From Big Medical Encyclopedia

BACTERIOPHAGE (a bacterium[a] + grech, phagos devouring; synonym: phage, bacterial virus) — the virus capable to infect a bacterial cell to be reproduced in it, forming numerous posterity and to cause its lysis which is followed by an exit of phage particles to the habitat of a bacterium.


For the first time the snap lysis of bacteria was described Η. F. Gamaleey in 1898. The English bacteriologist F. Twort in 1915 described a phenomenon of «vitreous» regeneration of stafilokokk and micrococci at their long cultivation on a medium. During regeneration bacterial colonies became transparent and eventually lost ability to resowings. The factor causing dissolution of colony was transferred from the infected colony to not infected and kept the activity at cultivation in 1 million times. F. Twort suggested that the agent causing vitreous regeneration of bacteria is the virus parasitizing in a bacterial cell. In 1917 the Canadian microbiologist of d' of F. d'Herelle irrespective of F. Twort described a similar phenomenon at dysenteric bacteria and appropriated to the agent causing death of cells, the name «bacteriophage». D'erell (see) laid the foundation of the modern doctrine about B. He considered that the phage represents the filtered virus, being an obligate parasite of bacteria. Rather exact method of titration of a phage was succeeded to develop D'erellya, using to-ry, he found out that in a cycle of interaction of a phage with a bacterium there are four phases: an attachment of a phage to a cell, penetration in a cell, reproduction in a cell and a lysis of a cell with release of descendants of a phage on Wednesday.

D'Erell's opening drew to itself attention of the medical world in connection with hope to use B. as means for treatment and prevention of infectious diseases (see. Fagoprofilaktika , phagotherapy ). However the majority of experiments and the researches conducted in clinic showed rather low therapeutic value of B. in comparison with antibiotics that first of all it is connected with a wide spread occurance of phagoresistant forms of bacteria and their bystry selection during the use of a phage in practice. Nevertheless B. can effectively be used in diagnosis of infectious diseases (see. Reaction of increase of a caption of phages , Phage diagnosis , Lysotypy ).

The principles of classification, the main properties and morphology of bacteriophages

Modern classification of phages is based first of all on their chemical structure, on type nucleinic to - you, concluded in a fibrous casing. By this criterion all a phage subdivide into DNA - and RNA-containing.

The second major classification sign of phages is the form of their interaction with a bacterial cell. According to this criterion a phage divide on virulent and moderate. Infection of bacteria with the first of them always comes to the end with a lysis of a cell and release on Wednesday of phage posterity. For moderate phages there are two alternative development cycles — a lysis of a cell, as well as at infection with a virulent phage, or establishment of special type of symbiotic relationship with a cell, a cut remains at the subsequent division of a bacterium and is transferred her to descendants (see. Lysogeny ). This state is, as a rule, caused by inclusion of a chromosome of a moderate phage in a chromosome of a bacterium, about a cut it is replicated. The bacteria bearing a moderate phage are called lysogenic, and the form B. which is present at a lysogenic bacterium — a prophase.

One of taxonomical signs of phages is their relation to sexual differentiation of bacteria. On this sign a phage it is possible to divide into three basic groups: 1) f-1, f-2, Qbeta, MS-2 attacking only men's bacteria; 2) T-3, T-7, Φ-ΙΙ, interacting with female cells (see. Conjugation at bacteria ); 3) indifferent to sexual differentiation of cells a phage.

Fig. 1. Morphology of various phages (for clarity see the scheme in fig. 3): and — a phage» T-4, the opaque filled head, a cover of a tail in an unreduced state, thorns and tail fibrilla are visible (X 156 000); — a phage of R-2, the normal particle, below — a particle with the reduced cover and a doll-head is above visible at the left (x 65 000); in — the phage of T-5, is visible thin, gyrose a tail, there is no sokratitelny cover, a basal plate and tail fibrilla (X 68 250); — a phage of R-22, the hexagonal basal plate with thorns attached to a head by a short neck is visible (X 64 500).

One of classification signs of phages is their morphology. Usually a phage divide on large and small; besides, they are distinguished in a form of a proteinaceous cover — a capsid. The groups T which are subdivided in turn on even and odd are among large phages most in detail studied virulent a coliphage (derivative of E. coli + a phage): T-1, T-2, T-3, T-4, T-5, T-6, T-7, and also moderate intestinal phage λ, R-1, R-2 and R-22 (fig. 1). H K-with RNA-phages f-2, MS-2, M-12, D the holding phage of FH-174, etc. belong to the category of minute phages.

Fig. 2. The scheme of the main morphological types of bacteriophages (and — e). The description in the text.

The main morphological types of phages are presented in fig. 2. Are most difficult arranged T-even a phage of E. coli: T-2, T-4, T-6, nek-ry of phages you. subtilis: SPO-1, SP-8, SP-82, etc. Fagi of T-even group have the extended hexagonal head, a rigid tail shoot 100 nanometers long, to dia. 2,5 nanometers and the sokratitelny cover attached to it (fig. 2, a). The tail shoot at nek-ry phages, napr, at a phage of T-2 (fig. 4), has additional structures which are necessary for implementation of separate stages of interaction of a phage with a cell. Intestinal a phage T-1, T-5 and a moderate phage λ also have a hexagonal head and a tail (fig. 2, b), however the last has no sokratitelny ability. Diameter of a head of a phage λ — apprx. 60 nanometers — a phage of T-5 — 90 nanometers, length of a tail — 160 and 200 nanometers respectively. The Fag relating to the third morphological group (fig. 2, c), have a large hexagonal head and very short tail appendage. Representatives of this group are DNA-containing a phage of T-3 and T-7, diameter of a head to-rykh reaches 50 — 60 nanometers. At last, there are tailless DNA-containing minute phages (fig. 2, d), the typical representative to-rykh is the phage of FH-174. The polygonal structure of a capsid with dia, apprx. 20 nanometers is inherent in this phage. Others tailless a phage, characterized small sizes of a capsid (20 — 35 nanometers), concern the RNA-containing group and are found in various representatives of intestinal group (f-2, f-7, MS-2, R-17, etc.), and also in Pseudomonas aeruginosa (fig. 2, e). Except polygonal or spherical, are found filamentary intestinal a phage, containing one-filamentous DNA (fig. 2, e). Length of threadlike phages reaches 800 nanometers, and to dia. — 6 nanometers. The majority of phages of this kind interacts with men's strains of E. coli, but also others are known filamentary a phage, the sorts Pseudomonas, active concerning representatives, capable to infect also female strains.

Identification of phages and calculation of quantity of phage particles can be carried out by a submicroscopy of the purified drugs or observing action of phages on population of sensitive bacteria.

Addition of a virulent phage to a suspension of bacteria with the subsequent incubation of mix at t ° 30 — 37 ° leads to dissolution of culture — to a lysis, to-ry is followed by a full enlightenment of the culture medium containing bacteria. Speed of approach of a lysis depends on plurality of an infection, i.e. on number of particles of the phage falling on one bacterium. If fagolizirovanny culture to incubate bigger time, then the secondary growth (repeated opacification of culture) due to reproduction of the selected resistant mutants preexisting in bacterial population can appear. The infection of bacteria moderate phages which is carried out in simulated condition leads to temporary reduction of a turbidity of culture, for the Crimea increase of a turbidity due to growth and reproduction of lizogenezirovanny bacteria soon follows.

Fig. 3. Growth of a phage of T-2 on culture of E. coli. Negative colonies of a phage (plaque) are visible.

Quantity of viable particles of a phage determine on solid nutrient mediums by various methods, from to-rykh the most widespread the method of Gratsia is (see. Gratsia method ). In this case on the surface of solid nutrient medium with the sowed lawn of sensitive bacteria sites of a lysis of culture are formed, to-rye call plaques of a phage, negative colonies or phage plaques (fig. 3).

The morphology of negative colonies is specific to various phages and is often used as a genetic tag in various researches.

Virulent phages usually form transparent negative colonies, and moderated — muddy. The number of colonies, or plaque-forming units (FIGHT) increased by a factor of cultivation of an initial suspension of a phage gives the size of a caption of a phage, i.e. quantity FIGHT in 1 ml of initial drug. Fag are characterized also by other size, namely plating effeciency, i.e. the relation of total number of particles of the phage which is present at drug (counted by means of a supermicroscope), to quantity FIGHT. Usually at optimal conditions the efficiency factor of crops of a phage fluctuates within 0,5 — 1,0.

Fig. 4. Scheme of a structure of a phage T-2: and. — head; — DNA; in — a core; — a cover; d — a basal plate; e — thorns; — tail fibrilla (on the scheme the collar is not allocated).

Structure and chemical composition of a phage

Since Schlesinger's works (M. Schlesinger) which are carried out in the thirties it became clear that B. represents nucleoproteid. In the subsequent it was established that any B. consists of the fibrous casing surrounding one molecule nucleinic to - you. The structure of a fibrous casing (capsid) is most studied at the T-even phages of colibacillus (T-2, T-4, T-6) belonging to the class of large DNA-containing of phages. The weight of a particle of a T-even phage is approximately equal 5 X 10^-16, from to-rykh about 60% it is the share of proteins. Research of structure and functional anatomy of T-even phages [Anderson (T. Anderson), 1953; S. Brenner, 1959; R. Horne, 1959; E. Kellenberger, 1966; B. F. Poglazov, 1970] established that the capsid consists of difficult organized elements, each of to-rykh performs a certain function at interaction of a phage with a cell. The thin analysis of structure of a phage became available after development of two main methodical receptions: disintegrations of a particle of a phage by means of physical. and chemical factors and the method of negative staining for a submicroscopy visualizing ultrastructure more than a dusting metal (Brenner, Horn, 1959). On the basis of these methods it is established that T-even a phage consist of the following elements (fig. 4): heads, a collar and the tail shoot which is coming to an end with a hexagonal basal plate with short thorns. Fibrilla is attached to a basal plate. The tail of a phage consists of a hollow cylindrical core and the sokratitelny cover surrounding it.

Fig. 5. Molecule DNA of a phage T-2 which left a head of the phage (in the center) subjected to osmotic shock. Above also the free ends of molecule DNA are below visible (the diffraction pattern; X 60 000).

Each of structural elements of a phage in turn consists of a large number of strictly oriented proteinaceous subunits (capsomeres). At transfer of the T-even phage from saline solution in a distilled water the head bursts (osmotic shock), DNA concluded in it is released and can be investigated by electronic and microscopic (fig. 5) and physical. - chemical methods. It is established that DNA of a T-even phage represents the linear two-spiral molecule length apprx. 50 microns consisting of 2 X 105 couples of the bases. DNA of the majority of phages contains in the structure usual nitrogen bases — adenine, thymine, guanine and tsitozin (And, T, C). Exception of this rule are DNA of T-even phages and DNA of nek-ry phages you. subtilis containing unusual (abnormal) nitrogen bases. So, instead of a usual tsitozin 5 oxymethylcytosine contain, to-ry can be in a glyukozilirovanny or neglyukozilirovanny form.

In DNA of phages you. subtilis (2C, SPO-1, SP-8, SP-82, etc.) thymine is replaced on 5 oxymethyluracil. So unusual structure of DNA of these phages gives them a number of the properties distinguishing them from usual DNA in particular allocates them with high immunological specificity [L. Levine, 1961; D. M. Goldfarb, L. A. Zamchuk, 1968; N. A. Braude, L. A. Zamchuk, 1972].

Content of separate nitrogen bases in DNA of various phages varies and depends on a type of a phage. Except DNA, in a head of phages of T-2, T-4 there is a so-called internal protein of a phage, to-ry is associated with its DNA. Polyamines are a part of this protein: spermidine and putrestsin, the acidsoluble polypeptide containing asparaginic to - that, glutaminic to - that both a lysine, and kislotonerastvorimy protein [A. Hershey, 1957].

Function of these components comes down probably to deacidizing of DNA and to creation of the matrix providing its condensation.

Fig. 6. The schedule of a one-stage growth cycle of a phage of T-4 (FIGHT — plaque-forming units): 1 — stage of latency; 2 — the period of increase (horizontal shooters characterize duration of stage of latency and the period of increase); 3 — quantity of the phage particles which left one infected bacterium (a vertical arrow).

Intracellular development cycle of a bacteriophage. The kinetics of a reproduction of B. is illustrated by experience of a one-stage growth cycle [Ellis, Delbryuk (Eliis, M. of Delbruck), 1939], in Krom decides a caption FIGHT depending on time after mixing of a certain quantity of particles of a T-even phage on bacterial suspension. The culture of E. coli containing 109 cells in 1 ml at catches a phage with average plurality 1. Mix 2 — 5 min. in the aerated environment incubate, and then transfer test to 0,9 ml of anti-phage serum (1: 100), the fagolizatama received by immunization of rabbits or concentrates of phages. During this time 80% of a phage are adsorbed on cells. The infected bacteria part with the fresh environment by 10 000 times (the first growth test tube) and in addition by 20 times (the second growth test tube). Both test tubes incubate at t ° 37 ° and periodically take samples, in to-rykh determine contents FIGHT by seeding by a method of Gratsia. Within the first 25 min. an incubation of such mix the number FIGHT remains to constants (stage of latency), then the sharp increase of quantity of a phage continuing apprx. 10 min. then the number FIGHT becomes constant (fig. 6) again is observed. Stage of latency and the period of increase of number FIGHT corresponds to one cycle of reproduction of a phage (lytic cycle) including events from interaction of a phage particle with a surface of a host cell to an exit of phage posterity in the environment. The lytic cycle can be divided into several stages conditionally.

Fig. 7. Adsorption of particles of a phage of T-5 on a surface of E. coli (diffraction pattern).

1. Adsorption. As a result of accidental collisions of particles of a phage with bacteria specific proteins of its capsid come to contact with superficial fagospetsifichesky receptors of a cell wall of a bacterium. Irreversible adsorption of particles of a phage (fig. 7) results. T-even a phage interact with bacterial receptors by means of specific proteins on the ends of tail threads. A certain ionic structure of the environment and presence of a cofactor of adsorption is necessary for successful adsorption, the Crimea for a phage of T-4 is tryptophane.

2. Penetration of a phage in a cell in essence represents «injection» nucleinic to - you a phage in a bacterium [Hersha, W. Hayes, 1952]. At T-even phages this process happens as follows: thorns of a basal plate of a tail shoot come to contact with a surface of a bacterium, the tail cover is reduced therefore the tail core «punctures» a cell wall and DNA flows in cytoplasm of a cell through a cavity in a tail core. Possibly, in the course of «piercing» of a cell wall plays a role of enzymes lysozyme (see), the inner peptidoglikanovy layer of a wall of a bacterium found as a part of proteins of a tail, dissolving. Mechanisms of adsorption and penetration of nucleic acids of other phages differ from those at T-even phages, however finally in all cases free nucleinic to - that appears in a cell.

3. Penetration of phage DNA into a cell marks the beginning of the so-called eklips-period, during to-rogo it is not possible to find full-fledged phage particles in the infected cell at its destruction by ultrasound or a lysozyme. At this time in a cell there is a number of processes which net result is education of predecessors of phage posterity. The genetic information which is contained in nucleinic to - those a phage, begins to be implemented with use of the decoding systems of a bacterium. In case of DNA-containing B. phage genes are transcribed with formation of fagospetsifichesky information RNA, to-rye determine synthesis of fagospetsifichesky proteins by the transkriptsionny device of a bacterium. In case of RNA-containing B. fagospetsifichesky proteins are synthesized under control of the phage RNA performing function i-RNK (see. RNA ). Also a number of the enzymes which are not a part of mature particles of a phage, but necessary for reorganization of the biochemical device of a bacterium according to needs of a phage belongs to number of fagospetsifichesky proteins, in addition to structural proteins of a capsid. Many B., having got into a cell, cause suppression of bacterial macromolecules (DNA, RNA, proteins) and degradation of nucleic acids of a bacterium. Products of degradation are used as substrates for synthesis of nucleic acids of a phage. Expression of a genome of B. happens over an eklipsperiod according to the accurate program defining the moment of inclusion and switching off of certain groups of phage genes (R. B. Hesin, M. M. Shemyakin, 1962; R. B. Hesin, 1970). In the bacteria infected with T-even phages at the first stages of the eklips-period the so-called early proteins representing group of the enzymes providing synthesis of phage DNA are formed. After the beginning of DNA replication of a phage synthesis of early proteins stops and begins synthesis of late proteins — structural components of phage particles.

Fig. 8. The scheme of intracellular development of a phage FH-174 (and — z). One-filamentous, circular «plus» - thread (light) DNA of the infecting phage (a) serves as a template for synthesis complementary (black) «minus» - threads (b), forming a two-filamentous replicative form (Russian Federation). On an early phase of an infection the replicative form of DNA is copied replicated) polukonservativno and symmetrically (in), creating affiliated rf-molecules DNA (g). On late phases of an infection asymmetric replication (a) during which «minus» - thread serves as a template and «plus» - DNA (e) thread which enters interaction with protein (zh) of a head of a phage, forming a full-fledged particle is synthesized only affiliated begins (and).

Replication (see) nucleic acids of phages it is in most cases carried out by the specific enzymes induced by a phage. At the T-even phages containing two-filamentous DNA, this process proceeds on the semi-tinned mechanism with participation of a fagospetsifichesky DNA polymerase — one of early fagoindutsirovanny proteins. In case of RNA-phages, and also the phages containing single-stranded DNA (e.g., a phage of FH-174), synthesis of nucleic acids includes an intermediate two-filamentous replicative form: at first on initial «plus» - threads phage nucleinic to - you are synthesized complementary by it «minus» - a molecule, edges then is used as a matrix for education «plus» - DNA-phage threads of posterity (fig. 8).

4. Assembly of phage particles. Eklips-period is replaced by a stage of a morphogenesis of particles of phage posterity. At this stage of stage of latency phage particles can be revealed in the infected cells. B.'s morphogenesis consists in assembly of a phage capsid from separate molecules of structural proteins of a phage. Assembly happens step by step; at first separate components of a capsid are formed, to-rye then combine in larger structures. The majority of stages of a morphogenesis occurs by the principle of self-assembly and in vitro during the mixing of separate components of a phage can be reproduced. However nek-ry stages of assembly are carried out with the participation of the specific proteins which are not structural components of a phage. The least clear moment of a morphogenesis of a phage is packaging of phage DNA in a capsid.

5. The cycle of reproduction of a phage comes to the end with an exit of phage particles of posterity from a cell to the environment. Exit is followed by a lysis of a bacterial cell, to-ry is expressed in an enlightenment of the bacterial culture infected with a phage. At T-even phages a lysis is carried out by two fagospetsifichesky enzymes, one of to-rykh the unknown of the nature, a so-called product of a gene of t, destroys a membrane, and another — the lysozyme — dissolves a cover of bacteria from within. The quantity of particles of phage posterity counting on one infected cell (a harvest of a phage) can be calculated on a curve of a one-stage growth cycle as the relation of number FIGHT after an exit of a phage to that prior to the beginning of stage of latency. The size of an exit of a phage varies at various phages from several tens to several thousand. In optimal conditions it is formed apprx. 200 particles of T-even phages. This size can be increased due to repeated adsorption of particles of a phage on the infected cells after the beginning of stage of latency (a phenomenon of a delay of a lysis).

The genetics of a bacteriophage

the Starting point in the analysis of the genetic organization of a phage, as well as any other organism, is allocation of the various mutants allowing carrying out the recombination analysis necessary for creation of the genetic map of a phage.

Mutations of a phage on morphology of negative colonies. The classical sign of a phage used in basic researches on molecular genetics and in the genetic analysis is the morphology of negative colonies of a phage. Negative colonies can vary in sizes, differ from each other in character of edge, transparency, the size of an aura around a zone of a lysis and so forth. All these changes often are result of changes fiziol, the conditions defining features of interaction of a phage with the bacterial owner. They can result also from mutations in a chromosome of a phage and serve as genetic markers at recombination analysis. The most studied type of the mutations leading to change of morphology of negative colonies is the so-called r-mutation at T-even phages. This type of a mutation was found Hersha in 1946. The normal (wild) type of a phage T-2 on a bacterial lawn of E. coli In creates the small negative colonies surrounded with a zone of an incomplete lysis. However among 103 — 104 such colonies of a phage of T-2 the single negative colonies having considerably the big sizes with accurately outlined edge are found. Hersha called such mutants fast-lyseing and designated them the letter «g» (from English rapid — bystry), wild type — a r+ symbol. Mutants like can be subdivided into three groups: r-I, r-II, r-III, to-rye are mapped in different sites of the genetic map of a phage of T-4 and phenotypical differ from each other on the nature of growth on three strains of E. coli (In, S, K). r-II the mutants of T-even phages which are forming on a strain of E. coli In typical r-colonies and not growing on a strain of E. coli of K-12 (λ) are the most studied from them.

From among other mutants, the phenotype to-rykh is characterized by change of morphology of negative colonies, it is necessary to mention muddy, or tu-mutants of a phage of T-4 (from English turbid — muddy), negative colonies to-rykh are surrounded with a muddy ring, and the star mutants described by Saymonds (N. Symonds, 1958). Similar types of mutations on morphology of plaques are described also at other phages, in particular at a phage λ, phages you. subtilis, etc.

Mutants of a phage on the range of action. This type of a mutation is connected with structural change of proteins of the receptor device of a phage and phenotypical is expressed in emergence of ability of a mutant to infect resistant options of bacteria, insensitive to wild type of a phage. For the first time mutants on the range of action were opened by S. Luria in 1945 during the studying of a phage of T-1, and then found also in other phages. S. Luria showed that the colibacillus usually sensitive to a phage of T-1 mutates, forming the resistant form designated by a Ton-r symbol (T — the name of group of phages, an opa — English one, resistant — resistant). Like it at E. coli it is possible to receive resistant mutants to T-even phages, napr, to T-2, i.e. to turn the wild type of colibacillus sensitive to a phage of T-2 (Tto-s), into resistant — Tto-r. Mutations such arise in the genes of colibacillus controlling synthesis of the receptors interacting with the adsorptive device of a phage. At crops of a phage of wild type on a resistant bacterial strain the majority of phage particles is not capable to infect bacteria, however on lawns of resistant cultures it is possible to find the single colonies of a phage arising at the expense of an infection of resistant cells mutant phage particles. Such mutants of a phage with the changed range of action designate an index h (from English host — the owner). Shtrezinger (G. Streisinger, 1956) established that the range of action of a phage of T-2 is controlled by the single gene defining a configuration of the proteinaceous subunit which is a part of tail fibrilla of a phage. Thus, the genotype can characterize a mutation on the range of action of an even phage as transition from h + a genotype to h. It is interesting that independently received h-mutants of a phage of T-2 do not recombine, and during the crossing it is not possible to receive the h+-recombinant capable to infect with E. coli Tto-S and not capable — E. coli Tto-r. On the other hand, if from h-mutants of a phage of T-2 to receive h+-revertant (see Reversion) and to cross them, then it is possible to receive h-recombinants. It indicates that initial structure of a gene — h of a phage T-2 is a h-form, and h observed in vitro + a genotype in essence is mutant.

Conditionally lethal mutants are characterized by the fact that in one conditions (restrictive, or limiting) they behave as flew, i.e. perish, and in others (permissive, or allowing) as wild type.

One of well studied classes of conditionally lethal mutants are so-called ts, or temperaturnochuvstvitelny, mutants, to-rye breed in the infected cells at t ° 28 — 30 ° and do not breed in them at t ° 42 — 45 °.

It is shown that mutations of this type can arise in any site of a chromosome of a phage of T-4, i.e. affect various functions of a phage. Further it was established that ts-mutants arise not only at T-even phages, but also at all other described bacterial viruses, as well as at viruses of animals and the person, and also at mushrooms and bacteria [Brown, Arber (A. Brown, W. Arber), 1964; W. Fattig and soavt., 1965]. The phenotype of ts-mu-tantov is result of the mutation changing finally primary structure of specific protein. It in turn leads to change of its secondary, tertiary and quarternary structure.

Protein gains hypersensitivity to ambient temperature and at increase in the last (42 — 45 °) is denatured, remaining active at t ° 28 °. The same protein at a phage of wild type keeps the structure and consequently, and activity in the range of t ° 42 — 45®.

Amber mutants (am) of a phage of T-4 are characterized by lack of growth on a strain of E. coli In, but are capable to create negative colonies on E. coli of K-12 (CR-63) [R. Epstein and soavt., 1963]. The strains of the bacterial owner «allowing» a reproduction of amber-mutants of a phage in them call permissive, and strains, in to-rykh a reproduction of mutants it is suppressed — not allowing, or not permissive. Studying of molecular mechanisms of amber-mutations showed that in DNA of such mutants there is a mutational change, a cut is transcribed (see. Transcription ) on i-RNK in the form of one of so-called a nonsense codons — UAG which is not distinguished by an anti-codon acceptor RNA (see. Genetic code , Broadcasting ). As a result at a transcription synthesis of polypeptide in that site where there is a nonsense codon stops [Stretton, Brenner (A. Stretton, S. Brenner), 1965]. Proceeding from this property, the amber-codon is a stop codon. However at infection of the permissive bacterial owner synthesis of polypeptide continues since is present at such bacterium acceptor RNA, is capable to broadcast edges («to comprehend») a stop codon and to include acceptable amino acid in this site of a polypeptide chain, recovering thereby functional activity of the synthesized protein. Thus, in the permissive bacterial owner the amber-mutation supressirutsya (see. Suppression ) and such bacterial strain is designated as su+, and the corresponding amber-mutation of a phage belongs to the type sus, T.e. a suppressor - sensitively to a class of mutations.

Property of bacteria to supressirovat an amber-mutation is defined by the genetic locus localized in its chromosome. Respectively not permissive bacterium is designated by a su-symbol.

Ochre mutation of a phage T-4 as well as amber-mutations, belong to the class a nonsense mutations. The Fag bearing ochre mutation breed in the permissive bacterial owner having supressiruyushchy genes which differ from genes, supressiruyushchy an amber-mutation. The nonsense codon at ochre mutation is a triplet of UAA [Brenner, J. Beckwith, 1965].

Feature of this class of conditionally lethal mutations is that they supressirutsya by suppressors, specific to them, though nek-ry amber-mutations supressirutsya as well ochre suppressors. As the amber-codon of UAG differs from UAA ochre codon only on one nitrogen base, they can pass each other as a result of a single mutational event.

The nonsense mutation at a phage of T-4 is the third known type so-called disgraces mutations, for a cut the senseless triplet At HECTARE which is not distinguished acceptor RNA in su - strains is characteristic. Studying of disgraces mutations allowed to come to conclusion that the UGA-triplet is, as well as codons of UAG and UAA, naming. Disgraces mutations it supressirutsya in the bacterial strains bearing a specific suppressor and does not supressirutsya amber-or ochre suppressors.

Crossing and a recombination of phages

For the first time the recombination of phages was carried out in 1946 by Delbryukom and W. Bailey and irrespective of them — Hersha.

For carrying out crossing of phages it is necessary to have genetically marked couple of parent phages differing from each other at least in two signs. The classical mutations used in early studies on a recombination of phages were h and. In the subsequent crossing and a recombination were carried out with use of the phages bearing mutations on all known signs.

Fig. 9. Morphology of the negative parent phages and their descendants received as a result of crossing of h+r and hr+ which is carried out in culture of E. coli of V. Pokazana negative colonies of parent phages of the T-2 group and two reciprocal recombinants (h+r + and hr).

Crossing of phages is carried out by infection of the sensitive bacterial owner with two parent phages genetically different from each other. Conditions of infection of a bacterium with both phages shall be such that almost all bacteria were at the same time infected with both types of phages. For this purpose plurality of an infection each phage shall be on average equal to 5. Two crossed phages apply Benzer's method to achievement of a synchronous infection of bacteria — Jacob, consisting in suppression of an intracellular cycle of a reproduction of a phage cyanide. Then, having reduced concentration of cyanide by cultivation of a suspension of the infected bacteria to inefficient, «include» it samm an intracellular phase of an infection, in the course the cut is carried out a recombination between parent phages. As a result of the subsequent killing in posterity find 4 classes of phages: both parent and two reciprocal classes of recombinants (fig. 9). Except the specified classes, rare options (2%) which are characterized by signs of both parent phages (heterozygote) are found. The relation of number of recombinants of phages in posterity to total number of particles of posterity characterizes the frequency of a recombination. This indicator is constant for two these markers of a phage under a condition if crossing is carried out in strictly reference conditions. However the frequency of a recombination varies depending on what markers of a phage are chosen for a recombination. Crossings of the phages bearing various genetic markers show that the frequency of a recombination varies from 0,01% at an intragenic recombination to 40% in case of the markers removed from each other. As the frequency of a recombination is proportional to distance between genes, by means of recombination analysis it was succeeded to establish the provision of one genes concerning others within a chromosome of a phage. Results of such analysis led to the conclusion that the genetic device of a phage is presented by one linkage group, i.e. consists of one chromosome. On the basis of result of two-factor crossings it was established that the markers of a chromosome of a phage which are most remote from each other are not linked that corresponded to idea of the linear organization of a chromosome of a T-even phage.

The genetic analysis which is carried out on the basis of three-factor crossing with the big ts set and am-mutations of a phage of T-4 found a stseplennost between extreme markers that indicates a tsirkulyarnost of a linkage map of genes at this phage. The decision concerning existence at a phage of one linkage group of genes received on the basis of genetic data corresponds physical. to the observations which established that in a head of a phage there is one molecule DNA. However idea of a tsirkulyarnost of the genetic map appeared in a contradiction with the elektronnomikroskopichesky data of Kleynshmidt (And. Kleinschmidt, 1962) which are unambiguously establishing existence of the free ends in the molecule DNA taken from a head of a phage of T-2. This contradiction was eliminated after opening in a chromosome of T-even phages of terminal redundancy and circular reorganizations.

The structure of a chromosome of a phage

the Chromosome of a phage consists of one molecule nucleinic to - you, edges is put into a proteinaceous cover of a head of a phage. At large T-even phages of colibacillus a pier. the weight of huge two-filamentous DNA is equal to about 1,2 X 10 8 dalton that corresponds to 2 x 10 5 steam of nucleotides.

It is a little known concerning the organization of a chromosome of a phage in a head. It is shown what during an intracellular development cycle of a phage of its DNA is condensed, and its volume in a head makes approximately Vis of the volume occupied in replikativny fund. DNA thread in a head is packed parallel to a longitudinal axis of a phage. Genetic and physical. researches established that the chromosome of phages of T-2 and T-4 which is in a head contains more DNA, than it is required for coding of functions of a vegetative phage since each chromosome which is in an individual phage particle has the same additional sequence of nucleotides on the ends. This additional area in a chromosome of a phage is called terminal redundancy of a chromosome and on extent makes apprx. 3% of a full genome [Shtrezinger, Edgar, Denkhardt (R. Edgar, G. Denhardt), 1967]. Moreover, individual chromosomes in population of a phage are not identical and are characterized by so-called circular shifts, i.e. their terminal redundancy contains various genetic areas which are accidentally received from various sites of a genome. It means that though each chromosome of a phage concluded in a proteinaceous head represents a line structure, any couple of genes in different particles can repeat on its ends (circular reorganization), creating thus a circular linkage group. The mechanism of formation of similar structure is defined by what DNA of even phages, being replicated in a cell, recombines forming polymers from several a genome equivalents [F. Frankel, 1966]. In the subsequent individual chromosomes result from cutting from this huge polymer called by concatenate (Latin of con together, catena a chain), the site of DNA of a certain length, however a little bigger, than a genome:

the Scheme of formation of a chromosome of a phage T-4 from concatenate (it is represented in the form of a straight line; figures designated genes; shooters specified sites of incision of chromosomes, and it is in bold — terminal redundancy).

Thus, in the infected cells there is a set of phage genomes with circular reorganizations in the field of terminal redundancy.

Fig. 10. The scheme of terminal redundancy and circular reorganizations in a chromosome of phages (Latin letters conditionally designated genes). Denaturation and renaturation of a lily family of molecules DNA of phages with terminal redundancy: and — without circular reorganization; at a thermal denaturation of such molecules DNA threads which at a renaturation will create linear chromosomes of initial length are formed; occasionally there is a pairing of molecules on the opposite ends in the field of terminal redundancy that leads to formation of seldom arising circular molecule DNA; — with circular reorganizations; separate polynucleotide threads at a renaturation reassotsiirut in the different ways at which circular molecules often form. I \native chromosomes (DNA) of phages; II \the denatured chromosomes (DNA) of phages; III \renaturirovanny chromosomes (DNA) of phages; IV \circular molecules (DNA) of phages; V \not coupled trailer sites in a circular chromosome (DNA) of phages.
Fig. 11. Genetic map of a phage of T-4. I \the genes controlling synthesis of proteinaceous subunits of a phage and their self-assembly; II \the genes controlling synthesis and DNA replication; e — the gene controlling synthesis of a lysozyme; td — the gene controlling synthesis of a phage timidilatsintetaza; rIIA and rIIb — the genes controlling synthesis of rII-protein; genes are allocated on the card in the form of the shaded and black segments; extent of black segments shows the minimum relative length of genes; figures (outside of the genetic map) — sequence number of a gene.

Opening of the phenomenon of terminal redundancy by Shtrezinger and circular reorganizations well explains how the line structure of DNA of a phage will be coordinated with the circular genetic map of a phage (fig. 10 and 11).

There was clear also a distinction in a behavior pattern of the genomes of the phages which are possessing circular reorganizations in the field of terminal redundancy and not possessing that (T-3, T-5 and T-7). The denaturation and the subsequent annealing (renaturation) of DNA bring in the first case as Thomas showed (S. of Thomas, 1963), and then and other researchers, to formation of a large number of circular genomes, to-rye seldom meet in case of DNA of the phages T-3, T-5, T-7 which do not have circular reorganizations.

Conditionally lethal mutations of a phage of T-4 of all described types, as well as at other phages, mention all signs and are localized in different sites of a chromosome. Allocation of a large number of ts-and am-mutants allowed to identify and map about 70 genes of a phage of T-4 that makes about 50% of possible number of genes in a chromosome of a phage. Identification of function of genes is carried out by the komplementatsionny test (see. Mutational analysis ) between a large number of conditionally lethal mutants and overseeing by existence or lack of various functions of a phage damaged by the corresponding mutations, napr, synthesis of DNA, synthesis of fagoindutsirovanny enzymes, structural proteins of a phage, a morphogenesis, lysis, emergence of phage antigens and so forth. The provision of separate genes in a chromosome of a phage relatively each other was established on the basis of the data obtained during recombination analysis (see. Genetic analysis ).

As a result it was succeeded to design genetic maps of various phages, but the most detailed of them are cards of phages λ and T-4 (fig. 11). If to estimate the extent of a chromosome of a phage of T-4 on the basis of indicators of percent of a recombination between markers, i.e. to express it in terms of the card (1 unit of the card is equal to 1% of a recombination), then it will turn out that the total length of a chromosome of the card will make about 800 pieces. From this it follows that one unit of the card of a phage of T-4 is equivalent to 200 couples of nucleotides. Such calculation demands amendments since it does not consider the phenomenon of high negative interference (see. Recombination ), the recombination influencing frequency within sites of a chromosome of a phage of T-4 which are close from each other. The similar result is received with phages of SPO 1, you. subtilis, etc.

There are two main functional classes of genes of a phage: 1) the genes controlling the early functions connected generally with synthesis of DNA, and 2) the genes controlling late functions, such as synthesis of structural components of a phage, its maturing, a morphogenesis and a lysis of a cell (fig. 11).

Genes defining similar functions are localized in a chromosome of a phage in the form of accumulations, napr, the genes defining a morphogenesis of a phage and formation of a capsid are located in the right upper part of a chromosome, and the majority of the genes connected with synthesis of DNA of a phage of T-4 — in left. In this tendency of topographical association of functionally related genes in a chromosome of a phage of T-4 there is a number of exceptions, napr, the gene 31 which is taking part in formation of a capsid, as well as the genes 34 — 38 controlling synthesis of threads of a tail of a phage are localized between the genes connected with control of synthesis of DNA.

The black segments designated in fig. 11 show the minimum length of genes established on frequencies of recombinations between the mutations localized in this gene. Figures outside of the genetic map of a phage of T-4 designate sequence number of a gene, rIIa, rIIb and td — the genes controlling synthesis of r-protein and a timidilatsintetaza respectively.

Bibliogr. Goldfarb I Will also rumple D. M. to L. A. Immunologiya of nucleic acids, M., 1968; Krylov V. N. The place and time of action of 11 genes of a bacteriophage of T4 at intracellular development of a phage, the Geneticist, t. 5, No. 2, page 136, 1967, bibliogr.; Poglazov B. Fch Assembly of biological structures, M., 1970; Stepanov A. I. both And l and x and N I am S. I N. Electronic and microscopic studying of ts-mutants of a phage of T4B, Geneticist, t. 6, Hv 7, page 114, 1968; Bernstein H., Edgar R. S. a. Denhardt G. H. Intragenic complementation among temperature sensitive mutants of bacteriophage T4D, Genetics, v. 51, p. 987, 1965, bibriogr.; Brenner S. Beckwith J. R. Ochre mutants, new class of suppressible nonsense mutants, J. molec. Biol., v. 13, p. 629, 1965; Brenner S., Stret-t ο n A. O. W. a. To a p 1 a n S. Genetic code, Nature (Lond.), v. 206, p. 994, 1965, bibliogr.; Delbruck M. Bailey W. T. Induced mutations in bacterial viruses, Cold Spr. Harb. Symp. quant. Biol., v. 11, p. 33, 1946; Edgar R. S. a. Lielausis I. Temperature-sensitive mutants of bacteriophage T4D, Genetics, v. 49, p. 649, 1964; Epstein R. H. a. o. Physiological studies of conditional lethal mutants of bacteriophage T4D, Cold Spr. Harb. Symp. quant. Biol., v. 28, p. 375, 1963; Frankel F. R. Studies on the nature of replicating DNA in T4-infected Escherichia coli, J. molec. Biol., v. 18, p. 127, 1966; Hayes W. The genetics of bacteria and their viruses, Oxford, 1968; Hershey A. D. Spontaneous mutations in bacterial viruses, Cold Spr. Harb. Symp. quant. Biol., v. 11, p. 67, 1946, bibliogr.; M a with H a t t i e L. A. a. o. Terminal repetition in permuted T2 bacteriophage DNA molecules, J. molec. Biol., v. 23, p.355,1967; Ritchie D. A. a. o. Terminal repetition in non-permuted T3 and T7 bacteriophage DNA molecules, ibid., p. 365; Salts Y. R ο n e n A. Neighbour effects in the mutation of ochre triplets in the T4r II gene, Mutation Res., v. 13, p. 109, 1971; Streisinger G., Edgar R. S. a. Denhardt G.H. Chromosome structure in phage T4, Proc. nat. Acad. Sci. (Wash.), v. 51, p. 775, 1964; Stret-t ο n A. O. W. a. Brenner S. Molecular consequences of the amber mutation and its suppression, J. molec. Biol., v. 12, p. 456, 1965; T h o m a s of Page A. The arrangements of nucleotide sequences in T2 and T5 DNA molecules, Cold Spr. Harb. Symp. quant. Biol., v. 28, p. 395, 1963; Z i p-ser D. A third class of suppressible polar mutants * J. molec. Biol., v. 29, p. 441, 1967.

D. M. Goldfarb.