DEOXYRIBONUCLEIC ACID (DNA; outdated names: dezoksipentozonukleinovy to - you, nuclear nucleinic to - you, thymonucleic to - you, animals nucleinic to - you) — nucleinic to - you, containing desoxyribose in quality of a carbohydrate component, and as one of the pirimidinovy bases — thymine, the Crimea in molecules ribonucleic to - t correspond a ribose and uracil. DNA represent linear polymers of deoxyribonucleotides in which sequence of nitrogen bases all hereditary information (is coded see. Genetic code ). Thus, DNA of this organism contains information on all signs of a look and features of an individual — it genotype (see) — also transfers this information to posterity, reproducing a certain sequence of the bases in a structure of individual DNA. As molecules DNA of very big sizes there is also a huge set of the possible unequal sequences from four various nucleotides, the number of different molecules DNA is almost infinite.
In the nature of DNA contain in all organisms except for the RNA-containing viruses. DNA are a typical component of a cellular kernel, in Krom they are in a complex with proteins, hl. obr. histones (see), forming the dezoksiribonukleoproteida making a basis tsitol, structures of chromatin and substance of chromosomes. DNA is found also in chlorolayers of a plant cell and in mitochondrions of animals and plants in which it codes a part of proteins of these structures thanks to what they possess a nek-swarm autonomy and only partially depend on DNA of a kernel.
DNA were for the first time open Mishr (F. Miescher, 1869) who called the received substance nuclein (Latin of nucleus a kernel). Afterwards it was shown that nuclein represents high-molecular, phosphorated to - that, being in a complex with proteins therefore began to distinguish actually nucleinic to - that [R. Altmann, 1889] and her complexes with proteins — nucleoproteids (see). Soon nucleinic to - that was received from the thymus (thymus gland) of a calf which appeared a rich source of this substance. The substance close on properties but differing from nucleinic to - you, received from a thymus gland, allocated from yeast. It became clear that nucleinic to - that from yeast contains adenine, guanine, tsitozin and uracil whereas nucleinic to - that, allocated from a thymus gland of a calf, instead of uracil contains thymine. As a carbohydrate component in barmy nucleinic to - those found pentose, and in drug from a thymus gland — to a dezoksipentoz. Depending on a source of receiving these nucleinic to - you received names thymonucleic and zimonukleinovy. As the first type nucleinic to - you found in animal objects, and the second — in vegetable, nucleinic to - you used also the names «animal» and «vegetable». However, when R. Feulgen with sotr. developed gistokhy, reaction on «animal» nucleinic to - that, it turned out that it is found in a kernel of both animal, and plant cells. On the other hand, «barmy» nucleinic to - that was found by Zh. Brashe in hl. obr. in cytoplasm of cells and plants, and animals. At last, availability of DNA at plants was proved by A. N. Belozersky chemically. The names «deoxyribonucleic to-that» (DNA) and «ribonucleic to - that» were offered (RNA) after by P. A. Levene it was established that the dezoksipentoza in thymonucleic to - those is desoxyribose (see), and pentose in zimonukleinovy to - those are ribose (see).
Ways of receiving
the Technique of release of DNA depends on structure and character of the used source (tissue of animals or plants, microorganisms, viruses). For laboratory and industrial receiving DNA usually use a thymus of a calf, and also sperm (milts) of fishes, a spleen of mammals, nuclear erythrocytes of birds.
The drugs DNA received usually in the form of the DNA sodium salt have an appearance of white fibers. For preservation of the DNA native properties processing of fabrics and cells is carried out in the cold, whenever possible quickly, in the conditions excluding or reducing action deoxyribonucleases (see), as a rule, DNA which are contained in fabrics and causing enzymatic disintegration. In addition to preservation of native properties, the major task is purification of DNA of other substances, first of all — from proteins and RNA. In this regard methods of receiving DNA differ with hl. obr. in the ways of deproteinization and purification of drugs of RNA impurity. Apply processing of cells and fabrics to these purposes various detergents (see), phenols (see) and other deproteinizirukltsy agents.
During the receiving DNA from animal and vegetable fabrics most often previously isolate fraction of cellular kernels or allocate dezoksiribonukleoproteida, washing them salt solutions in fiziol, concentration in the presence of EDTA or other connections connecting the bivalent cations necessary for manifestation of enzymatic activity of deoxyribonucleases. After removal of ground mass of proteins drugs in addition deproteinize chloroform with octanol or phenol. Quite often they are processed also ribonucleases (see) and proteolytic enzymes, usually pronazy (see. Peptide-hydrolase ).
For receiving DNA from bacteria usually use method of Mar mess which consists in washing of bacterial weight of 0,15 M of NaCl, containing 0,015 M sodium citrate, killing at 60 ° and pH 8,0 in 0,15 M of NaCl containing EDTA and 2% dodetsilsulfat sodium, deproteinization by their chloroform containing isoamyl alcohol, reprecipitation by alcohol, repeated repeated deproteinization, processing by ribonuclease and sedimentation by isopropyl alcohol. This method in various modifications is also successfully applied to receiving DNA from animal and vegetable fabrics and the isolated cellular structures, napr, mitochondrions.
Chemical composition and physical and chemical properties
represent DNA polybasic strong to - you which alkaline salts form in water very viscous transparent colloid solutions stiffening at concentration higher than 0,25% DNA solutions are characterized by the anomalous (structural) viscosity which is explained by the extended form of molecules and in a flow possess double refraction (see).
Chemically DNA represent the high-molecular polymers of monodeoxyribonucleotides (mononucleotides) which are monomers of which molecules DNA are constructed. Each mononucleotide of DNA consists of the remains phosphoric to - you, 2-D-desoxyribose and purine or pirimidinovy nitrogen base. The carbohydrate and phosphatic rest is identical in all DNA monomers, nitrogen base can be presented by adenine (A), guanine (G), tsitoziny (C) or thymine (T). In DNA of different organisms there is a nek-swarm a quantity of so-called minor bases, e.g. 5 methylcytosines, partially replacing tsitozin. At the highest animals and the person the maintenance of this basis reaches 1,5%, at the higher plants of 5 — 7%, at bacteria — no more than 0,6%. In DNA of bacteria also 6 methyladenine and sometimes other metilirovanny nitrogen bases meet. In DNA of T-even bacteriophages (T2, T4 and T6) tsitozin it is completely replaced with 5 oxymethylcytosine, in DNA of the SP01 and SP8 viruses thymine is replaced with 5 oxymethyluracil, and at a phage of PBS1 — uracil.
In mononucleotides 2-D-desoxyribose is attached by a glycosidic linkage through the first carbon atom to a nitrogen atom in the 9th provision of purine base (adenine or guanine) or in the 3rd provision of the pirimidinovy basis (a tsitozin or thymine). The rest phosphoric to - you are attached by radio communication to 5 '-mu or 3 '-mu to carbon atom of desoxyribose. Thus, the mononucleotide remains are connected among themselves through phosphoric to - that, edges is connected with 5 '-C - atom of desoxyribose of one nucleotide and with 3' - C - atom of desoxyribose of the next nucleotide etc. (scheme 1).
DNA from various sources differ from each other based on the ratio of the nitrogen bases which are their part, i.e. on nucleotide structure, however the nucleotide composition of all DNA submits to certain patterns — Chargaff's rules, according to the Crimea: 1) the number of molecules of adenine is equal to number of molecules of thymine; 2) the number of molecules of guanine is equal to number of molecules of a tsitozin; 3) the number of molecules of purine bases is equal to number of molecules of the pirimidinovy bases; 4) number 6-of amino groups is equal in molecule DNA to number 6-of ketogroups, i.e. the sum adenine + tsitozin is equal to the sum guanine + thymine. Having written down Chargaffa governed alphabetic references, we will receive the following expressions: 1) And — T; 2) G — C; 3) And + = T + C; 4) And + C == + T. These rules are valid and if the given nitrogen bases are replaced with their metilirovanny or other derivative (minor bases). Thus, the nucleotide composition of DNA is characterized by a molar ratio [+ C] / [And + T] (a factor of specificity) or percent of HZ-couples, i.e. ([+ C] / [And + T+G+Ts]) h100. The size of this indicator is identical to DNA of various bodies and fabrics of one organism and practically does not differ at different types of animals and plants within one class. It is rather close at the higher plants and animal (vertebrata) — from 0,55 to 0,93. At bacteria, according to A.S. Spirin and A. N. Belozersky, the size of a factor of specificity fluctuates from 0,35 to 2,73 or from 26,8 to 74,2% of HZ-couples.
The X-ray crystallographic analysis of DNA showed that the purine and pirimidinovy bases of the nucleotide remains of DNA lie in one plane, a perpendicular longitudinal axis of a molecule whereas cycles of desoxyribose are in the plane, almost perpendicular that, in a cut cycles of the bases lie. Distances between nitrogen bases of separate nucleotides make 3,4 A. According to these data and with Chargaff Dzh's rules. Watson and T. Shout constructed model of molecule DNA (scheme 2). Further researches confirmed their correctness. Establishment of a structure of molecule DNA was the largest opening in the area molecular biology (see). According to Watson's model — Shout, molecule DNA represents the double helix constructed of two polynucleotide chains directed antiparallelno i.e. if in one chain the rest phosphoric to - you connect separate nucleotides from 5 '-to 3 '-C - to atoms from below up, then in other chain these bonds are directed from top to down. Each chain consists of a carbohydrate and phosphorus skeleton, the nitrogen bases attached to a carbohydrate component are oriented inside and connected among themselves by in pairs hydrogen bindings, namely And — and — with Ts. Adenin with thymine are connected to T by two H-bonds whereas guanine with tsitoziny are connected still by the third hydrogen binding (scheme 3). The double helix is twirled to the right, and to a full spiral turn there correspond 10 couples of the nucleotide remains occupying distance in 34 A — the V-form. The B-form is steady in the environment with high humidity (97% of a saturated steam). All molecule DNA represents the rigid, not branching linear polymer. In the conditions of low humidity (from 76% of saturation) the double helix of DNA accepts an A-form, in a cut the full spiral turn occupies distance in 28 A, and also the provision of the plane changes, in a cut nitrogen bases, and number of the bases on a full round are located (one round contains Also nucleotides).
In DNA chromatin forms complexes with histones (see). Such nucleohistones are in a sverkhspiralizovanny state, and the superspiral has radius 50 A and distance between rounds 120 A. In chromosomes and partially in chromatin such superspirals of a dezoksiribonukleoproteid are twirled in a spiral of the highest orders with dia. 250 and 500 A.
Mol. the weight (weight) of DNA is not identical and depends on sources of receiving DNA sample. In addition, even at the most careful and sparing procedures of release of DNA the nek-swarm of degradation and its pier is exposed. weight can be lower, than in cells. The drugs received by modern methods from tissues of animals and plants have a pier. weight 6•10 6 — 10•10 6 , however true pier. the weight of DNA of animals and plants as show methods of definition a pier. weight on viscosity and on length of molecules (1A to double-helix DNA in a B-form there correspond 197 units a pier. weight), much higher and can reach tens of billions. Thus, molecules DNA of chromosomes are the largest molecules from all known biopolymers.
At some viruses, napr, at bacteriophages of F X174, fd and M13, DNA is presented by one polynucleotide chain closed in a ring and having rather small pier. weight — 1,7•10 6 . At the majority of the DNA viruses represents a double helix, linear or closed in a ring; quite often such forms pass each other, and these molecules have the so-called «sticky ends», the nucleotide sequences containing one-filamentous complementary each other by means of which the molecule becomes isolated in a ring. Strong absorption in an ultra-violet part of a range at wavelength apprx. 260 nanometers is characteristic of DNA. Ud. absorption of high-polymeric DNA in the solution containing higher than 10 - 3 The m of NaCl, at pH 7,0 makes apprx. 6000 on 1 g • atom of phosphorus. DNA are rather easily depolymerized under the influence of some chemical connections, ultrasound, the ionizing and ultra-violet radiation; heating of DNA with divorced mineral to-tami leads to eliminating of purines (adenine and guanine) and education «apurinovy to - you», containing only the pirimidinovy bases. Heating of DNA solutions, and also their alkalifying, etc. cause the denaturation of DNA consisting in melting of a double helix (a rupture of hydrogen and hydrophobic bindings) and discrepancy of polynucleotide chains. The denaturation is followed by decrease in viscosity of solution and increase in absorption in ultraviolet on what it is possible to control this process. Temperature of melting (temperature, at a cut is denatured a half of DNA) that higher, than bigger percent of HZ-couples contains in DNA; this indicator can serve for definition of nucleotide composition of DNA. It is established that t ° pl it is linearly connected with composition of DNA: 1 ° there correspond 2,5 molar % of HZ-couples. Homogeneous drugs DNA (e.g., virus DNA) are characterized by melting with sharp transition whereas heterogeneous drugs give rather wide fusion zone that can serve as a measure of heterogeneity of DNA. At rapid cooling after a denaturation of DNA does not recover the native properties, however at slow cooling polynucleotide chains reassotsiirutsya by the principle of a complementarity and thus there is a renaturation of molecules DNA. At slow cooling with the denatured DNA in the presence of RNA the DNA and RNA polynucleotide threads can be associated by the principle of a complementarity of couples of guanine with tsitoziny and adenine with uracil (instead of thymine), forming two-filamentous hybrids of DNA — RNA. The method of hybridization is widely applied to a research of a complementarity and structure of two types nucleinic to - t, and also DNA from different sources. Studying of a renaturation of DNA showed that DNA of the higher organisms contain the repeating sequences which can be divided into very often repeating sequences and rather often repeating. Besides, there are also unique sequences. Regulatory genes, apparently, belong to the repeating sequences (see. Gene ), and also the genes coding ribosomal RNA acceptor RNA and histones. Structural genes, as a rule, belong to the unique sequences that is proved for such active genes as genes of a globin in erythroblasts and genes of fibroin in shelkootdelitelny gland of a silkworm. At the lowest organisms (prokariot) — viruses and bacteria, and also does not contain in mitochondrions of DNA or almost does not contain the repeating sequences. In DNA of a number of organisms sites are found, in each of nucleotide chains of which there are sequences of the bases repeating further, but upside-down. As such sequences are read equally since both ends as, e.g., the word «flood», they received the name of palindromes. Palindromes in DNA and in the RNA synthesized on their matrix can form crosswise structures, fiziol which role, perhaps, is connected with initiation (beginning) of synthesis of RNA or proteins.
By method of molecular hybridization it is shown that in nuclear DNA of a fruit fly of Drosophila melanogaster apprx. 75% of all DNA is presented by the unique sequences, apprx. 15% — it is very frequent (to 1 000 000 times) repeating and apprx. 10% — it is rather frequent (1000 — 100 000 times) the repeating nucleotide sequences. Very often repeating sequences are located hl. obr. in the dense chromatin cytologic described as heterochromatin; they meet most often in the so-called satellite DNA usually different from the ground mass of DNA on nucleotide structure and density of chloride caesium separated from it at equilibrium centrifuging in a gradient. Such satellites contain almost at all eukaryotes and make from 1% to a half of all mass of a genome. Even at closely related types the amount of satellite DNA can significantly differ. Rather often repeating sequences are distributed between hetero - and euchromatin. A considerable part of a dezoksiribonukleoproteid of chromatin consists of the alternating sites of the repeating and unique sequences of DNA. Noticeable amounts of DNA containing rather often repeating sequences are also in the chromatin associated with kernels and coding ribosomal RNA.
Primary structure of DNA difficult gives in to studying already because molecules DNA have the huge sizes. Nek-ry information on the sequence of nucleotides is given by studying of pirimidinovy blocks. During the processing of DNA concentrated ant to - eliminating of purine bases and further hydrolysis of DNA happens that, containing diphenylamine. In a molecule the pirimidinovy sequences remaining in the blocks representing the oligodezoksinukleotida deprived of purine monomers remain. Such blocks divide with the help chromatography (see) into isoplates — the oligomers containing identical number of the nucleotide remains then in turn divide them and analyze. In this way study the purine blocks received by processing of DNA a hydrazine. However the greatest progress in studying of structure of DNA is made as a result of use of the deoxyribonucleases splitting certain sequences of nucleotides, and in particular restriktaz (see. Deoxyribonucleases ), having narrow specificity concerning the short nucleotide sequences in 6 — 7 nucleotides. More detailed information concerning the nucleotide sequence in the molecules DNA representing structural genes is received by the analysis of the nucleotide sequence in the RNA and proteins corresponding to them. It was succeeded to establish the sequence of nucleotides in small molecules of satellite DNA at the higher organisms, also nucleotide sequence in quite considerable sites of DNA is found out from some viruses, bacteria, etc.
Methylation of nitrogen bases as a part of DNA happens after synthesis of a molecule and belongs to so-called post-synthetic changes or modifications.
U E. coli is methylated the adenine which is just in that short sequence of nucleotides which «is learned» by restriktazy R1. Apparently, restriktaza selectively destroy the alien DNA getting to a bacterium, the sequences in own DNA «recognized» by them are protected by methyl groups.
Methods of quantitative and qualitative test and a research
are obliged by the Majority of staining chemical reactions of DNA to the carbohydrate component — to desoxyribose (see). Under action to - the t of DNA easily chips off purine bases, and the aldehydic group of desoxyribose is released. As a result of further action to - you desoxyribose undergoes transformations with formation of ω-oxylevulinic aldehyde:
responsible, apparently, for formation of coloring with reactants on DNA.
More often than other reactions to detection and quantitative definition of DNA apply heating with diphenylamine in concentrated acetic to - those in the presence of the concentrated chamois to - you (see. Dishe method ). This reaction is usually applied in Burton's modification (To. Burton) at 30 ° in the presence of acetaldehyde. Less sensitive staining reactions with cysteine, with tryptophane or an indole, and also with a carbazole are less often applied. Sometimes apply also staining reaction with n-nitrophenylhydrazine. The flyuorimetrichesky method is very sensitive (see. Flyuorimetriya ), allowing to define to 3•10 - 9 of DNA.
Quantitative definition of DNA requires its preliminary separation from RNA and (whenever possible) from other substances preventing the applied reaction. For these purposes usually use Schmidt and Tanngauzer's method (G. Schmidt, S. J. Thannhauser) in various modifications. The principle of a method consists in sedimentation nucleinic to - t together with proteins trichloroacetic or chloric to - that, washing of acidsoluble phosphoric connections, extraction of lipids and extraction nucleinic to - t by means of hydrolysis of 5% trichloroacetic to - that at 90 ° within 15 — 20 min. Proteins at the same time remain in draft; from the solution containing nucleinic to - you and subjected to hydrolysis of 0,3 — 1,0 N by the alkali causing disintegration of RNA to nucleotides, DNA besiege acidulation trichloroacetic or chloric to - that. The deposit is washed and DNA extract hot chloric to - that. Content of DNA is determined by phosphorus, by spektrofotometrichesk or by means of specific staining reactions, but the spectrophotometric method is to the simplest and bystry DNA for definition after its separation from other substances which are characterized by a maximum of absorption at 260 nanometers.
During the definition of nucleotide composition of DNA the last is subjected to hydrolysis chloric to - that and the chipped-off purine and pirimidinovy bases divide paper chromatography or on ion exchangers. Good results are yielded also by a chromatography in a thin coat on derivatives cellulose (see), etc. As in usual double-helix DNA the nucleotide structure submits to certain rules (Chargaffa governed), it can be expressed as percentage of HZ-couples. Molar percent of HZ-couples calculates, using temperature of melting of DNA (temperature, at a cut is denatured a half of DNA), on a formula: percent of HZ-couples = 2,44 (t pl - 69,3 °). The coefficients specified in a formula are calculated empirically and vary depending on ionic strength, ionic structure and the size pH of solution. Good results during the definition of nucleotide composition of DNA are yielded by a method ultracentrifuging (see) in a gradient of density of chloride caesium. The floating density of DNA at the same time is linearly connected with the molar maintenance of HZ-couples (change of maintenance of HZ-couples for 1% changes floating density to 0,001 units) and is determined by the equation: the molar maintenance of HZ-couples = 10,2-(ρ-1,660), where ρ — the floating density of the studied drug DNA. For pure drugs DNA the nucleotide structure can be determined also by an absorption spectrum in 0,1 M acetic to - those on the formula offered by Frederik (E. Fredericq).
Contents in cells and fabrics
the Content of DNA in bodies and tissues of animals and the person fluctuates over a wide range and, as a rule, that is higher, than more cellular kernels are necessary per unit mass fabric. Especially DNA (apprx. 2,5% of crude weight) in the thymus consisting hl is a lot of. obr. from lymphocytes with large kernels. It is a lot of DNA in a spleen (0,7 — 0,9%), it is not enough (0,05 — 0,08%) in a brain and muscles where nuclear substance makes considerably a smaller share. At early stages of embryonic development these bodies contain more DNA, but contents it decreases in the course of ontogenesis in process of a differentiation. However the amount of DNA on one cellular kernel containing a diploid set of chromosomes is almost constant for each biol. look. Respectively the amount of DNA in kernels of sex cells are twice lower. For the same reason various fiziol, and patol, factors almost do not influence the content of DNA in fabrics, and at starvation, e.g., abundance of DNA even increases due to decrease in concentration of other substances (proteins, carbohydrates, lipids, RNA). At all mammals the amount of DNA in a diploid kernel is almost identical and makes apprx. 6•10 - 12 at birds — apprx. 2,5•10 - 12 at different types of fishes, amphibians and protozoa it fluctuates in considerable limits.
Content of DNA in bacteria is quite big and reaches several percent in terms of dry weight; in viruses it can reach 50%. At the same time the absolute amount of DNA in a bacterial cell are on average two orders lower, than in a cellular kernel of the higher organisms, and in the DNA-containing viruses it is two orders lower.
At bacteria one huge molecule DNA forms the gynophore corresponding to a chromosome of the higher organisms. So, at Escherichia coli colibacillus a pier. the weight of such ring-shaped double-helix molecule reaches apprx. 2,5-109 and length exceeding 1,2 mm. This huge molecule is densely packed in small «nuclear area» of a bacterium and connected to a bacterial membrane.
In chromosomes of the higher organisms (eukaryotes) of DNA is in a complex with proteins, hl. obr. histones; each chromosome contains, apparently, one molecule DNA up to several centimeters long and a pier. weighing up to several tens billions. Such huge molecules find room in a cellular kernel and in mitotic chromosomes of several micrometers. A part of DNA remains not connected with proteins; sites of untied DNA alternate with DNA blocks, connected with histones. It is shown that such blocks contain about two molecules of histones of 4 types: H2a, H2b, H3 and H4 (see. Histones ).
In addition to a cellular kernel, DNA contains in mitochondrions and in chlorolayers. The amount of such DNA is usually small and makes a small share of the general DNA of a cell. However in oocytes and at early stages of embryonic development of animals an overwhelming part of DNA is localized in cytoplasm, hl. obr. in mitochondrions. Contains in each mitochondrion on some molecules DNA. At animals a pier. the weight of mitochondrial DNA makes apprx. 10 7 ; its double-helix molecules are closed in a ring and are in two main forms: supertwisted and opened ring. In mitochondrions and in DNA chlorolayers is not in a complex with proteins, she is associated with membranes and reminds bacterial DNA. Small amounts of DNA are found also in membranes and some other structures of cells, however their features and biol, a role remain not clear.
In process biol, synthesis of DNA on a matrix of similar molecule DNA is formed the same molecule, and the amount of DNA doubles. Therefore process of biosynthesis of DNA received the name of reduplication or replications (see).
The principle of a complementarity (complementarity), according to Watson and Shout, is put in the structure of DNA.
J. Watson and T. By shout it was postulated that DNA replication shall happen in the semi-conservative way, i.e. by untwisting of a double helix and synthesis new, complementary initial polynucleotide chains on each thread. This mechanism was also proved experimentally by introduction to a DNA matrix of heavy nitrogen (radioactive label) and the analysis of DNA of succeeding generations by means of centrifuging in a gradient of density of chloride caesium or method of an autoradiography.
DNA is synthesized from dezoksinukleozidtrifosfat which connect in a polynucleotide chain to eliminating of a pyrophosphate. This reaction proceeds on a matrix of the one-chained preformed DNA under the influence of enzyme of a DNA polymerase, and the synthesized dezoksiribopolinukleotidny chain of affiliated DNA is strictly complementary a matrix chain. The DNA polymerase for the first time allocated from E. coli, is well studied. Its pier. weight makes 110 000 dalton, under the influence of trypsin it breaks up to 2 fragments — active and inactive. Matrix DNA, obligatory presence of all four dezoksinukleozidtrifosfat and ions of Mg are necessary for course of the reaction catalyzed by a DNA polymerase 2+ . Balance of reaction is strongly displaced towards synthesis, the optimum size pH 7,5; reaction is inhibited by a pyrophosphate: concentration of a pyrophosphate 2•10 - 3 The m oppresses synthetic reaction for 50%. It is shown that double-helix molecule DNA is inactive as a matrix, however the site complementary is necessary for it for initiation of replication on an active matrix of one-chained DNA of a polynucleotide chain with the free 3rd '-OH-end of a ribose, serving as a priming for growth of again synthesized chain. This priming consists of the ribonukleotidny remains which are removed upon completion of synthesis of a complementary chain of DNA. The DNA polymerase consistently attaches the dezoksiribonukleotidny remains connecting hydrogen bindings to the complementary bases of a matrix chain to the 3rd '-OH-end of a priming. Growth of the synthesized chain happens in the direction 5' - OH —> to 3 '-OH-ends, antiparallelno a matrix chain. DNA replication leads to doubling of amount of genetic material of a cell and, as a rule — to cellular division. Therefore replication happens that more often than time of generation of a virus or a bacterium is shorter and what cells at the higher organisms share more often. Rate of replication is high at embryos, in particular during crushing, and is slowed down in process of development and a differentiation. In general rate of replication corresponds to mitotic activity of fabric (see. Mitosis ) and therefore it is low in not sharing cells, napr, in cells of a brain or muscles, and is rather high in often sharing cells of marrow or tumors. DNA replication takes place and at the endomitoses leading to polyploidization of kernels. Replication happens not during actually mitosis, and in an interkinetichesky phase during the synthetic S-period of a cellular cycle between the periods of G1 and G2.
At bacteria and viruses replication begins in one point of molecule DNA. In each chromosome of the higher organisms of such points usually happens on some cells. In a point of the beginning of synthesis of DNA one or two replicative forks can be formed. In the first case replication proceeds in one direction; two forks which move on molecule DNA in opposite directions are usually formed. Such bidirectional replication is shown by an autoradio graphical method on ring DNA of bacteria, and also at the higher organisms. In process of advance of replicative forks the affiliated double-helix molecules DNA consisting half of old chains and half from complementary to them new chains of DNA are formed.
R. Okazaki of biosynthesis of DNA at bacteria showed that at first rather short fragments of dezoksiribopolinukleotidny chains up to 1000 nucleotide remains long which then are sewed among themselves by DNA-ligase enzyme (polinukleotidligazy) are synthesized. One of two chains of DNA at the same time grows continuously, and another falteringly. Formation of fragments of Okazaki is shown also at the higher organisms. It is shown that the separation and untwisting of two polynucleotide chains of a double helix of DNA necessary for replication is carried out by means of the special DNA-connecting protein.
Replication of virus and several ring molecules DNA has some features. So, one-chained DNA of the F X174 virus at first synthesizes on the matrix a complementary chain — so-called minus chain. This chain becomes isolated in a ring of DNA-ligase and forms biologically active replicative form of DNA of a bacteriophage. A. Kornberg and sotr. this reaction sequence was reproduced out of an organism, and thus synthetic biologically active replicative form of DNA was for the first time received. At ring molecules DNA of mitochondrions presence of a small fragment length apprx. 450 nucleotide remains, complementary one («easy») chain of double-helix molecule DNA is revealed. Other («heavy») chain in this site is displaced and forms a so-called D-loop. The called fragment serves as the initial site of the synthesized «heavy» chain of DNA, the «easy» chain is synthesized on the released «heavy» chain of initial DNA. Replication happens asymmetric in one direction and begins with preeducated fragments. In DNA of Papova viruses, napr, the SV 40 virus and a virus of papilloma, replication goes in two directions at once. At bacteria replication, most likely, begins in the place of an attachment of DNA to a membrane. At the higher organisms of DNA of chromosomes it is also connected with an inner membrane of a kernel, however value of this communication in the course of replication is not clear yet.
In addition to DNA replication, in an organism there is a DNA repair, i.e. recovery of the damaged, destroyed or changed sites of polynucleotide chains. Gaps in one of polynucleotide chains of DNA, apparently, reparirutsya under the influence of DNA-ligase. More difficult damages, napr, formation of dimer of thymine under the influence of ultra-violet radiation, are liquidated as follows: the damaged site containing dimeasures of thymine «is cut out» by means of endonuclease (usually it is an oligonucleotide, three - or a tetranucleotide), and the gap is filled with the normal nucleotide block. In the course of a reparation a number of enzymes participates: endo-, ekzo-I and ekzo-II nucleases and DNA polymerase. Interpretation of mechanisms of damage and DNA repair will undoubtedly lead to more effective prevention and therapy of the diseases caused by radiation and chemical mutagens.
During the studying of a mutant E. coli sensitive to uv radiation became clear that it is defective also concerning a DNA polymerase. However at this mutant (PolA-) DNA replication continued. On this basis there was an assumption that the polymerase described by A. Kornberg participates in a reparation and does not participate in replication. Soon from a mutant of PolA-other DNA polymerase similar on the mechanism of action with earlier known, but other than it on a nek-eye was allocated to properties. The DNA polymerase of II began to be considered responsible for replication. Then the DNA polymerase of III on the properties reminding a DNA polymerase of I was allocated. Thus, three DNA polymerases are revealed, and, apparently, the DNA polymerase of III is necessary for replication.
In the oncogenous RNA-containing viruses (oncornaviruses) the enzyme catalyzing synthesis of a complementary chain of DNA on a matrix i.e. process, the return to process of transfer of information from DNA to RNA is found. This enzyme received the name «RNA-dependent DNA Polymerase» or «return transcriptase». Discovery of this enzyme meant success of science about malignant tumors — oncology. Earlier it was established that at malignant regeneration of cells under the influence of oncogenous viruses there is an inclusion of DNA of a virus in a chromosome of a cell of the owner. However the RNA-containing Oncogenous viruses dropped out of this pattern. It turned out that they contain the return transcriptase, edges right after infection on virus RNA synthesized virus DNA, edges and was implemented into a chromosome of a cell of the owner.
In some cases, napr, in oocytes for ribosomal DNA, amplification (multiplication) of certain sites of DNA takes place. The mechanism of amplification is not absolutely clear; apparently, there is a replication of certain sites of DNA containing cistrons of those RNA which are strenuously synthesized in these conditions.
The catabolism of DNA does not represent any features. In an intestinal path and in DNA fabrics are hydrolyzed under the influence of deoxyribonucleases; the formed nucleotides are hydrolyzed by nucleotidases, and the formed purine and pirimidinovy bases and sugar are split in the usual ways.
A biological role
Cytogenetic researches in 20 — the 30th 20 century demonstrated that transfer and storage of ancestral features are connected with chromosomes (see), being in nuclear substance. The fact that hereditary substance is DNA, but not protein became clear as a result of the researches conducted in the 40th 20 century on bacteria and bacteriophages (see. Gene ).
In 1944 Mr. of Avery, Mac-Laud and IAC Carti (O. T. of Avery, S. M. of MacLeod, M. of McCarty) established the nature of a transforming factor at bacteria. DNA was it. Process transformations (see) consists, undoubtedly, of a number of stages: reversible sorption of molecules DNA bacterial cell; implementations of these molecules in a cell; integration of a molecule of someone else's DNA into a chromosome of a cell, splitting of the formed complex structure and its transition to recombinants (see. Recombination ).
At a research of bacterial viruses (see. Bacteriophage ) under a supermicroscope or by means of the radioactive label entered into protein or into DNA of a bacteriophage it was shown that a virus, being fixed on. surfaces of a bacterial cell, enters into it only molecule DNA, leaving outside the fibrous casing. The molecule DNA of a virus which got to a cell, bearing in itself all hereditary information (genome) of a virus causes education in a cell of new virus particles, their reproduction and death of a cell from a lysis.
Some, so-called moderate, a phage do not cause strong indications of infection in a part of bacterial cells, however their DNA, getting to a cell, strongly contacts a genome of the bacterium, being integrated with DNA of a bacterial cell. Many generations of such bacteria bear in themselves a bacteriophage in the hidden look, without showing signs of disturbance of life activity. However under unfavorable conditions and at action of any disturbing factors, napr, the ionizing or ultra-violet radiation, the virus in such bacteria begins to breed and causes a lysis (death) of bacteria (see. Lysogeny ). DNA of a virus so strongly contacts DNA of bacteria that infection with the virus got from lysogenic bacteriums is followed by transfer together with DNA of a virus of a part of DNA of bacteria, about a cut some hereditary properties of these bacteria which are absent and at again infected bacteria, and at the virus are transferred. This phenomenon similar to transformation received the name transductions (see).
The sequence of nucleotides in a chain of DNA corresponds in complementary to it the sequence of nucleotides in molecule RNA — so-called. transcription (see). This process is carried out with the participation of enzyme of a RNA polymerase. The genetic information rewritten to DNA on RNA eventually defines primary structure (the sequence of the amino-acid remains in a molecule of protein under construction). By means of a submicroscopy it was succeeded to see growth of chains of RNA on a matrix of DNA, i.e. work of a gene at the level of a transcription.
In implementation process or expressions of genes coding of genetic information takes place. It is shown that three consistently located nucleotide rests (triplet) in a chain of DNA code a complementary triplet in a chain of RNA which in turn controls inclusion of one, strictly certain amino acid in a polypeptide chain of the synthesized protein. It is established that the polypeptide chain is synthesized kolinearno with DNA, i.e. according to a linear arrangement of triplets of DNA. It is known what triplets code inclusion of each amino acid (see. Genetic code ).
The sequence of nucleotides of DNA coding formation of a certain polypeptide chain represents a structural gene, or cistron. Change even of one couple of nucleotides in cistron (point mutation) can lead to structural change of protein and loss by it biol, activities. Such point mutations (see. Mutation ) can represent transitions (replacement of couple of nucleotides of HZ by AT or on the contrary), transversions (replacement of AT on THAT or HZ on TsG, i.e. movement of the complementary bases from one chain in another), inserts of couple of nucleotides or their deletion (loss). Transversions and transitions lead usually to replacement of one amino acid in a polypeptide chain under construction whereas an insert and deletions cause change of an order of reading and lead to deep disturbance of structure of protein. The insert or deletion at once of three couples of nucleotides, i.e. the whole triplet, recovers the sequence of reading, as served one of the most important proofs of a tripletnost of a code.
At the higher organisms the amount of DNA on a genome are enough millions of proteins for coding. Actually the number of genes at the person and the highest animals at least 10 times less also is, apparently, between 10 000 and 100 000. The huge amount of excess DNA, thus, does not bear structural genes and performs other functions. It turned out that a part of DNA does not participate in process of a transcription at all, and the prevailing part of the RNA synthesized on a matrix of DNA at the higher organisms undergoes disintegration in a cellular kernel, without participating in synthesis of cellular proteins. In this regard G. P. Georgiev stated a hypothesis, according to a cut the operon (the sequence of the genes controlling synthesis of the enzymes participating in a catalysis of all stages of the same process) at the higher organisms contains a large number of the regulatory genes located at the beginning of reading. The huge molecule RNA which is synthesized on such operon breaks up in the course of its transfer in cytoplasm where only actually information RNA containing structural genes and coding synthesis of cellular proteins arrives. Other part of this RNA has regulatory functions and breaks up in a kernel.
Feature of the higher organisms is also the differentiation of cells and fabrics. Genes which are contained in DNA of each diploid cell of the same organism (genome) qualitatively are also quantitatively absolutely identical, however the fact that different fabrics and cells are sharply various on the structure, a structure and functions, is explained by the fact that in them unequal proteins are synthesized. Thus, in addition to regulation of activity of the operating genes, at a differentiation switching off or blocking of the most part of genes takes place, and usually active is a small part of a genome, and only one or several proteins, napr, synthesis of hemoglobin in reticulocytes are in certain cases synthesized. Mechanisms of a differentiation are in many respects not clear, however is shown that the proteins which are a part of dezoksiribonukleoproteid chromatin (see), have the expressed effect on a transcription. Histones suppress this process, and acid proteins can activate it. Inactive sites of chromatin cytologic are represented by more dense, and in the course of a transcription, on the contrary, chromatin looks more friable and DNA threads, apparently, partially separate from histones. By various methods it is shown that the transcription of DNA occurs in the loosened sites of chromatin, in the so-called padded stools representing inflation of chromosomes in the field of the operating genes.
Histochemical methods of detection in fabrics
In a basis gistokhy, methods of identification nucleinic to - t lie reactions to all components which are their part. In the growing fabrics there is a bystry updating of purines, pyrimidines, phosphoric connections and sugars. It use for selective identification of DNA in them an autoradio graphical method (see. Autoradiography ) by means of 3H-thymidine. DNA forms salts with alkaline and heavy metals. The remains phosphoric to - you which are usually connected with nuclear proteins (most often histones) at replacement of the last easily enter chemical reactions with the main dyes. Can be for this purpose used safranin Oh, Janus green In, toluidine blue, thionine, azur And yes some other dyes which divorced solutions in acetic to - those selectively paint chromatin. For quantitative gistokhy, definitions of DNA the method using gallocyanine - chrome alum which has two valuable qualities is recommended. Gallotsianinkhromovy alum gives steady coloring, the edge does not change at dehydration and an enlightenment of cuts in a xylol. Coloring can be carried out at any pH value from 0,8 to 4,3, however it is recommended to work at an optimum pH value for this dye — 1,64 since at it there is the maximum specific identification of DNA. During the coloring gallocyanine - the DNA chrome alum connects to dye in a stoichiometric relationship, and the relation dye: DNA makes 1:3,7.
Feylgen's reaction is considered the most widespread reaction to DNA. It is preposted after soft hydrolysis of the fixed fabric in 1 N of HCl at 60 ° therefore from a dezoksiribozofosfat purines, and then and pyrimidines are chipped off, releasing thereby reactive aldehydic groups which are painted by Schiff's reactant in red color. Time of hydrolysis depends by nature an object and a method of fixing. Obtaining good results requires in each separate case time of hydrolysis to select experimentally.
For check of specificity of reaction of Feylgen there is a method of enzymatic and acid extraction of DNA. Zymolysis of DNA is carried out by a deoxyribonuclease at concentration of fermental drug of 2 mg on 100 ml 0,01 M of the pH 7,6 tris-buffer; solution before the use is dissolved a dist, water in the ratio 1:5. It is recommended to incubate cuts at 37 ° during the 2nd hour. In a different way removals of DNA processing gistokhy serves, than drugs of 5% water solution trichloroacetic to - you within 15 min. at 90 ° or 10% are hotter (70 °) chloric to - that within 20 min. then Feylgen's reaction shall yield negative takes.
Bibliography: Ashmarin I.P. Molecular biology, M., 1974; Breslers. E. Molecular biology, L., 1973, bibliogr.; Georgiev G. P. About structure of units of a transcription in cells of eukaryotes, in book: Usp. biol, chemistry, under the editorship of B. N. Stepanenko, t. 14, page 3, M., 1973, bibliogr.; Davidson J. Biochemistry of nucleic acids, the lane with English, M., 1976; The Cellular kernel, Morphology, physiology, biochemistry, under the editorship of I. B. Zbarsky and G. P. Georgiev, M., 1972; Lille R. D. Patologicheskaya of the technician and a practical histochemistry, the lane with English, M., 1969, bibliogr.; Methods of a research of nucleic acids, the lane with English, under the editorship of. A. N. Belozersky, M., 1970; Pearce E. A histochemistry, the lane with English, M., 1962; The Structure of DNA and the provision of organisms in an oistema, under the editorship of. A. N. Belozersky and A.S. Antonov, M., 1972; At otson J. Molecular biology of a gene, the lane with English, M., 1967; Chemistry and biochemistry of nucleic acids, under the editorship of. And. B. Zbarsky and S. S. Debov, L., 1968; Nucleic acids, Meth. Enzymol., v. 12, pt A — In, 1967 — 1968, v. 21, pt D, 1971, v. 29, pt E, 1974; Progress in nucleic acid research and molecular biology, ed. by J. N. Davidson a. W. E. Cohn, v. 1 — 16, N. Y. — L., 1963 — 1976; Walker P. M of B. Repetitive DNA in higher organisms, Progr. Biophys, molec, biol., v. 23, p. 145, 1971, bibliogr.
I. B. Zbarsky; A. G. Ufimtseva (gist.).