymerase begins to synthesize DNA chains complementary to the template - this is called elongation(Fig. 8). After one such cycle, the number of copies of the required fragments doubled. However, nothing prevents you from doing it again. And not just once, but dozens of times! And with each repetition, the number of copies of our DNA fragment will double, because the newly synthesized molecules will also serve as templates (Fig. 9)! (In fact, the efficiency of PCR is rarely so high that the number of copies doubles, but ideally this is the case, and real numbers are often close to this.)

Figure 9. With each PCR cycle, the amount of target DNA doubles.

It is very easy to see the results of PCR: it is enough to carry out electrophoresis of the reaction mixture after PCR, and a bright band with the obtained copies will be visible.

Previously, heat-inactivated polymerase with each cycle had to be added all the time, but soon it was proposed to use a thermostable polymerase from thermophilic bacteria that can withstand such heat, which greatly simplifies PCR (the most commonly used Taq polymerase from bacteria Thermus aquaticus ).

To avoid strong evaporation of water from the reaction mixture, oil is added to cover it from above and/or a heated lid is used. thermal cycler- a device in which PCR is carried out. It quickly changes the temperature of the test tubes, and they do not have to constantly shift from one thermostat to another. To prevent non-specific synthesis even before heating and the actual start of the cycles, I often use PCR with a “hot start”: all DNA and polymerase are separated from each other by a paraffin layer, which melts at a high temperature and allows them to interact already under the right conditions. Sometimes modified polymerases are used that do not work at low temperatures.

There is much more to say about the various subtleties of PCR, but the most important thing to say is about alternative methods for determining results to classical phoresis. For example, a fairly obvious option is to add substances that fluoresce in the presence of DNA to the reaction tube before starting the reaction. Then, by comparing the initial fluorescence with the final one, one can see whether a significant amount of DNA was synthesized or not. But this method is not specific: we will not be able to determine in any way whether necessary a fragment, or is it some primers sticking together and building up to unpredictable sequences.

The most interesting option is PCR "real time" ("real-time PCR"). There are several implementations of this method, but the idea is the same everywhere: you can directly observe the accumulation of PCR products (by fluorescence) during the reaction. Accordingly, real-time PCR requires a dedicated instrument that can excite and read fluorescence in each tube. The simplest solution is to add to the test tube the same substances that fluoresce in the presence of DNA, but the disadvantages of this method have already been described above.

This is strictly referred to as "real-time fluorescence PCR" or "quantitative PCR". - Ed.

Figure 11. Example of fluorescence accumulation curves in real-time PCR: dependence of the fluorescence intensity (in several test tubes - for each its own curve) on the cycle number.

The most popular implementation of this approach is the method of fluorophore cleavage by destroying the probe (TaqMan Assay; Fig. 10). In this case, the reaction mixture should contain one more component - a special single-stranded DNA probe: a DNA molecule complementary to the sequence of the fragment being amplified located between primers. At the same time, one of its ends should be chemically attached fluorophore(fluorescent molecule), and to the other - a quencher (a molecule that absorbs the energy of the fluorophore and "quenching" the fluorescence). When such a probe is in solution or complementary to the target sequence, the fluorophore and quencher are relatively close to each other and no fluorescence is observed. However, due to the 3´-exonuclease activity that Taq polymerase possesses (that is, it cleaves the DNA that it “stumbles upon” during synthesis and synthesizes a new one in its place), the probe is destroyed during the synthesis of the second strand, the fluorophore and quencher due to diffusions move away from each other, and fluorescence appears.

As the number of copies increases exponentially during PCR, so does the fluorescence. However, this does not last long, since at some point the efficiency of the reaction begins to fall due to the gradual inactivation of the polymerase, the lack of some components, etc. (Fig. 11). By analyzing the fluorescence growth graphs, one can understand a lot about the PCR process, but, most importantly, one can find out how many DNA templates were originally: this is the so-called quantitative PCR(quantitative PCR, qPCR).

It is impossible to enumerate all the applications of PCR in science. Isolation of a DNA fragment, sequencing, mutagenesis... PCR is one of the most popular methods for non-scientific purposes (video 1). It is widely used in medicine for the early diagnosis of hereditary and infectious diseases, paternity determination, in investigations to establish identity, and much more.

Natural cellular processes in vitro

All major molecular biological processes can be easily carried out in vitro(that is, in a test tube). An example is given above: PCR is analogous to DNA replication. To do this, simply mix the necessary reagents under suitable conditions: transcription requires a DNA template, RNA polymerase and ribonucleotides, translation requires mRNA, ribosome subunits and amino acids, reverse transcription requires an RNA template, reverse transcriptase(aka revertase) and deoxyribonucleotides. These methods are widely used in various fields of biology, when it is necessary, for example, to obtain pure RNA of a specific gene. In this case, you must first reverse transcribe its (gene) mRNA, amplify it using PCR, and then use in vitro- transcription get a lot of mRNA. The first stage is necessary due to the fact that before the formation of mature mRNA in the cell, splicing And processing RNA (in eukaryotes; in bacteria, in this sense, everything is simpler) - preparation for work as a matrix for protein synthesis. Sometimes this can be avoided if the entire coding sequence of the gene is located in one exon.

DNA sequencing

It can be said that the most important methods of manipulating DNA have already been described. The next step is to determine the actual nucleotide sequence of the chain in the molecule - sequencing. The determination of the DNA nucleotide sequence is extremely important for many fundamental and applied problems. It occupies a special place in science: to analyze the results of genome sequencing, in fact, a new science was created - bioinformatics. Sequencing is now used by molecular biologists, geneticists, biochemists, microbiologists, botanists and zoologists, and, of course, evolutionists: almost all modern systematics is based on its results. Sequencing is widely used in medicine as a method of searching for hereditary diseases and studying infections. (See, for example, " Clarification of the "pedigree" of arthropods" And " Sk true anecdote: Negro, Chinese and Craig Venter ... ». - Ed.)

In fact, chronologically, the methods were invented in a completely different order. For example, Sanger sequencing was developed in 1977, and PCR, as mentioned above, only in 1983.

There are many different sequencing techniques, but all methods can be divided into two categories: "classic" and new generation. Now, in fact, only one “classical” method is used - Sanger sequencing, or the terminator method. Compared to new methods, it has an important advantage: the read length, that is, the number of nucleotides in a sequence that can be obtained at a time, is higher - up to 1000 nucleotides. At the same time, the most "good" in this regard "new" sequencing method - 454-, or pyrosequencing - this parameter does not exceed 500 nucleotides. It is the length of the read that limits the possibilities of new methods: it turns out to be extremely difficult to “assemble” the whole genome from fragments of several tens of nucleotides in size. At a minimum, this requires supercomputers, and some places in the genome are simply impossible to resolve if they contain highly repetitive sequences. In this case, comparing the obtained fragments with the already existing whole genome can help, but in this way it is impossible to read the genome of the organism for the first time ( de novo). (See also: " Code of life: to read is not to understand ». - Ed.)

English biochemist and luminary of molecular biology, twice winner of the Nobel Prize in Chemistry: for determining the amino acid sequence of insulin (1955) and for developing a DNA sequencing method (1980). - Ed.

There is a new generation method that allows you to read several thousand mon, but with large errors (Pacific Biosciences). 454/Roche today can read more than 500 Mon; the young “semiconductor sequencing” can already do the same. - Ed.

Both of the sequencing methods mentioned above have already been described in sufficient detail on the "biomolecule": I strongly advise you to familiarize yourself. For example, I will tell you about another common fast and cheap method (per one read nucleotide) - the method implemented in Illumina sequencers (video 2). Its main drawback is the reading of fragments of a very short length, no more than 100 nucleotides, and the resulting difficulty in reading the genome from scratch.

Three stages can be distinguished in this method: preparation of a library of fragments (1), creation of clusters (2), and sequencing itself (3).

Video 2. There are several good videos on the Internet that describe the Illumina sequencing process, for example, on the company's official website (Technology tab). However, they are all in English.

Quite a lot has already been said about the methods of working with nucleic acids and their study. It's time to find out how you can find out how the cell works - in particular, try to determine the function of the gene and the protein that it encodes.

In vitro-mutagenesis

To study the function of a protein, it is very important to learn how to introduce into it mutations. For example, if you have an organism with a non-functioning enzyme, you can understand by biochemical differences what a normal protein does. There are different ways to create a completely non-working gene (both arbitrary from the entire genome, and completely specific - then it's called knockout this gene). One of these methods is the insertion of some DNA fragment into the genome: if this insertion falls on a gene, then it (more precisely, most likely, the protein that it encodes) will cease to function normally.

However, there are ways to very precisely change the sequence of a gene and, accordingly, a protein. About one of these methods - site-specific mutagenesis- I'll tell you. Its essence is to change a specific (usually one) nucleotide in the sequence. To use it, you first need to clone this gene in a plasmid. After that, it is necessary to carry out, as it were, PCR with one primer. Moreover, this primer should just include the sequence that we want to change - already in the form we need. For example, in fig. 14, instead of the letter A, which should have been opposite T in the parent chain, the primer contains C. After the synthesis of the second DNA strand of the plasmid containing the primer, a mutation will be introduced into it - A will be replaced by C. Such plasmids are introduced into cells in which when dividing, the two chains will end up in different daughter cells. Thus, half of the progeny cells will have the original version of the plasmid, and half will have the mutant one. Then, accordingly, half of the cells will produce a normal protein encoded by this gene, and half will produce a mutant one. In the case shown in Fig. 14, instead of one amino acid (asparagine) there will be another (alanine). By analogy, random mutations can be introduced using a special DNA polymerase that introduces an increased number of errors.

Figure 14. Scheme of site-specific mutagenesis.C.elegans, - that is, about turning off the gene in all (almost) cells of this worm.

Such a striking effect is achieved by introducing double-stranded RNA molecules (dsRNA) into the cell, one of the chains in each of which is complementary to the mRNA section of the “turned off” gene. This opens up amazing possibilities for studying the functions of genes. Previously, to disable genes, it was necessary to create "knockout" animals (which scientists still have to do, for example, with mice - see " Nobel Prize in Physiology or Medicine awarded for technology to knock out mice ». - Ed.), in which the studied gene basically absent from the genome. However, the creation of knockouts is quite difficult, and turning the gene back on in such organisms is no longer possible. With the help of RNA interference, it is very easy to turn off a gene, just as it is to turn it on, having stopped introducing the corresponding dsRNA into the body.

There are three main ways to introduce dsRNA into the body. The most obvious is the injection of their solution into the animal. They also use "soaking" nematodes in an RNA solution. However, it turned out that you can do everything much easier: feed these molecules to nematodes! Moreover, it is especially convenient that it also works great if the nematodes are fed with bacteria ( E. coli) that synthesize these dsRNAs (Fig. 15).

Figure 15. Systemic RNA interference. Worm C.elegans expresses a green fluorescent (luminous) protein in the cells of the pharynx (ph) and the muscles of the body wall (bm). Left- initial appearance. On right- during RNA interference with the help of "feeding" by bacteria, the gene is inactivated.

In principle, the fact that RNA molecules from the intestines are distributed to almost all tissues is quite surprising. It is known that a protein channel is responsible for the entry of RNA molecules into intestinal cells. sid-1, . However, it is not known for certain how RNA is distributed throughout the body of the worm, most likely with the participation of a protein. rsd-8 Interestingly, all known proteins involved in systemic RNA interference in C.elegans, are also present in humans, but such an effective system of artificial suppression of gene activity at the systemic level in humans cannot be observed. If it were possible to use systemic RNAi in humans, it could be a way to fight a huge range of diseases, from the common cold to cancer.

By the way, the use of RNA interference specifically on human cell culture made it possible to reveal that many human genes contribute development of the influenza virus: Molecular double-dealing: human genes work for the flu virus ». - Ed.

Studying Gene Expression: DNA Microarrays

When studying the function of a gene, it is very important to find out when and in what tissues of the body it works (is expressed), as well as together with what other genes. If you want to know this about a small number of genes and tissues, then you can do it very simply: isolate RNA from tissue, reverse transcription (that is, synthesize cDNA - complementary DNA) and then perform quantitative PCR. Depending on whether PCR has passed, we will find out if there is mRNA of the gene under study in the tissue.

However, if it is necessary to do the same for many tissues and many genes, then this technique becomes very long and costly. In that case, use DNA microarrays. These are small plates on which DNA molecules are applied and attached, complementary to the RNA of the studied genes, and it is known in advance where on them (the plates) which molecule is located. One way to create a chip is to synthesize DNA molecules directly on it using a robot.

To study gene expression using chips, it is also necessary to synthesize their cDNA and label it with a fluorescent dye (without separating the cDNA of different genes). This mixture is applied to the microchip, ensuring that the cDNA hybridizes with the DNA molecules on the chip. After that, they look at where the fluorescence is observed and compare it with the location of the DNA molecules on the chip. If the place of fluorescence coincides with the position of the DNA molecule, then this gene is expressed in this tissue. In addition, by labeling cDNA from different tissues with different dyes, it is possible to study the expression of several (usually not more than 2) tissues on one chip at once: the fluorescence color can be used to determine in which of the tissues it is expressed (if it is expressed in several tissues at once, it will turn out to be mixed). color) (Fig. 16).

However, in recent years, instead of chips, mass sequencing of the entire cDNA from tissue (the creation of so-called transcriptomes), which has been greatly simplified due to the development of sequencing methods. This turns out to be cheaper and more efficient, since knowing the complete sequences of all mRNAs provides more information than just the mere fact of their presence or absence.

We have reviewed the basic methods of molecular biology. I hope that it has become a little clearer to you how molecular biological research is done, what Nobel Prizes are for, and how they can help in some applied problems. But most of all, I hope that you, too, have seen the beauty of the ideas behind them, and perhaps you would like to learn more about some of these techniques.

Literature

1. Nobel laureates: J. Watson, F. Crick, M. Wilkins; Wikipedia: 454-sequencing (high-throughput DNA pyrosequencing). Cold Spring Harbor Symposia on Quantitative Biology. 71 , 95-100.

1 question. Purpose, tasks and methods of molecular biology. The term "Molecular Biology" itself was first used by the English. scientists W. Astbury to research related to elucidating the relationship between the molecular structure and the physical and biological properties of fibrillar (fibrous) proteins, such as collagen, blood fibrin, or contractile muscle proteins. The term "molecular biology" has been widely used since the early 1950s. 20th century Molecular biology is a complex of biological sciences that studies the mechanisms of storage, transmission and implementation of genetic information, the structure and functions of irregular biopolymers (proteins and nucleic acids). William Thomas Astbury () British physicist, molecular biologist

Molecular biology studies the basic properties and manifestations of life at the molecular level. Finds out how and to what extent the growth and development of organisms, the storage and transmission of hereditary information, the conversion of energy in living cells and other phenomena are due to the structure and properties of biologically important macromolecules (mainly proteins and nucleic acids). A distinctive feature of molecular biology is the study of the phenomena of life on inanimate objects or those objects that have the most primitive manifestations of life. These are biological formations from the cellular level and below: subcellular organelles, such as isolated cell nuclei, mitochondria, ribosomes, chromosomes, cell membranes; further systems standing on the border of animate and inanimate nature, viruses, including bacteriophages, and ending with the molecules of the most important components of living matter, nucleic acids.

Subject of MB research Mechanisms of storage, transfer and realization of genetic information, structure and functions of irregular biopolymers Object of MB research are subcellular organelles viruses (bacteriophages). nucleus mitochondria ribosomes chromosomes systems that stand on the border of animate and inanimate nature

Tasks of molecular biology Search for solutions to the problem of the molecular basis of malignant growth Ways to prevent and overcome hereditary diseases Elucidation of the molecular basis of biological catalysis, i.e. the action of enzymes Deciphering the molecular mechanisms of action of hormones, toxic and medicinal substances Elucidation of the details of the molecular structure and functioning of biological membranes involved in the regulation processes of penetration and transport of substances More distant tasks - knowledge of the nature of nervous processes, memory mechanisms

In the light of the ideas of molecular biology, life can be considered as the result of a combination of three flows: the flow of matter, which finds its expression in the phenomena of metabolism, i.e., assimilation and dissimilation; the flow of energy, which is the driving force for all manifestations of life; a flow of information that permeates not only the entire diversity of the processes of development and existence of each organism, but also a continuous series of successive generations. It is the idea of ​​the flow of information, introduced into the doctrine of the living world by the development of molecular biology, that leaves its own specific, unique imprint on it.

Molecular biology is the final stage in the direction in the study of living objects, which is called "reductionism", i.e., the desire to reduce complex life functions to phenomena occurring at the molecular level and therefore accessible to study by the methods of physics and chemistry. The successes achieved in molecular biology testify to the effectiveness of this approach. At the same time, it should be taken into account that under natural conditions, a cell, tissue, organ, and the whole organism are systems of increasing complexity. These systems are formed from lower-level components by integrating them into a whole that acquires new properties.

Methods of molecular biology. Since molecular biology is a complex of biological sciences, it uses the methods of these sciences: ultracentrifugation, X-ray diffraction analysis, electron microscopy, nuclear magnetic resonance, polymerase chain reaction method. In addition, molecular biology uses the methods of other sciences - for example, physics: the use of computers, synchrotron, or bremsstrahlung, radiation, laser technology.

Electron microscopy An electron microscope is a device that makes it possible to obtain an image of objects with a maximum magnification of millions of times, thanks to the use, unlike an optical microscope, of an electron beam instead of a light flux. To obtain an image in an electron microscope, special magnetic lenses are used that control the movement of electrons in the instrument column using a magnetic field.

Fluorescent (luminescent) microscopy is based on the ability of certain substances to luminesce, i.e., glow when illuminated with invisible ultraviolet or blue light. When luminescence is excited by blue light, its color can be from green to red; if luminescence is excited by ultraviolet radiation, then the glow can be in any part of the visible spectrum. The device of a luminescent microscope and the rules for working with it differ from a transmitted light microscope in the following: The presence of a powerful light source in the illuminator, emitting mainly in the short-wave (ultraviolet, blue) part of the spectrum (high-pressure mercury-quartz lamp or halogen quartz lamp). Availability of a system of light filters: 1. Exciting filters pass only that part of the spectrum that excites luminescence; 2. The heat-shielding light filter protects other light filters, preparation and optics of the luminescent microscope from overheating. 3. "Locking" filters are located between the eyepiece. These light filters absorb exciting radiation and transmit luminescence light from the preparation to the observer's eye.

Centrifugation Centrifugation Separation of inhomogeneous systems (eg liquid-solid particles) into density fractions using centrifugal forces. Centrifugation is carried out in apparatuses called centrifuges. Centrifuge - a device that creates high centrifugal forces due to the rotation of the rotor from 200 rpm to rpm. Centrifugation in biology is used to separate the sediment of cellular structures from the solution. To study macromolecular substances (proteins, nucleic acids, etc.) and biological systems, ultracentrifuges with a rotor speed of 2000 rpm to rpm (up to 2500 rpm) are used.

Electrophoresis Electrophoresis is the movement of particles of the dispersed phase of colloidal or protein solutions in a liquid or gaseous medium under the action of an external electric field. It was first discovered by professors of Moscow University P. I. Strakhov and F. F. Reiss in 1809. In biology, electrophoresis is used to separate macromolecules.

The polymerase chain reaction (PCR, PCR) was invented in 1983 by the American scientist Cary Mullis. Subsequently, he received the Nobel Prize for this invention. The basis of the PCR method is the repeated doubling of a certain section of DNA. As a result, sufficient amounts of DNA are produced for visual detection. Molecular cloning (Gene cloning) is the production of a large number of identical DNA molecules using living organisms (bacteria or viruses). Immunocytochemical methods allow localization and identification of cellular and tissue components (antigens) based on their binding to antibodies. The binding site is determined using labeled antibodies or secondary labeling. The PCR method makes it possible to determine the presence of the causative agent of the disease, even if only a few DNA molecules of the causative agent are present in the sample.

Molecular biology has historically emerged as a branch of biochemistry. The birth date of molecular biology is considered to be April 1953, when an article by James Watson and Francis Crick appeared in the English journal Nature, proposing a spatial model of the DNA molecule. 2 question. History of development of molecular biology. This fundamental discovery was prepared by a long stage of research into the genetics and biochemistry of viruses and bacteria. In 1928, Frederick Griffith was the first to show that an extract of heat-killed disease-causing bacteria could transfer the trait of pathogenicity to benign bacteria.

Another discovery important for the methodology was the discovery at the beginning of the 20th century of bacteriophage viruses. In the 50s of the 20th century, it was shown that bacteria have a primitive sexual process, they are able to exchange extrachromosomal DNA - a plasmid. The structure of a bacteriophage Bacteriophages on the surface of a bacterial cell

The further development of molecular biology was accompanied by both the development of its methodology, in particular, the invention of a method for determining the nucleotide sequence of DNA (W. Gilbert and F. Sanger, 1980 - Nobel Prize in Chemistry), and new discoveries in the field of research into the structure and functioning of genes. By the beginning of the 21st century, data were obtained on the primary structure of the entire DNA of a person and a number of other organisms, the most important for medicine, agriculture and scientific research, which led to the emergence of several new areas in biology: genomics, bioinformatics, etc.).

Disclosure of the structure and mechanism of the biological function of DNA, all types of RNA and ribosomes, disclosure of the genetic code; discovery of reverse transcription, i.e., DNA synthesis on an RNA template; study of the mechanisms of functioning of respiratory pigments; the discovery of the three-dimensional structure and its functional role in the action of enzymes, the discovery of the principle of matrix synthesis and the mechanisms of protein biosynthesis; disclosure of the structure of viruses and the mechanisms of their replication, the primary and, in part, the spatial structure of antibodies; isolation of individual genes, chemical and then biological (enzymatic) gene synthesis, including human, outside the cell (in vitro); transfer of genes from one organism to another, including into human cells; discovery of the phenomena of "self-assembly" of some biological objects of ever-increasing complexity, starting from nucleic acid molecules and moving on to multicomponent enzymes, viruses, ribosomes, etc.; elucidation of allosteric and other basic principles of regulation of biological functions and processes. 3 question. The most important achievements of molecular biology and its connection with other sciences. The most important achievements of molecular biology:

Today, the field of interest of molecular biologists covers a wide range of fundamental scientific issues. As before, the leading role is occupied by the study of the structure of nucleic acids and protein biosynthesis, the study of the structure and functions of various intracellular structures and cell surfaces.

MOLECULAR BIOLOGY Late Lat. molecula, diminutive of lat. moles mass; biology) is a biomedical science that studies the phenomena of life at the level of biological macromolecules - proteins and nucleic acids, such simple systems as cell-free structures, viruses, and, as a limit, at the cell level. Most of these objects are inanimate or endowed with elementary manifestations of life. M.'s position. in the system of biol, sciences is determined by ideas about the structural levels of living matter, i.e., evolutionarily established forms of life, starting with prebiotic steps and ending with complex systems: small organic molecules - macromolecules - cell and subcellular structures - the body, etc., respectively to-rym are built and levels of knowledge. Historically M. b. formed as a result of the study of biological macromolecules, whereby M. would. considered as a section of biochemistry (see). M. b. is at the same time the boundary science which arose at a joint of biochemistry, biophysics (see), organic chemistry (see), cytology (see) and genetics (see). M.'s idea b. consists in disclosing the elementary mechanisms of the main processes of vital activity - heredity (see), variability (see), movement, etc. - through the study of biol, macromolecules. Molecular biol. ideas found fertile ground especially in genetics - molecular genetics arose (see), and it was here that results were achieved, to-rye contributed to M.'s development. and recognition of its principles. M.'s representations. have heuristic (cognitive) value, since biol, macromolecules - proteins (see) and nucleic acids (see) exist and act at all levels of development of living matter. For this reason, M.'s borders would. difficult to define: it turns out to be an all-pervading science.

The very name "molecular biology" belongs to the English. crystallographer Astbury (W. T. Astbury). The formal date of M.'s occurrence. consider 1953, when J. Watson and F. Crick established the structure of DNA and expressed a later confirmed assumption about the mechanism of its replication underlying heredity. But at least since 1944, since the works of Avery (O. Th. Avery), facts have accumulated that indicate the genetic role of DNA; N. K. Koltsov expressed the idea of ​​matrix synthesis in a very clear form as early as 1928; The study of the molecular basis of muscle contraction began with the works of V. A. Engelgardt and M. N. Lyubimova, published in 1939-1942. M. b. also developed in the field of evolutionary doctrine and systematics. In the USSR A. N. Belozersky was the initiator of studying nucleic to - t and researches on molecular bases of evolution.

A distinctive feature of M. b. lies in the nature of the observations, in its methodological techniques and the design of the experiment. M. b. forced biologists to take a fresh look at the material basis of life. For molecular biol. researches comparison biol, functions with chemical is characteristic. and physical characteristics (properties) of biopolymers and, in particular, their spatial structure.

To understand the patterns of the structure of nucleic acids and their behavior in a cell, the principle of complementarity of bases in two-stranded structures of nucleic acids, established in 1953 by J. Watson and F. Crick, is of paramount importance. Recognition of the importance of spatial relationships has found its expression in the idea of complementarity of surfaces of macromolecules and molecular complexes, which is a necessary condition for the manifestation of weak forces acting only at short distances and contributing to the creation of morphol, biol diversity. structures, their functional mobility. These weak forces are involved in the formation of complexes such as enzyme - substrate, antigen - antibody, hormone - receptor, etc., in the phenomena of self-assembly of biol, structures, for example, ribosomes, in the formation of nitrogenous base pairs in nucleic acid molecules and in that similar processes.

M. b. directed the attention of biologists to simple objects standing at the boundaries of life, introduced ideas and precise methods of chemistry and physics into the arsenal of biol, research. The mutation process was interpreted at the molecular level as the loss, insertion and displacement of DNA segments, the replacement of a pair of nitrogenous bases in functionally significant segments of the genome (see Mutation). The phenomena of a mutagenesis (see) were, thus, translated into chemical. language. Thanks to M.'s methods. the molecular bases of such genetic processes in prokaryotes as recombination (see), transduction (see), transformation (see), transfection, sexduction were revealed. Significant progress has been made in studying the structure of chromatin and eukaryotic chromosomes; improvement of methods of cultivation and hybridization of animal cells created the possibility of development of genetics of somatic cells (see). The regulation of DNA replication found its expression in F. Jacob and S. Brenner's replicon concept.

In the field of protein biosynthesis, the so-called. the central postulate characterizing the following movement of genetic information: DNA -> messenger RNA -> protein. According to this postulate, the protein is a kind of information valve that prevents the return of information to the level of RNA and DNA. In the process of M.'s development. in 1970, H. Temin and D. Baltimore discovered the phenomenon of reverse transcription (in nature, DNA synthesis occurs in oncogenic RNA-containing viruses using a special enzyme - reverse transcriptase). Syntheses of proteins and nucleic acids occur according to the type of matrix syntheses, for their occurrence a matrix (template) is necessary - the initial polymer molecule, which predetermines the sequence of nucleotides (amino acids) in the synthesized copy. Such templates for replication and transcription are DNA, and for translation - messenger RNA. The genetic code (see) formulates a way of "recording" of hereditary information in messenger RNA, in other words, it coordinates the sequence of nucleotides in nucleic acids and amino acids in proteins. Transcription is associated with protein biosynthesis - the synthesis of messenger RNA on a DNA template catalyzed by RNA polymerases; translation - protein synthesis on the messenger RNA associated with the ribosome, proceeding according to a very complex mechanism, dozens of auxiliary proteins and transport RNAs participate in Krom (see Ribosomes). The regulation of protein synthesis is most studied at the level of transcription and is formulated in the presentation of F. Jacob and J. Monod on the operon, repressor proteins, allosteric effect, positive and negative regulation. Diverse in content and even less complete than the previous sections of M. b. there are a number of fundamental and applied problems. These include repair of damage to the genome caused by short-wave radiation, mutagens (see) and other influences. A large independent area is the study of the mechanism of action of enzymes, based on ideas about the three-dimensional structure of proteins and the role of weak chem. interactions. M. b. found out many details of the structure and development of viruses, especially bacteriophages. The study of hemoglobins in persons suffering from sickle cell anemia (see) and other hemoglobinopathies (see), marked the beginning of the study of the structural basis of "molecular diseases", congenital "errors" of metabolism (see Hereditary diseases). The latest branch of M. b. is genetic engineering (see. ) - develops methods for constructing hereditary structures in the form of recombinant DNA molecules.

In molecular biol. experiments find application various methods of chromatography (see) and ultracentrifugation (see), x-ray diffraction analysis (see), electron microscopy (see), molecular spectroscopy (electron paramagnetic and nuclear magnetic resonance). The use of synchrotron (magnetic bremsstrahlung) radiation, neutron diffraction, Mössbauer spectroscopy, and laser technology has begun. Model systems and obtaining mutations are widely used in experiments. The use of radioactive and (to a lesser extent) heavy isotopes in M. would be. the usual analytical method, as well as the application of mathematical methods and computers. If earlier molecular biologists were guided by ch. arr. on the physical methods created for the study of polymers nebiol. origin, now there is an ever-increasing trend towards the use of chemical. methods.

For M.'s development. in the USSR, the resolution of the Central Committee of the CPSU and the Council of Ministers of the USSR “On measures to accelerate the development of molecular biology and molecular genetics and the use of their achievements in the national economy”, published on May 20, 1974, was of great importance. of Molecular Genetics at the State Committee for Science and Technology of the Council of Ministers of the USSR and the Academy of Sciences of the USSR, the Scientific Council on Problems of Molecular Biology of the Academy of Sciences of the USSR, similar councils of the Academy of Sciences of the Union Republics and branch academies. The journal "Molecular Biology" (since 1967) and an abstract journal with the same name are published. Researches on M. would. are conducted in institutes of the Academy of Sciences of the USSR, the Academy of Medical Sciences of the USSR, the republican academies of sciences, Glavmikrobioprom, in higher educational institutions of the country. In the socialist countries, there are many laboratories of this profile. In Europe, there are the European Molecular Biology Organization (EMBO), the European Molecular Biology Laboratory (EMBL) in Heidelberg, and the European Molecular Biology Conference (EMBC). There are large specialized laboratories in the USA, France, Great Britain, Germany and other countries.

Special periodicals devoted to the problems of M., abroad: "Journal of Molecular Biology", "Nucleic Acids Research", "Molecular Biology Reports", "Gene".

Reviews on M. b. published in the VINITI Molecular Biology series, Progress in Nucleic Acids Research and Molecular Biology, Progress in Biophysics and Molecular Biology, Annual Rewiew of Biochemistry, Cold Spring Harbor Symposia on Quantitative Biology.

Bibliography: Ashmarin I. P. Molecular biology, L., 1977; Belozersky A. N. Molecular biology - a new stage in the knowledge of nature, M., 1970; Bresler S. E. Molecular biology, L., 1973; Koltsov N. K. Hereditary molecules, Bull. Moscow about-va test. nature, div. biol., v. 70, c. 4, p. 75, 1965; October and science, ed. A.P. Aleksandrova and others, p. 393, 417, M., 1977; Severin S. E. Modern problems of physical and chemical biology, in the book: 250 years of the Academy of Sciences of the USSR, p. 332, M., 1977; Watson J. Molecular biology: gene, trans. from English, M., 1978; Engelhardt V. A. Molecular biology, in the book: Development of biol, in the USSR, ed. B. E. Bykhovsky, p. 598, M., 1967.

The development of biochemistry, biophysics, genetics, cytochemistry, many sections of microbiology and virology around the beginning of the 40s of the XX century. closely led to the study of life phenomena at the molecular level. The successes achieved by these sciences, simultaneously and from different sides, led to the realization of the fact that it is at the molecular level that the main control systems of the body function and that the further progress of these sciences will depend on the disclosure of the biological functions of the molecules that make up the bodies of organisms, their participation in the synthesis and disintegration, mutual transformations and reproduction of compounds in the cell, as well as the exchange of energy and information that occurs in this case. Thus, at the junction of these biological disciplines with chemistry and physics, a completely new branch arose - molecular biology.

Unlike biochemistry, the attention of modern molecular biology is focused mainly on the study of the structure and function of the most important classes of biopolymers - proteins and nucleic acids, the first of which determine the very possibility of metabolic reactions, and the second - the biosynthesis of specific proteins. It is clear, therefore, that it is impossible to make a clear distinction between molecular biology and biochemistry, the corresponding branches of genetics, microbiology, and virology.

The emergence of molecular biology was closely associated with the development of new research methods, which have already been discussed in the relevant chapters. Along with the development of electron microscopy and other methods of microscopic technique, the methods of fractionation of cellular elements developed in the 1950s played an important role. They were based on improved methods of differential centrifugation (A. Claude, 1954). By this time, there were already quite reliable methods for the isolation and fractionation of biopolymers. This includes, in particular, the method of protein fractionation by electrophoresis proposed by A. Tiselius (1937; Nobel Prize, 1948), methods for isolating and purifying nucleic acids (E. Kay, A. Downs, M. Sevag, A. Mirsky, and others. ). At the same time, various methods of chromatographic analysis were developed in many laboratories of the world (A. Martin and R. Sing, 1941; Nobel Prize, 1952), subsequently significantly improved.

X-ray diffraction analysis played an invaluable service in deciphering the structure of biopolymers. The basic principles of X-ray diffraction analysis were developed at King's College London University under the leadership of W. Bragg by a group of researchers, which included J. Bernal, A. Londsdale, W. Astbury, J. Robertson and others.

Special mention should be made of the studies of Protoplasm Biochemistry (1925-1929), Professor of Moscow State University A. R. Kizel, which were of great importance for the subsequent development of molecular biology. Kizel dealt a blow to the firmly rooted notion that any protoplasm is based on a special protein body - plates, which allegedly determines all its most important structural and functional features. He showed that plates are a protein that is found only in myxomycetes, and then at a certain stage of development, and that no permanent component - a single skeletal protein - exists in protoplasm. Thus, the study of the problem of the structure of protoplasm and the functional role of proteins took the right path and received scope for its development. Kisel's research has won worldwide recognition, stimulating the study of the chemistry of the constituent parts of the cell.

The term "molecular biology", first used by the English crystallographer, Professor of the University of Leeds W. Astbury, probably appeared in the early 1940s (before 1945). The fundamental X-ray diffraction studies of proteins and DNA, carried out by Astbury in the 1930s, served as the basis for the subsequent successful deciphering of the secondary structure of these biopolymers. In 1963, J. Bernal wrote: "A monument to him will be erected by the whole of molecular biology - the science that he named and really founded" * , In the literature, this term appeared for the first time, perhaps in 1946 in the article by W. Astbury "Progress of X-ray diffraction analysis of organic and fibrillar compounds", published in the English journal "Nature" ** . In his Harvey Lecture, Astbury (1950) noted: “I am pleased that the term molecular biology is now quite widely used, although it is unlikely that I was the first to propose it. I liked it and I have long tried to spread it” ***. Already in 1950 Astbury was clear that molecular biology deals primarily with the structure and conformation of macromolecules, the study of which is of decisive importance for understanding the functioning of living organisms.

* (biogr. Mem. Fellows Roy. Soc, 1963, v. 9, 29.)

** (W. T. Astbury. Progress of X-ray analysis of organic and fiber structures.- Nature,. 1946, v. 157, 121.)

*** (W. T. Astbury. Adventures in Molecular Biology. Thomas Springfield, 1952, p. 3.)

Molecular biology has faced and faces, in fact, the same tasks as biology as a whole - the knowledge of the essence of life and its basic phenomena, in particular, such as heredity and variability. Modern molecular biology is primarily intended to decipher the structure and function of genes, the ways and mechanisms of realization of the genetic information of organisms at different stages of ontogenesis and at different stages of its reading. It is designed to reveal the subtle mechanisms of regulation of gene activity and cell differentiation, to elucidate the nature of mutagenesis and the molecular basis of the evolutionary process.

Establishing the genetic role of nucleic acids

For the development of molecular biology, the following discoveries were of the greatest importance. In 1944, American researchers O. Avery, K. McLeod (Nobel Prize, 1923) and M. McCarthy showed that DNA molecules isolated from pneumococci have transforming activity. After hydrolysis of these DNAs by deoxyribonuclease, their transforming activity completely disappeared. Thus, for the first time, it was convincingly proved that it is DNA, and not protein, that is endowed with genetic functions in a cell.

In fairness, it should be noted that the phenomenon of bacterial transformation was discovered much earlier than the discovery of Avery, McLeod and McCarthy. In 1928, F. Griffith published an article in which he reported that after adding killed cells of an encapsulated virulent strain to non-virulent (non-encapsulated) pneumococci, the resulting mixture of cells becomes fatal for mice. Moreover, live pneumococcal cells isolated from animals infected with this mixture were already virulent and possessed a polysaccharide capsule. Thus, in this experiment it was shown that under the influence of some components of the killed pneumococcal cells, the non-encapsulated form of bacteria turns into a capsule-forming virulent form. Sixteen years later, Avery, McLeod, and McCarthy replaced killed whole pneumococcal cells with their deoxyribonucleic acid in this experiment and showed that it was DNA that had transforming activity (see also chapters 7 and 25). The significance of this discovery is difficult to overestimate. It stimulated the study of nucleic acids in many laboratories around the world and forced scientists to focus on DNA.

Along with the discovery of Avery, McLeod, and McCarthy, by the beginning of the 1950s, a fairly large amount of direct and indirect evidence had already accumulated that nucleic acids play an exceptional role in life and carry a genetic function. This, in particular, was indicated by the nature of DNA localization in the cell and the data of R. Vendrelli (1948) that the DNA content per cell is strictly constant and correlates with the degree of ploidy: in haploid germ cells, DNA is half that in diploid somatic cells. The pronounced metabolic stability of DNA also testified in favor of the genetic role of DNA. By the beginning of the 1950s, a lot of various facts had accumulated, indicating that most of the known mutagenic factors act mainly on nucleic acids and, in particular, on DNA (R. Hotchkiss, 1949; G. Ephrussi-Taylor, 1951; E. Freese , 1957 and others).

Of particular importance in establishing the genetic role of nucleic acids was the study of various phages and viruses. In 1933, D. Schlesinger found DNA in the bacteriophage of Escherichia coli. Since the isolation of tobacco mosaic virus (TMV) in the crystalline state by W. Stanley (1935, Nobel Prize, 1946), a new stage in the study of plant viruses began. In 1937 - 1938. employees of the Rothamsted Agricultural Station (England) F. Bowden and N. Peary showed that many plant viruses isolated by them are not globulins, but are ribonucleoproteins and contain nucleic acid as an obligatory component. At the very beginning of the 40s, the works of G. Schramm (1940), P. A. Agatov (1941), G. Miller and W. Stanley (1941) were published, indicating that a noticeable chemical modification of the protein component does not lead to loss of TMV infectivity. This indicated that the protein component could not be the carrier of the hereditary properties of the virus, as many microbiologists continued to believe. Convincing evidence in favor of the genetic role of nucleic acid (RNA) in plant viruses was obtained in 1956 by G. Schramm in Tübingen (FRG) and H. Frenkel-Konrath in California (USA). These researchers almost simultaneously and independently of each other isolated RNA from TMV and showed that it, and not protein, has infectivity: as a result of infection of tobacco plants with this RNA, normal viral particles were formed and multiplied in them. This meant that RNA contained information for the synthesis and assembly of all viral components, including the viral protein. In 1968, I. G. Atabekov established that protein plays a significant role in the very infection of plants - the nature of the protein determines the spectrum of host plants.

In 1957, Frenkel-Konrat for the first time carried out the reconstruction of the TMV from its constituent components - RNA and protein. Along with normal particles, he received mixed "hybrids" in which the RNA was from one strain and the protein from another. The heredity of such hybrids was completely determined by RNA, and the progeny of the viruses belonged to the strain whose RNA was used to obtain the initial mixed particles. Later, the experiments of A. Gierer, G. Schuster and G. Schramm (1958) and G. Witman (1960 - 1966) showed that the chemical modification of the TMV nucleic component leads to the appearance of various mutants of this virus.

In 1970, D. Baltimore and G. Temin found that the transfer of genetic information can occur not only from DNA to RNA, but vice versa. They found in some oncogenic RNA-containing viruses (oncornaviruses) a special enzyme, the so-called reverse transcriptase, which is capable of synthesizing complementary DNA on RNA chains. This major discovery made it possible to understand the mechanism of insertion of the genetic information of RNA-containing viruses into the host genome and to take a fresh look at the nature of their oncogenic action.

Discovery of nucleic acids and study of their properties

The term nucleic acids was introduced by the German biochemist R. Altman in 1889, after these compounds were discovered in 1869 by the Swiss physician F. Miescher. Misher extracted the pus cells with dilute hydrochloric acid for several weeks and obtained almost pure nuclear material in the remainder. He considered this material to be a characteristic "substance of cell nuclei and called it nuclein. In its properties, nuclein differed sharply from proteins: it was more acidic, did not contain sulfur, but it contained a lot of phosphorus, it was readily soluble in alkalis, but did not dissolve in dilute acids.

Misher sent the results of his observations on nuclein to F. Goppe-Seyler for publication in a journal. The substance he described was so unusual (at that time only lecithin was known of all biological phosphorus-containing compounds) that Goppe-Seyler did not believe Misher's experiments, returned the manuscript to him and instructed his employees N. Plosh and N. Lyubavin to check his conclusions on other material . Miescher's work "On the chemical composition of pus cells" was published two years later (1871). At the same time, the works of Goppe-Seyler and his collaborators were published on the composition of pus cells, erythrocytes of birds, snakes, and other cells. Over the next three years, nuclein was isolated from animal cells and yeast.

In his work, Misher noted that a detailed study of different nucleins can lead to the establishment of differences between them, thereby anticipating the idea of ​​specificity of nucleic acids. While studying salmon milk, Misher found that the nuclein in them is in the form of salt and is associated with the main protein, which he called protamine.

In 1879, A. Kossel began to study nucleins in the laboratory of Goppe-Seyler. In 1881, he isolated hypoxanthine from nuclein, but at that time he still doubted the origin of this base and believed that hypoxanthine could be a degradation product of proteins. In 1891, among the products of nuclein hydrolysis, Kossel discovered adenine, guanine, phosphoric acid, and another substance with the properties of sugar. For research on the chemistry of nucleic acids, Kossel was awarded the Nobel Prize in 1910.

Further progress in deciphering the structure of nucleic acids is associated with the research of P. Levin and colleagues (1911 - 1934). In 1911, P. Levin and V. Jacobs identified the carbohydrate component of adenosine and guanosine; they found that these nucleosides contain D-ribose. In 1930, Lewin showed that the carbohydrate component of deoxyribonucleosides is 2-deoxy-D-ribose. From his work, it became known that nucleic acids are built from nucleotides, i.e., phosphorylated nucleosides. Levin believed that the main type of bond in nucleic acids (RNA) is the 2", 5" phosphodiester bond. This notion turned out to be wrong. Thanks to the work of the English chemist A. Todd (Nobel Prize, 1957) and his collaborators, as well as the English biochemists R. Markham and J. Smith, it became known in the early 50s that the main type of bond in RNA is 3", 5" - phosphodiester bond.

Lewin showed that different nucleic acids can differ in the nature of the carbohydrate component: some of them contain the sugar deoxyribose, while others contain ribose. In addition, these two types of nucleic acids differed in the nature of one of the bases: pentose-type nucleic acids contained uracil, and deoxypentose-type nucleic acids contained thymine. Deoxypentose nucleic acid (in modern terminology, deoxyribonucleic acid - DNA) was usually easily isolated in large quantities from the thymus (sweet gland) of calves. Therefore, it was called thymonucleic acid. The source of pentose-type nucleic acid (RNA) was mainly yeast and wheat germ. This type was often referred to as yeast nucleic acid.

In the early 1930s, the notion that plant cells were characterized by a yeast-type nucleic acid was rather firmly rooted, while thymonucleic acid was characteristic only of the nuclei of animal cells. The two types of nucleic acids, RNA and DNA, were then called plant and animal nucleic acids, respectively. However, as the early studies of A. N. Belozersky showed, such a division of nucleic acids is unjustified. In 1934, Belozersky first discovered thymonucleic acid in plant cells: from pea seedlings, he isolated and identified the thymine-pyrimidine base, which is characteristic of DNA. Then he discovered thymine in other plants (soybean seeds, beans). In 1936, A. N. Belozersky and I. I. Dubrovskaya isolated DNA preparatively from horse chestnut seedlings. In addition, a series of studies carried out in England in the 1940s by D. Davidson and co-workers convincingly showed that plant nucleic acid (RNA) is contained in many animal cells.

The widespread use of the cytochemical reaction for DNA developed by R. Felgen and G. Rosenbeck (1924) and the reaction of J. Brachet (1944) for RNA made it possible to quickly and unambiguously resolve the issue of the preferential localization of these nucleic acids in the cell. It turned out that DNA is concentrated in the nucleus, while RNA is predominantly concentrated in the cytoplasm. Later, it was found that RNA is contained both in the cytoplasm and in the nucleus, and in addition, cytoplasmic DNA was identified.

As for the question of the primary structure of nucleic acids, by the mid-1940s, P. Levin's idea was firmly established in science, according to which all nucleic acids are built according to the same type and consist of the same so-called tetranucleotide blocks. Each of these blocks, according to Lewin, contains four different nucleotides. The tetranucleotide theory of the structure of nucleic acids largely deprived these biopolymers of specificity. Therefore, it is not surprising that at that time all the specifics of living things were associated only with proteins, the nature of the monomers of which is much more diverse (20 amino acids).

The first gap in the theory of the tetranucleotide structure of nucleic acids was made by the analytical data of the English chemist J. Gouland (1945 - 1947). When determining the composition of nucleic acids by the base nitrogen, he did not obtain an equimolar ratio of bases, as it should have been according to Lewin's theory. Finally, the tetranucleotide theory of the structure of nucleic acids collapsed as a result of the research of E. Chargaff and his collaborators (1949 - 1951). To separate the bases released from DNA as a result of its acid hydrolysis, Chargaff used paper chromatography. Each of these bases was accurately determined spectrophotometrically. Chargaff noticed significant deviations from the equimolar ratio of bases in DNA of different origins and for the first time definitely stated that DNA has a pronounced species specificity. This ended the hegemony of the concept of protein specificity in the living cell. Analyzing DNA of different origins, Chargaff discovered and formulated unique patterns of DNA composition, which entered science under the name of Chargaff's rules. According to these rules, in all DNA, regardless of origin, the amount of adenine is equal to the amount of thymine (A = T), the amount of guanine is equal to the amount of cytosine (G = C), the amount of purines is equal to the amount of pyrimidines (G + A = C + T), the amount bases with 6-amino groups is equal to the number of bases with 6-keto groups (A + C = G + T). At the same time, despite such strict quantitative correspondences, DNA of different species differ in the value of the A+T:G+C ratio. In some DNA, the amount of guanine and cytosine prevails over the amount of adenine and thymine (Chargaff called these DNA GC-type DNA); other DNAs contained more adenine and thymine than guanine and cytosine (these DNAs were called AT-type DNA). The data obtained by Chargaff on the composition of DNA played an exceptional role in molecular biology. It was they that formed the basis for the discovery of the structure of DNA, made in 1953 by J. Watson and F. Crick.

Back in 1938, W. Astbury and F. Bell, using X-ray diffraction analysis, showed that the base planes in DNA should be perpendicular to the long axis of the molecule and resemble, as it were, a stack of plates lying on top of each other. With the improvement of the technique of X-ray diffraction analysis, by 1952 - 1953. accumulated information that made it possible to judge the length of individual bonds and the angles of inclination. This made it possible to represent with the greatest probability the nature of the orientation of the rings of pentose residues in the sugar-phosphate backbone of the DNA molecule. In 1952, S. Farberg proposed two speculative models of DNA, which represented a single-stranded molecule folded or twisted on itself. A no less speculative model of the structure of DNA was proposed in 1953 by L. Pauling (Nobel Prize winner, 1954) and R. Corey. In this model, three twisted strands of DNA formed a long helix, the core of which was represented by phosphate groups, and the bases were located outside of it. By 1953, M. Wilkins and R. Franklin obtained clearer X-ray diffraction patterns of DNA. Their analysis showed the complete failure of the models of Farberg, Pauling and Corey. Using Chargaff's data, comparing different combinations of molecular models of individual monomers and X-ray diffraction data, J. Watson and F. Crick in 1953 came to the conclusion that the DNA molecule must be a double-stranded helix. Chargaff's rules severely limited the number of possible ordered combinations of bases in the proposed DNA model; they suggested to Watson and Crick that there must be a specific base pairing in the DNA molecule - adenine with thymine, and guanine with cytosine. In other words, adenine in one strand of DNA always strictly corresponds to thymine in the other strand, and guanine in one strand necessarily corresponds to cytosine in the other. Thus, Watson and Crick were the first to formulate the extremely important principle of the complementary structure of DNA, according to which one DNA strand complements another, i.e., the base sequence of one strand uniquely determines the base sequence in the other (complementary) strand. It became obvious that already in the very structure of DNA lies the potential for its exact reproduction. This model of DNA structure is currently generally accepted. Crick, Watson and Wilkins were awarded the Nobel Prize in 1962 for deciphering the structure of DNA.

It should be noted that the idea of ​​a mechanism for the exact reproduction of macromolecules and the transmission of hereditary information originated in our country. In 1927, N. K. Koltsov suggested that during cell reproduction, the reproduction of molecules occurs by exact autocatalytic reproduction of the existing parent molecules. True, at that time Koltsov endowed this property not with DNA molecules, but with molecules of a protein nature, the functional significance of which was then unknown. Nevertheless, the very idea of ​​autocatalytic reproduction of macromolecules and the mechanism of transmission of hereditary properties turned out to be prophetic: it became the guiding idea of ​​modern molecular biology.

Conducted in the laboratory of A. N. Belozersky by A. S. Spirin, G. N. Zaitseva, B. F. Vanyushin, S. O. Uryson, A. S. Antonov and others variety of organisms fully confirmed the patterns discovered by Chargaff, and full compliance with the molecular model of the structure of DNA proposed by Watson and Crick. These studies have shown that the DNA of different bacteria, fungi, algae, actinomycetes, higher plants, invertebrates and vertebrates have a specific composition. Differences in the composition (the content of AT-base pairs) are especially pronounced in microorganisms, turning out to be an important taxonomic feature. In higher plants and animals, species variations in the composition of DNA are much less pronounced. But this does not mean that their DNA is less specific. In addition to the composition of bases, specificity is largely determined by their sequence in DNA chains.

Along with the usual bases, additional nitrogenous bases were found in DNA and RNA. Thus, G. White (1950) found 5-methylcytosine in the DNA of plants and animals, and D. Dunn and J. Smith (1958) found methylated adenine in some DNA. For a long time, methylcytosine was considered a hallmark of the genetic material of higher organisms. In 1968, A. N. Belozersky, B. F. Vanyushin and N. A. Kokurina found that it can also be found in the DNA of bacteria.

In 1964, M. Gold and J. Hurwitz discovered a new class of enzymes that carry out the natural modification of DNA - its methylation. After this discovery, it became clear that minor (contained in small amounts) bases arise already on the finished DNA polynucleotide chain as a result of specific methylation of cytosine and adenine residues in special sequences. In particular, according to B. F. Vanyushin, Ya. I. Buryanov, and A. N. Belozersky (1969), adenine methylation in E. coli DNA can occur in terminating codons. According to A. N. Belozersky and colleagues (1968 - 1970), as well as M. Meselson (USA) and V. Arber (Switzerland) (1965 - 1969), methylation gives unique individual features to DNA molecules and, in combination with the action of specific nucleases, is part of a complex mechanism that controls the synthesis of DNA in the cell. In other words, the nature of methylation of a particular DNA predetermines the question of whether it can multiply in a given cell.

Almost at the same time, the isolation and intensive study of DNA methylases and restriction endonucleases began; in 1969 - 1975 the nucleotide sequences recognized in DNA by some of these enzymes have been established (X. Boyer, X. Smith, S. Lynn, K. Murray). When different DNAs are hydrolyzed by a restriction enzyme, rather large fragments with identical "sticky" ends are cleaved. This makes it possible not only to analyze the structure of genes, as is done in small viruses (D. Nathans, S. Adler, 1973 - 1975), but also to construct various genomes. With the discovery of these specific restriction enzymes, genetic engineering has become a tangible reality. Embedded in small plasmid DNA genes of various origins are already easily introduced into various cells. So, a new type of biologically active plasmids was obtained, giving resistance to certain antibiotics (S. Cohen, 1973), ribosomal genes of a frog and Drosophila were introduced into Escherichia coli plasmids (J. Morrow, 1974; X. Boyer, D. Hogness, R. Davis , 1974 - 1975). Thus, real ways are open for obtaining fundamentally new organisms by introducing and integrating various genes into their gene pool. This discovery can be directed to the benefit of all mankind.

In 1952, G. White and S. Cohen discovered that the DNA of T-even phages contains an unusual base, 5-hydroxymethylcytosine. Later, from the works of E. Volkin and R. Sinsheimer (1954) and Cohen (1956), it became known that hydroxymethylcytosine residues can be completely or partially glucosidated, as a result of which the phage DNA molecule is protected from the hydrolytic action of nucleases.

In the early 1950s, from the works of D. Dunn and J. Smith (England), S. Zamenhof (USA) and A. Wacker (Germany), it became known that many artificial base analogues can be included in DNA, sometimes replacing up to 50% thymine. As a rule, these substitutions lead to errors in DNA replication, transcription and translation and to the appearance of mutants. Thus, J. Marmur (1962) found that the DNA of some phages contains oxymethyluracil instead of thymine. In 1963, I. Takahashi and J. Marmur discovered that the DNA of one of the phages contains uracil instead of thymine. Thus, another principle, according to which nucleic acids were previously separated, collapsed. Since the time of P. Levin's work, it has been believed that thymine is the hallmark of DNA, and uracil is the hallmark of RNA. It became clear that this sign is not always reliable, and the fundamental difference in the chemical nature of the two types of nucleic acids, as it seems today, is only the nature of the carbohydrate component.

In the study of phages, many unusual features of the organization of nucleic acids have been uncovered. Since 1953, it has been believed that all DNA are double-stranded linear molecules, while RNA is only single-stranded. This position was significantly shaken in 1961, when R. Sinsheimer discovered that the DNA of the phage φ X 174 is represented by a single-stranded circular molecule. However, later it turned out that in this form this DNA exists only in a vegetative phage particle, and the replicative form of the DNA of this phage is also double-stranded. In addition, it turned out to be quite unexpected that the RNA of some viruses can be double-stranded. This new type of macromolecular organization of RNA was discovered in 1962 by P. Gomatos, I. Tamm and other researchers in some animal viruses and in plant wound tumor virus. Recently, V. I. Agol and A. A. Bogdanov (1970) established that in addition to linear RNA molecules, there are also closed or cyclic molecules. They detected cyclic double-stranded RNA, in particular, in the encephalomyelocarditis virus. Thanks to the works of X. Deveaux, L. Tinoko, T. I. Tikhonenko, E. I. Budovsky and others (1960 - 1974), the main features of the organization (folding) of genetic material in bacteriophages became known.

In the late 1950s, the American scientist P. Doty found that heating causes DNA denaturation, which is accompanied by the breaking of hydrogen bonds between base pairs and the separation of complementary chains. This process has the character of a "spiral-coil" phase transition and resembles the melting of crystals. Therefore, Doty called the process of thermal denaturation of DNA DNA melting. With slow cooling, renaturation of molecules occurs, i.e., the reunification of complementary halves.

The principle of renaturation in 1960 was used by J. Marmur and K. Schildkraut to determine the degree of "hybridizability" of DNA of different microorganisms. Subsequently, E. Bolton and B. McCarthy improved this technique by proposing the method of the so-called DNA-agar columns. This method turned out to be indispensable in studying the degree of homology of the nucleotide sequence of different DNA and elucidating the genetic relationship of different organisms. The denaturation of DNA discovered by Doty in combination with the chromatography on methylated albumin described by J. Mandel and A. Hershey * (1960) and centrifugation in a density gradient (the method was developed in 1957 by M. Meselson, F. Stahl and D. Winograd) is widely used for separation, isolation and analysis of individual complementary DNA strands For example, W. Shibalsky (USA), using these techniques to separate the DNA of the lambda phage, showed in 1967 - 1969 that both phage chains are genetically active, and not one, as this was considered to be (S. Spiegelman, 1961). It should be noted that for the first time the idea of ​​the genetic significance of both DNA strands of the lambda phage was expressed in the USSR by SE Bresler (1961).

* (For their work on the genetics of bacteria and viruses, A. Hershey, together with M. Delbrück and S. Luria, were awarded the Nobel Prize in 1969.)

To understand the organization and functional activity of the genome, the determination of the DNA nucleotide sequence is of paramount importance. The search for methods for such determination is carried out in many laboratories around the world. Since the late 1950s, M. Beer and his collaborators have been trying to establish the DNA sequence using electron microscopy in the USA, but so far without success. In the early 1950s, from the first works of Sinsheimer, Chargaff, and other researchers on the enzymatic degradation of DNA, it became known that different nucleotides in a DNA molecule are distributed, although not randomly, but unevenly. According to the English chemist C. Barton (1961), pyrimidines (more than 70%) are concentrated mainly in the form of the corresponding blocks. A. L. Mazin and B. F. Vanyushin (1968 - 1969) found that different DNAs have different degrees of pyrimidine cohesion and that in the DNA of animal organisms it increases markedly as it moves from lower to higher. Thus, the evolution of organisms is also reflected in the structure of their genomes. That is why, for understanding the evolutionary process as a whole, the comparative study of the structure of nucleic acids is of particular importance. Analysis of the structure of biologically important polymers and, first of all, DNA is extremely important for solving many particular problems of phylogenetics and taxonomy.

It is interesting to note that the English physiologist E. Lankester, who studied the hemoglobins of mollusks, anticipated the ideas of molecular biology exactly 100 years ago, wrote: “Chemical differences between different species and genera of animals and plants are as important for clarifying the history of their origin as their form. If we could clearly establish the differences in the molecular organization and functioning of organisms, we would be able to understand the origin and evolution of different organisms much better than on the basis of morphological observations " * . The significance of biochemical studies for taxonomy was also emphasized by VL Komarov, who wrote that "the basis of all even purely morphological features, on the basis of which we classify and establish species, are precisely biochemical differences" ** .

* (E. R. Lankester. Uber das Vorcommen von Haemoglobin in den Muskeln der Mollusken und die Verbreitung desselben in den lebendigen Organismen.- "Pfluger" s Archiv fur die gesammte Physiol., 1871, Bd 4, 319.)

** (V. L. Komarov. Selected works, vol. 1. M.-L., Publishing House of the Academy of Sciences of the USSR, 1945, p. 331.)

A. V. Blagoveshchenskii and S. L. Ivanov, back in the 1920s, took the first steps in our country to elucidate certain questions of the evolution and systematics of organisms on the basis of a comparative analysis of their biochemical composition (see Chapter 2). Comparative analysis of the structure of proteins and nucleic acids is now becoming an increasingly tangible tool for taxonomists (see Chapter 21). This method of molecular biology allows not only to clarify the position of individual species in the system, but also makes it necessary to take a fresh look at the very principles of classification of organisms, and sometimes to revise the entire system as a whole, as happened, for example, with the systematics of microorganisms. Undoubtedly, in the future, the analysis of the structure of the genome will occupy a central place in the chemosystematics of organisms.

Of great importance for the development of molecular biology was the deciphering of the mechanisms of DNA replication and transcription (see Chapter 24).

Protein biosynthesis

An important shift in solving the problem of protein biosynthesis is associated with advances in the study of nucleic acids. In 1941, T. Kasperson (Sweden) and in 1942, J. Brachet (Belgium) drew attention to the fact that tissues with active protein synthesis contain an increased amount of RNA. They concluded that ribonucleic acids play a decisive role in protein synthesis. In 1953, E. Gale and D. Fox seem to have received direct evidence of the direct involvement of RNA in protein biosynthesis: according to their data, ribonuclease significantly suppressed the incorporation of amino acids in bacterial cell lysates. Similar data were obtained by V. Olfri, M. Delhi and A. Mirsky (1953) on liver homogenates. Later, E. Gale rejected the correct idea he had expressed about the leading role of RNA in protein synthesis, mistakenly believing that the activation of protein synthesis in a cell-free system occurred under the influence of some other substance of an unknown nature. In 1954, P. Zamechnik, D. Littlefield, R. B. Khesin-Lurie and others found that the most active incorporation of amino acids occurs in RNA-rich fractions of subcellular particles - microsomes. P. Zamechnik and E. Keller (1953 - 1954) found that the incorporation of amino acids was noticeably enhanced in the presence of the supernatant under conditions of ATP regeneration. P. Sikevitz (1952) and M. Hoagland (1956) isolated a protein fraction (pH 5 fraction) from the supernatant, which was responsible for the sharp stimulation of the inclusion of amino acids in microsomes. Along with proteins, a special class of low molecular weight RNAs, now called transfer RNAs (tRNAs), was found in the supernatant. In 1958, Hoagland and Zamechnik, as well as P. Berg, R. Sweet and F. Allen, and many other researchers found that each amino acid requires its own special enzyme, ATP, and specific tRNA to activate. It became clear that tRNAs perform exclusively the function of adapters, i.e. devices that find a place on the nucleic matrix (mRNA) for the corresponding amino acid in the emerging protein molecule. These studies fully confirmed the adapter hypothesis of F. Crick (1957), which provided for the existence in the cell of polynucleotide adapters necessary for the correct arrangement of the amino acid residues of the synthesized protein on the nucleic matrix. Much later, the French scientist F. Chapville (1962) in the laboratory of F. Lipman (Nobel Prize, 1953) in the USA very ingeniously and unequivocally showed that the location of an amino acid in a synthesized protein molecule is completely determined by the specific tRNA to which it is attached. Crick's adaptor hypothesis was developed by Hoagland and Zamechnik.

By 1958, the following main stages of protein synthesis became known: 1) activation of an amino acid by a specific enzyme from the “pH 5 fraction” in the presence of ATP with the formation of aminoacyl adenylate; 2) attachment of an activated amino acid to a specific tRNA with the release of adenosine monophosphate (AMP); 3) binding of aminoacyl-tRNA (tRNA loaded with an amino acid) to microsomes and incorporation of amino acids into a protein with tRNA release. Hoagland (1958) noted that guanosine triphosphate (GTP) is required at the last stage of protein synthesis.

Transfer RNAs and gene synthesis

After the discovery of tRNAs, active searches for their fractionation and determination of the nucleotide sequence began. The American biochemist R. Holly achieved the greatest success. In 1965, he established the structure of alanine tRNA from yeast. Using ribonucleases (guanyl RNase and pancreatic RNase), Holly divided the nucleic acid molecule into several fragments, determined the nucleotide sequence in each of them separately, and then reconstructed the sequence of the entire alanine tRNA molecule. This way of analyzing the nucleotide sequence is called the block method. Holly's merit consisted mainly in the fact that he learned to divide the RNA molecule not only into small pieces, as many did before him, but also into large fragments (quarters and halves). This gave him the opportunity to properly assemble individual small pieces together and thereby recreate the complete nucleotide sequence of the entire tRNA molecule (Nobel Prize, 1968).

This technique was immediately adopted by many laboratories around the world. Over the next two years, the primary structure of several tRNAs was deciphered in the USSR and abroad. A. A. Baev (1967) and co-workers established the nucleotide sequence in yeast valine tRNA for the first time. To date, more than a dozen different individual tRNAs have been studied. A peculiar record in determining the nucleotide sequence was set in Cambridge by F. Senger and G. Brownlee. These researchers developed a surprisingly elegant method for separating oligonucleotides and sequencing the so-called 5 S (ribosomal) RNA from E. coli cells (1968). This RNA consists of 120 nucleotide residues and, unlike tRNA, does not contain additional minor bases, which greatly facilitate the analysis of the nucleotide sequence, serving as unique landmarks for individual fragments of the molecule. At present, thanks to the use of the Sanger and Brownlee method, work on the study of the sequence of long ribosomal RNAs and some viral RNAs is being successfully advanced in the laboratory of J. Ebel (France) and other researchers.

A. A. Baev and colleagues (1967) found that valine tRNA cut in half restores its macromolecular structure in solution and, despite a defect in the primary structure, has the functional activity of the original (native) molecule. This approach - the reconstruction of a cut macromolecule after the removal of certain fragments - turned out to be very promising. It is now widely used to elucidate the functional role of individual sections of certain tRNAs.

In recent years, great success has been achieved in obtaining crystalline preparations of individual tRNAs. Many tRNAs have already been crystallized in several laboratories in the USA and England. This made it possible to study the structure of tRNA using X-ray diffraction analysis. In 1970, R. Bock presented the first X-ray patterns and three-dimensional models of several tRNAs that he had created at the University of Wisconsin. These models help determine the localization of individual functionally active sites in tRNA and understand the basic principles of the functioning of these molecules.

Of paramount importance for revealing the mechanism of protein synthesis and solving the problem of the specificity of this process was the deciphering of the nature of the genetic code (see Chapter 24), which, without exaggeration, can be considered as the leading achievement of the natural sciences of the 20th century.

R. Holly's discovery of the primary structure of tRNA gave impetus to the work of G. Korana * (USA) on the synthesis of oligonucleotides and directed them towards the synthesis of a specific biological structure - a DNA molecule encoding alanine tRNA. The first steps in the chemical synthesis of short oligonucleotides made by the Qur'an almost 15 years ago culminated in 1970 with the first gene synthesis. Koran and his collaborators first chemically synthesized short fragments of 8-12 nucleotide residues from individual nucleotides. These fragments with a given nucleotide sequence formed spontaneously double-stranded complementary pieces with an overlap of 4–5 nucleotides. Then these ready-made pieces were joined end-to-end in the right order using the enzyme DNA ligase. Thus, in contrast to the replication of DNA molecules, according to A. Kornberg ** (see Chapter 24), the Qur'an managed to re-create a natural double-stranded DNA molecule according to a pre-planned program in accordance with the tRNA sequence described by Holly. Similarly, work is now underway on the synthesis of other genes (M. N. Kolosov, Z. A. Shabarova, D. G. Knorre, 1970 - 1975).

* (For the study of the genetic code, G. Koran and M. Nirenberg were awarded the Nobel Prize in 1968.)

** (For the discovery of polymerase and DNA synthesis A. Kornberg, and for the synthesis of RNA S. Ochoa in 1959 was awarded the Nobel Prize.)

Microsomes, ribosomes, translation

In the mid-1950s, it was believed that microsomes were the center of protein synthesis in the cell. The term microsomes was first introduced in 1949 by A. Claude to refer to the fraction of small granules. Later it turned out that not the entire fraction of microsomes, consisting of membranes and granules, but only small ribonucleoprotein particles, is responsible for protein synthesis. These particles in 1958 were called ribosomes by R. Roberts.

Classical studies of bacterial ribosomes were carried out by A. Tisier and J. Watson in 1958-1959. Bacterial ribosomes turned out to be somewhat smaller than plant and animal ones. J. Littleton (1960), M. Clark (1964), and E. N. Svetailo (1966) showed that the ribosomes of the chloroplasts of higher plants and mitochondria belong to the bacterial type. A. Tisier and others (1958) found that ribosomes dissociate into two unequal subunits containing one RNA molecule each. In the late 50s, it was believed that each ribosomal RNA molecule consists of several short fragments. However, AS Spirin in 1960 was the first to show that RNA in subparticles is represented by a continuous molecule. D. Waller (1960), having separated ribosomal proteins using starch gel electrophoresis, found that they are very heterogeneous. At first, many doubted Waller's data, since it seemed that the ribosome protein should be strictly homogeneous, like, for example, the TMV protein. At present, as a result of the research of D. Waller, R. Trout, P. Traub and other biochemists, it has become known that the composition of the actual ribosomal particles includes more than 50 proteins that are completely different in structure. AS Spirin in 1963 was the first to unfold ribosomal subparticles and show that ribosomes are a compactly twisted ribonucleoprotein strand, which can unfold under certain conditions. In 1967 - 1968 M. Nomura completely reconstructed a biologically active subunit from ribosomal RNA and protein and even obtained ribosomes in which protein and RNA belonged to different microorganisms.

The role of ribosomal RNA is still unclear. It is assumed that it is that unique specific matrix on which, during the formation of a ribosomal particle, each of the numerous ribosomal proteins finds a strictly defined place (AS Spirin, 1968).

A. Rich (1962) discovered aggregates of several ribosomes interconnected by a strand of mRNA. These complexes were called polysomes. The discovery of polysomes allowed Rich and Watson (1963) to suggest that the synthesis of the polypeptide chain occurs on the ribosome, which, as it were, moves along the mRNA chain. As the ribosome moves along the mRNA chain, information is read out in the particle and the protein polypeptide chain is formed, and new ribosomes alternately attach to the released read end of the mRNA. From the data of Rich and Watson, it followed that the significance of polysomes in a cell lies in the mass production of protein by successive reading of the matrix by several ribosomes at once.

As a result of the research of M. Nirenberg, S. Ochoa, F. Lipman, G. Korana and others in 1963 - 1970. it became known that along with mRNA, ribosomes, ATP and aminoacyl-tRNA, a large number of various factors take part in the translation process, and the translation process itself can be conditionally divided into three stages - initiation, translation itself and termination.

Translation initiation means the synthesis of the first peptide bond in the complex ribosome - template polynucleotide - aminoacyl-tRNA. Such initiatory activity is possessed not by any aminoacyl-tRNA, but by formylmethionyl-tRNA. This substance was first isolated in 1964 by F. Senger and K. Marker. S. Bretcher and K. Marker (1966) showed that the initiatory function of formylmethionyl-tRNA is due to its increased affinity for the peptidyl center of the ribosome. For the start of translation, some protein initiation factors are also extremely important, which were isolated in the laboratories of S. Ochoa, F. Gro and other research centers. After the formation of the first peptide bond in the ribosome, translation itself begins, i.e., the sequential addition of an aminoacyl residue to the C-terminus of the polypeptide. Many details of the translation process were studied by K. Monroe and J. Bishop (England), I. Rykhlik and F. Shorm (Czechoslovakia), F. Lipman, M. Bretcher, W. Gilbert (USA) and other researchers. In 1968, A. S. Spirin proposed an original hypothesis to explain the mechanism of the ribosome. The driving mechanism that ensures all spatial movements of tRNA and mRNA during translation is the periodic opening and closing of ribosome subparticles. The translation termination is encoded in the readable matrix itself, which contains the termination codons. As shown by S. Brenner (1965 - 1967), triplets UAA, UAG and UGA are such codons. M. Capecci (1967) also identified special protein termination factors. AS Spirin and LP Gavrilova described the so-called "non-enzymatic" protein synthesis in ribosomes (1972 - 1975) without the participation of protein factors. This discovery is important for understanding the origin and evolution of protein biosynthesis.

Regulation of gene and protein activity

After the problem of the specificity of protein synthesis, the problem of regulation of protein synthesis, or, what is the same, regulation of gene activity, turned out to be in the first place in molecular biology.

The functional inequivalence of cells and the repression and activation of genes associated with it have long attracted the attention of geneticists, but until recently the real mechanism for controlling gene activity remained unknown.

The first attempts to explain the regulatory activity of genes were associated with the study of histone proteins. Even the Steadman spouses * in the early 40s of the XX century. suggested that it is histones that can play the main role in this phenomenon. Subsequently, they obtained the first clear data on differences in the chemical nature of histone proteins. At present, the number of facts testifying in favor of this hypothesis is increasing every year.

* (E. Stedman, E. Stedman. The basic proteins of cell nuclei.- Philosoph. Trans. Roy. soc. London, 1951, v. 235, 565 - 595.)

At the same time, an increasing amount of data is accumulating, indicating that the regulation of gene activity is a much more complex process than the simple interaction of gene sections with histone protein molecules. In 1960 - 1962 in the laboratory of R. B. Khesin-Lurie, it was found that the phage genes begin to be read non-simultaneously: the T2 phage genes can be divided into early genes, the functioning of which occurred in the first minutes of infection of a bacterial cell, and late ones, which began to synthesize mRNA after the completion of the work of early genes.

In 1961, the French biochemists F. Jacob and J. Monod proposed a scheme for the regulation of gene activity, which played an exceptional role in understanding the regulatory mechanisms of the cell in general. According to the scheme of Jacob and Monod, in addition to structural (informational) genes, DNA also contains genes-regulators and genes-operators. The regulator gene encodes the synthesis of a specific substance - a repressor, which can attach both to the inducer and to the operator gene. The operator gene is linked to structural genes, while the regulator gene is located at some distance from them. If there is no inductor in the environment, for example, lactose, then the repressor synthesized by the regulator gene binds to the operator gene and, blocking it, turns off the work of the entire operon (a block of structural genes together with the operator that controls them). Enzyme formation does not occur under these conditions. If an inductor (lactose) appears in the medium, then the product of the regulator gene, the repressor, binds to lactose and removes the block from the operator gene. In this case, the work of the structural gene encoding the synthesis of the enzyme becomes possible, and the enzyme (lactose) appears in the medium.

According to Jacob and Monod, this regulation scheme is applicable to all adaptive enzymes and can take place both during repression, when the formation of the enzyme is suppressed by an excess of the reaction product, and during induction, when the introduction of a substrate causes the synthesis of the enzyme. For studies of the regulation of gene activity, Jacob and Monod were awarded the Nobel Prize in 1965.

Initially, this scheme seemed too far-fetched. However, later it turned out that the regulation of genes according to this principle takes place not only in bacteria, but also in other organisms.

Since 1960, a prominent place in molecular biology has been occupied by studies of the organization of the genome and the structure of chromatin in eukaryotic organisms (J. Bonner, R. Britten, W. Olfrey, P. Walker, Yu. S. Chentsov, I. B. Zbarsky and others .) and regulation of transcription (A. Mirsky, G. P. Georgiev, M. Bernstiel, D. Goll, R. Tsanev, R. I. Salganik). For a long time, the nature of the repressor remained unknown and controversial. In 1968, M. Ptashne (USA) showed that a protein is a repressor. He isolated it in the laboratory of J. Watson and found that the repressor really has an affinity for the inductor (lactose) and at the same time "recognizes" the operator gene of the lac operon and specifically binds to it.

In the last 5 - 7 years, data have been obtained on the presence of another control cell of gene activity - the promoter. It turned out that next to the operator site, to which the product synthesized on the gene-regulator - the protein substance of the repressor, is attached, there is another site, which should also be attributed to the members of the regulatory system of gene activity. A protein molecule of the enzyme RNA polymerase is attached to this site. In the promoter region, mutual recognition of the unique nucleotide sequence in DNA and the specific configuration of the RNA polymerase protein must occur. The implementation of the process of reading genetic information with a given sequence of genes of the operon adjacent to the promoter will depend on the recognition efficiency.

In addition to the scheme described by Jacob and Monod, there are other mechanisms of gene regulation in the cell. F. Jacob and S. Brenner (1963) established that the regulation of bacterial DNA replication is controlled in a certain way by the cell membrane. The experiments of Jacob (1954) on the induction of various prophages convincingly showed that under the influence of various mutagenic factors in the cell of lysogenic bacteria, selective replication of the prophage gene begins, and replication of the host genome is blocked. In 1970, F. Bell reported that small DNA molecules can pass from the nucleus into the cytoplasm and be transcribed there.

Thus, gene activity can be regulated at the level of replication, transcription, and translation.

Significant progress has been made in studying the regulation of not only the synthesis of enzymes, but also their activity. A. Novik and L. Szilard pointed out the phenomena of regulation of the activity of enzymes in the cell back in the 1950s. G. Umbarger (1956) found that in the cell there is a very rational way to suppress the activity of the enzyme by the end product of the feedback chain of reactions. As established by J. Monod, J. Change, F. Jacob, A. Purdy and other researchers (1956 - 1960), the regulation of enzyme activity can be carried out according to the allosteric principle. The enzyme or one of its subunits, in addition to affinity for the substrate, has an affinity for one of the products of the reaction chain. Under the influence of such a signal product, the enzyme changes its conformation in such a way that it loses activity. As a result, the entire chain of enzymatic reactions is switched off at the very beginning. D. Wieman and R. Woodward (1952; Nobel Prize winner, 1965) pointed out the essential role of protein conformational changes in enzymatic reactions, and in a certain sense, the presence of an allosteric effect.

Structure and function of proteins

As a result of the work of T. Osborn, G. Hofmeister, A. Gurber, F. Schulz and many others at the end of the 19th century. Many animal and vegetable proteins have been obtained in crystalline form. Around the same time, using various physical methods, the molecular weights of some proteins were established. So, in 1891, A. Sabaneev and N. Alexandrov reported that the molecular weight of ovalbumin is 14,000; in 1905, E. Reid found that the molecular weight of hemoglobin is 48,000. The polymeric structure of proteins was discovered in 1871 by G. Glasivetz and D. Gaberman. The idea of ​​a peptide bond of individual amino acid residues in proteins was put forward by T. Curtius (1883). Work on the chemical condensation of amino acids (E. Schaal, 1871; G. Schiff, 1897; L. Balbiano and D. Traschiatti, 1900) and the synthesis of heteropolypeptides (E. Fisher, 1902 - 1907, Nobel Prize, 1902) led to the development of the basic principles the chemical structure of proteins.

The first crystalline enzyme (urease) was obtained in 1926 by J. Sumner (Nobel Prize, 1946), and in 1930 J. Northrop (Nobel Prize, 1946) obtained crystalline pepsin. After these works, it became clear that enzymes are of a protein nature. In 1940, M. Kunits isolated crystalline RNase. By 1958, more than 100 crystalline enzymes and over 500 non-crystalline enzymes were already known. Obtaining highly purified preparations of individual proteins contributed to the deciphering of their primary structure and macromolecular organization.

Of great importance for the development of molecular biology in general and human genetics, in particular, was the discovery by L. Pauling (1940) of abnormal hemoglobin S, isolated from the erythrocytes of people with a severe hereditary disease, sickle cell anemia. In 1955 - 1957 W. Ingram used the "fingerprint" method developed by F. Sanger (spots formed by individual peptides during chromatography on paper) to analyze the products of hydrolysis of hemoglobin S with alkali and trypsin. In 1961, Ingram reported that hemoglobin S differs from normal hemoglobin only in the nature of one amino acid residue: in normal hemoglobin, a glutamic acid residue is in the seventh position of the chain, and in hemoglobin S, a valine residue. Thus, Pauling's assumption (1949) that sickle cell anemia is a disease of a molecular nature was fully confirmed. An inherited change in just one amino acid residue in each half of the hemoglobin macromolecule leads to the fact that hemoglobin loses its ability to dissolve easily at a low oxygen concentration and begins to crystallize, which leads to disruption of the cell structure. These studies clearly showed that the structure of a protein is a strictly defined amino acid sequence that is encoded in the genome. The works of K. Anfinsen (1951) testified to the exceptional importance of the primary structure of a protein in the formation of a unique biologically active conformation of a macromolecule. Anfinsen showed that the biologically active macrostructure of pancreatic ribonuclease, which is lost as a result of restoration, is predetermined by the amino acid sequence and can reappear spontaneously during the oxidation of SH groups of cysteine ​​residues with the formation of disulfide crosslinks in strictly defined places of the peptide chain of the enzyme.

To date, the mechanism of action of a large number of enzymes has been studied in detail and the structure of many proteins has been determined.

In 1953, F. Sanger established the amino acid sequence of insulin. : This protein consists of two polypeptide chains connected by two disulfide crosslinks. One of the chains contains only 21 amino acid residues, while the other contains 30 residues. Sanger spent about 10 years deciphering the structure of this relatively simple protein. In 1958 he was awarded the Nobel Prize for this outstanding research. After the creation by V. Stein and S. Moore (1957) of an automatic analyzer of amino acids, the identification of products of partial hydrolysis of proteins accelerated significantly. In 1960, Stein and Moore already reported that. that they were able to determine the sequence of ribonuclease, the peptide chain of which is represented by 124 amino acid residues. In the same year, in the laboratory of G. Schramm in Tübingen (Germany), F. Anderer and others determined the amino acid sequence in the TMV protein. Then the amino acid sequence was determined in myoglobin (A. Edmunson) and α- and β-chains of human hemoglobin (G. Braunitzer, E. Schroeder, etc.), lysozyme from egg protein (J. Jollet, D. Keyfield). In 1963, F. Shorm and B. Keil (Czechoslovakia) established the sequence of amino acids in the chymotrypsinogen molecule. In the same year, the amino acid sequence of trypsinogen was determined (F. Shorm, D. Walsh). In 1965, K. Takahashi established the primary structure of ribonuclease T1. Then the amino acid sequence was determined for several more proteins.

As is known, the final proof of the correctness of the definition of a particular structure is its synthesis. In 1969, R. Merifield (USA) was the first to carry out the chemical synthesis of pancreatic ribonuclease. Using the method of synthesis he developed on a solid-phase carrier, Merifield added one amino acid after another to the chain in accordance with the sequence that was described by Stein and Moore. As a result, he received a protein that was identical in quality to pancreatic ribonuclease A. For the discovery of the structure of ribonuclease, V. Stein, S. Moore and K. Anfinsen were awarded the Nobel Prize in 1972. This natural protein synthesis opens up tremendous prospects, pointing to the possibility of creating any proteins in accordance with a pre-planned sequence.

From X-ray studies by W. Astbury (1933) it followed that the peptide chains of protein molecules are twisted or stacked in some strictly defined way. Since that time, many authors have expressed various hypotheses about the ways in which protein chains are folded, but until 1951, all models remained speculative constructions that did not correspond to experimental data. In 1951, L. Pauling and R. Corey published a series of brilliant works in which the theory of the secondary structure of proteins, the theory of the α-helix, was finally formulated. Along with this, it also became known that proteins also have a tertiary structure: the α-helix of the peptide chain can be folded in a certain way, forming a rather compact structure.

In 1957, J. Kendrew and his collaborators first proposed a three-dimensional model of the structure of myoglobin. This model was then refined over several years, until the final work appeared in 1961 with a characterization of the spatial structure of this protein. In 1959, M. Perutz and colleagues established the three-dimensional structure of hemoglobin. Researchers spent more than 20 years on this work (the first x-rays of hemoglobin were obtained by Perutz in 1937). Since the hemoglobin molecule consists of four subunits, having deciphered its organization, Perutz thereby first described the quaternary structure of the protein. For their work on the determination of the three-dimensional structure of proteins, Kendrew and Perutz were awarded the Nobel Prize in 1962.

The creation of a spatial model of the structure of hemoglobin by Perutz ALLOWED. to come closer to understanding the mechanism of functioning of this protein, which, as is known, carries out oxygen transport in animal cells. Back in 1937, F. Gaurowitz came to the conclusion that the interaction of hemoglobin with oxygen, air should be accompanied by a change in the structure of the protein. In the 1960s, Perutz and co-workers discovered a noticeable shift in the hemoglobin chains after its oxidation, caused by the shift of iron atoms as a result of binding with oxygen. On this basis, ideas about the "breathing" of protein macromolecules were formed.

In 1960, D. Phillips and his collaborators began X-ray diffraction studies of the lysozyme molecule. By 1967, they were more or less able to establish the details of the organization of this protein and the localization of individual atoms in its molecule. In addition, Phillips found out the nature of the addition of lysozyme to the substrate (triacetylglucosamine). This made it possible to recreate the mechanism of this enzyme. Thus, knowledge of the primary structure and macromolecular organization made it possible not only to establish the nature of the active centers of many enzymes, but also to fully reveal the mechanism of functioning of these macromolecules.

The use of electron microscopy methods helped to reveal the principles of the macromolecular organization of such complex protein formations as collagen, fibrinogen, contractile muscle fibrils, etc. At the end of the 1950s, models of the muscular contractile apparatus were proposed. Of exceptional importance for understanding the mechanism of muscle contraction was the discovery by V. A. Engelgardt and M. N. Lyubimova (1939) of the ATPase activity of myosin. This meant that the act of muscle contraction is based on a change in the physicochemical properties and macromolecular organization of the contractile protein under the influence of adenosine triphosphoric acid (see also Chapter 11).

Virological research has been essential to understanding the principles of assembling biological structures (see Chapter 25).

Unresolved issues

The main advances in modern molecular biology have been achieved mainly as a result of the study of nucleic acids. However, even in this area far from all problems have been resolved. Great efforts will be required, in particular, to decipher the entire nucleotide sequence of the genome. This problem, in turn, is inextricably linked with the problem of DNA heterogeneity and requires the development of new advanced methods for fractionation and isolation of individual molecules from the total genetic material of the cell.

Until now, efforts have mainly been focused on the separate study of proteins and nucleic acids. In the cell, these biopolymers are inextricably linked with each other and function mainly in the form of nucleoproteins. Therefore, the need to study the interaction of proteins and nucleic acids has now become particularly acute. The problem of recognition of certain sections of nucleic acids by proteins is brought to the fore. Steps have already been outlined towards studying such an interaction of these biopolymers, without which a complete understanding of the structure and functions of chromosomes, ribosomes, and other structures is unthinkable. Without this, it is also impossible to understand the regulation of gene activity and finally decipher the principles of the work of protein-synthesizing mechanisms. After the work of Jacob and Monod, some new data appeared on the regulatory significance of membranes in the synthesis of nuclear material. This poses the problem of a deeper study of the role of membranes in the regulation of DNA replication. In general, the problem of regulation of gene activity and cell activity in general has become one of the most important problems of modern molecular biology.

The current state of biophysics

In close connection with the problems of molecular biology, the development of biophysics proceeded. Interest in this area of ​​biology was stimulated, on the one hand, by the need for a comprehensive study of the effect of various types of radiation on the body, and, on the other hand, by the need to study the physical and physico-chemical foundations of life phenomena occurring at the molecular level.

Obtaining accurate information about molecular structures and the processes taking place in them became possible as a result of the use of new fine physical and chemical methods. Based on the achievements of electrochemistry, it was possible to improve the method of measuring bioelectric potentials by using ion-selective electrodes (G. Eisenman, B.P. Nikolsky, Khuri, 50-60s). Increasingly, infrared spectroscopy (with the use of laser devices) is coming into practice, which makes it possible to study the conformational changes in proteins (I. Plotnikov, 1940). Valuable information is also provided by the method of electron paramagnetic resonance (E. K. Zavoisky, 1944) and the biochemiluminescent method (B. N. Tarusov et al., 1960), which make it possible, in particular, to judge the transport of electrons during oxidative processes.

By the 1950s, biophysics was already gaining a strong position. There is a need to train qualified specialists. If in 1911 in Europe only the University of Pécs, in Hungary, had a chair of biophysics, then by 1973 such chairs exist in almost all major universities.

In 1960, the International Society of Biophysicists was organized. In August 1961, the first International Biophysical Congress took place in Stockholm. The second congress was held in 1965 in Paris, the third - in 1969 in Boston, the fourth - in 1972 in Moscow.

In biophysics, there is a clear distinction between two areas of different content - molecular biophysics and cellular biophysics. This distinction also receives an organizational expression: separate departments of these two areas of biophysics are being created. At Moscow University, the first department of biophysics was created in 1953 at the Faculty of Biology and Soil Science, and a little later the Department of Biophysics appeared at the Faculty of Physics. Departments were organized on the same principle in many other universities.

Molecular biophysics

In recent years, the connection between molecular biophysics and molecular biology has been increasingly strengthened, and it is now sometimes difficult to determine where the dividing line between them lies. In a general attack on the problem of hereditary information, such cooperation between biophysics and molecular biology is inevitable.

The main direction in the research work is the study of the physics of nucleic acids - DNA and RNA. The use of the above methods and, above all, X-ray diffraction analysis contributed to the deciphering of the molecular structure of nucleic acids. Currently, intensive research is underway to study the behavior of these acids in solutions. Particular attention is paid to the "helix-coil" conformational transitions, which are studied by changes in viscosity, optical and electrical parameters. In connection with the study of the mechanisms of mutagenesis, studies are being developed to study the effect of ionizing radiation on the behavior of nucleic acids in solutions, as well as the effect of radiation on the nucleic acids of viruses and phages. The effect of ultraviolet radiation, some spectral regions of which are known to be well absorbed by nucleic acids, was subjected to a comprehensive analysis. A large share in this kind of research is the detection of active radicals of nucleic acids and proteins by the method of electron paramagnetic resonance. With the use of this method, the emergence of a whole independent direction is associated.

The problem of encoding DNA and RNA information and its transmission during protein synthesis has long been of interest to molecular biophysics, and physicists have repeatedly expressed certain considerations on this subject (E. Schrödinger, G. Gamow). The deciphering of the genetic code caused numerous theoretical and experimental studies on the structure of the DNA helix, the mechanism of sliding and twisting of its threads, and the study of the physical forces involved in these processes.

Molecular biophysics provides considerable assistance to molecular biology in studying the structure of protein molecules with the help of X-ray diffraction analysis, which was first used in 1930 by J. Bernal. It was as a result of the use of physical methods in combination with biochemical (enzymatic methods) that the molecular conformation and the sequence of amino acids in a number of proteins were revealed.

Modern electron microscopic studies, which revealed the presence of complex membrane systems in cells and its organelles, stimulated attempts to understand their molecular structure (see Chapters 10 and 11). The chemical composition of membranes and, in particular, the properties of their lipids are studied in vivo. It was found that the latter are capable of overoxidation and non-enzymatic reactions of chain oxidation (Yu. A. Vladimirov and F. F. Litvin, 1959; B. N. Tarusov et al., 1960; I. I. Ivanov, 1967), leading to membrane dysfunction. To study the composition of membranes, methods of mathematical modeling also began to be used (V. Ts. Presman, 1964 - 1968; M. M. Shemyakin, 1967; Yu. A. Ovchinnikov, 1972).

Cellular biophysics

A significant event in the history of biophysics was the formation in the 50s of clear ideas about the thermodynamics of biological processes, as a result of which the assumptions about the possibility of independent energy generation in living cells, contrary to the second law of thermodynamics, finally disappeared. Understanding the operation of this law in biological systems is associated with the introduction by the Belgian scientist I. Prigogine (1945) * into biological thermodynamics of the concept of open systems exchanging energy and matter with the external environment. Prigogine showed that positive entropy is formed in living cells during working processes in accordance with the second law of thermodynamics. The equations he introduced determined the conditions under which the so-called stationary state arises (previously it was also called dynamic equilibrium), in which the amount of free energy (negentropy) entering the cells with food compensates for its consumption, and positive entropy is output. This discovery reinforced the general biological idea of ​​the inseparable connection between the external and internal environment of cells. It marked the beginning of a real study of the thermodynamics of living systems, including the modeling method (A. Burton, 1939; A. G. Pasynsky, 1967).

* (The general theory of open systems was first put forward by L. Bertalanffy in 1932.)

According to the basic principle of biothermodynamics, a necessary condition for the existence of life is stationarity in the development of its biochemical processes, for the implementation of which it is necessary to coordinate the rates of numerous metabolic reactions. On the basis of the new biophysical thermodynamics, a trend has emerged that singles out external and internal factors that ensure this coordination of reactions and make it stable. Over the past two decades, a large role in maintaining the stationary state of the system of inhibitors and especially antioxidants has been revealed (B. N. Tarusov and A. I. Zhuravlev, 1954, 1958). It has been established that the reliability of stationary development is associated with environmental factors (temperature) and the physicochemical properties of the cell environment.

Modern principles of biothermodynamics have made it possible to give a physicochemical interpretation of the mechanism of adaptation. According to our data, adaptation to environmental conditions can occur only if, when they change, the body is able to establish stationarity in the development of biochemical reactions (B.N. Tarusov, 1974). The question arose of developing new methods that would allow assessing the stationary state in vivo and predicting its possible violations. The introduction of cybernetic principles of self-regulating systems into biothermodynamics and research into the processes of biological adaptation promises great benefit. It became clear that in order to solve the problem of the stability of the steady state, it is important to take into account the so-called perturbing factors, which include, in particular, non-enzymatic reactions of lipid oxidation. Recently, studies of the processes of peroxidation in the lipid phases of living cells and the growth of active radical products that disrupt the regulatory functions of membranes have been expanding. The source of information about these processes is both the detection of active peroxide radicals and peroxide compounds of biolipids (A. Tappel, 1965; I. I. Ivanov, 1965; E. B. Burlakova, 1967 and others). To detect radicals, biochemiluminescence is used, which occurs in the lipids of living cells during their recombination.

On the basis of physicochemical ideas about the stability of the steady state, biophysical ideas arose about the adaptation of plants to changes in environmental conditions as a violation of inhibitory antioxidant systems (B. N. Tarusov, Ya. E. Doskoch, B. M. Kitlaev, A. M. Agaverdiev , 1968 - 1972). This opened up the possibility of evaluating such properties as frost resistance and salt tolerance, as well as making appropriate predictions in the selection of agricultural plants.

In the 1950s, an ultra-weak glow was discovered - biochemiluminescence of a number of biological objects in the visible and infrared parts of the spectrum (B. N. Tarusov, A. I. Zhuravlev, A. I. Polivoda). This became possible as a result of the development of methods for registering superweak light fluxes using photomultipliers (L. A. Kubetsky, 1934). Being the result of biochemical reactions occurring in a living cell, biochemiluminescence makes it possible to judge important oxidative processes in the electron transfer chains between enzymes. The discovery and study of biochemiluminescence is of great theoretical and practical importance. So, B. N. Tarusov and Yu. B. Kudryashov note the great role of the products of oxidation of unsaturated fatty acids in the mechanism of the occurrence of pathological conditions that develop under the influence of ionizing radiation, in carcinogenesis and other violations of the normal functions of the cell.

In the 1950s, in connection with the rapid development of nuclear physics, radiobiology, which studies the biological effect of ionizing radiation, emerged from biophysics. The production of artificial radioactive isotopes, the creation of thermonuclear weapons, atomic reactors, and the development of other forms of practical use of atomic energy have posed with all their acuteness the problem of protecting organisms from the harmful effects of ionizing radiation, and developing the theoretical foundations for the prevention and treatment of radiation sickness. To do this, it was necessary first of all to find out which components of the cell and links of metabolism are the most vulnerable.

The object of study in biophysics and radiobiology was the elucidation of the nature of the primary chemical reactions that occur in living substrates under the influence of radiation energy. Here it was important not only to understand the mechanisms of this phenomenon, but also to be able to influence the process of exchanging physical energy for chemical energy, to reduce its coefficient of "useful" action. Work in this direction was initiated by the studies of the school of N. N. Semenov (1933) in the USSR and D. Hinshelwood (1935) in England.

An important place in radiobiological research was occupied by the study of the degree of radiation resistance of various organisms. It was found that increased radioresistance (for example, in desert rodents) is due to the high antioxidant activity of cell membrane lipids (M. Chang et al., 1964; N. K. Ogryzov et al., 1969). It turned out that tocopherols, vitamin K and thio compounds play an important role in the formation of the antioxidant properties of these systems (II Ivanov et al., 1972). In recent years, studies of the mechanisms of mutagenesis have also attracted much attention. For this purpose, the effect of ionizing radiation on the behavior of nucleic acids and proteins in vitro, as well as in viruses and phages is studied (A. Gustafson, 1945 - 1950).

The struggle for a further increase in the effectiveness of chemical protection, the search for more effective inhibitors and principles of inhibition remain the main tasks of biophysics in this direction.

Progress has been made in the study of excited states of biopolymers, which determine their high chemical activity. The most successful was the study of excited states arising at the primary stage of photobiological processes - photosynthesis and vision.

Thus, a solid contribution has been made to understanding the primary activation of the molecules of plant pigment systems. The great importance of the transfer (migration) of the energy of excited states without loss from activated pigments to other substrates has been established. A major role in the development of these ideas was played by the theoretical works of A. N. Terenin (1947 and later). A. A. Krasnovsky (1949) discovered and studied the reaction of reversible photochemical reduction of chlorophyll and its analogues. There is now a general belief that in the near future it will be possible to reproduce photosynthesis under artificial conditions (see also Chapter 5).

Biophysicists continue to work on uncovering the nature of muscle contraction and the mechanisms of nerve excitation and conduction (see Chapter 11). Research into the mechanisms of the transition from an excited state to a normal state has also become of current importance. The excited state is now considered as a result of an autocatalytic reaction, and inhibition is considered as a consequence of a sharp mobilization of inhibitory antioxidant activity as a result of molecular rearrangements in compounds such as tocopherol (I. I. Ivanov, O. R. Kols, 1966; O. R. Kols, 1970).

The most important general problem of biophysics remains the knowledge of the qualitative physical and chemical features of living matter. Properties such as the ability of living biopolymers to selectively bind potassium or polarize electric current cannot be preserved even with the most careful removal from the body. Therefore, cellular biophysics continues to intensively develop criteria and methods for the lifetime study of living matter.

Despite the youth of molecular biology, the progress it has made in this area is truly stunning. In a relatively short period of time, the nature of the gene and the basic principles of its organization, reproduction and functioning have been established. Moreover, not only in vitro reproduction of genes has been carried out, but for the first time the complete synthesis of the gene itself has been completed. The genetic code has been completely deciphered and the most important biological problem of the specificity of protein biosynthesis has been resolved. The main ways and mechanisms of protein formation in the cell have been identified and studied. The primary structure of many transport RNAs, specific adapter molecules that translate the language of nucleic templates into the language of the amino acid sequence of the synthesized protein, has been completely determined. The amino acid sequence of many proteins has been fully deciphered and the spatial structure of some of them has been established. This made it possible to elucidate the principle and details of the functioning of enzyme molecules. The chemical synthesis of one of the enzymes, ribonuclease, was carried out. The basic principles of the organization of various subcellular particles, many viruses and phages have been established, and the main ways of their biogenesis in the cell have been unraveled. Approaches to understanding the ways of regulation of gene activity and elucidation of the regulatory mechanisms of vital activity have been discovered. Already a simple list of these discoveries indicates that the second half of the 20th century. was marked by tremendous progress in biology, which is due primarily to an in-depth study of the structure and functions of biologically important macromolecules - nucleic acids and proteins.

Achievements in molecular biology are already being used in practice today and bring tangible results in medicine, agriculture and some industries. There is no doubt that the return of this science will increase every day. However, the main result should still be considered that under the influence of the successes of molecular biology, confidence in the existence of unlimited possibilities on the way to revealing the most secret secrets of life has strengthened.

In the future, apparently, new ways of studying the biological form of the motion of matter will be opened - biology will move from the molecular level to the atomic level. However, now there is, perhaps, not a single researcher who could realistically predict the development of molecular biology even for the next 20 years.

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