[The resistance of the DNA of cyanophage LPP-3 to the action of different restriction endonucleases]. / Rezistentnost' DNK tsianofaga LPP-3 k deistviiu razlichnykh éndonukleaz restriktsii.

Data on the study of structure peculiarities of cyanophage LPP-3 DNA are presented in the work. The length of cyanophage DNA calculated by means of the enzymatic hydrolysis by restrictases is 40 +/- 3.5 thou. pairs of bases. Cyanophage LPP-3 DNA was hydrolysed by more than 50 different restrictases. As a result of screening it was found out that the great number of restrictases, which recognized hexanucleotide sequences did not hydrolyze DNA of cyanophage LPP-3. A considerable deviation of the number of the observed sites of restriction from their theoretically expected number for restrictases Hae III and Cfr 131 was established. Restrictases-isoschisomeres with different sensitivity to the methylation of the recognition sites--Msp I, Hpa II and Sau 3A, MboI and DpnI were used to check the availability of methylated bases in LPP-3 DNA. Absence of methylated adenine in the site GATC and methylated cytosine in the second position of the site CCGG were established. The results obtained permit supposing that the expressed counterselection by the sites of recognition of many restriction endonucleases takes place in cyanophage LPP-3 DNA. It is supposed that apparently, this method of protection of its genome in LPP-3 is one of most important but the inconsiderable percentage of site-specific methylation of the virus DNA cannot be completely excluded.

[Features of genome expression of phage transposon D3112 of Pseudomonas aeruginosa in Escherichia coli bacteria: dependence of bacterial phenotype on copy number of D3112 genome]. / Osobennosti vyrazheniia genoma faga-transpozona D3112 Pseudomonas aeruginosa v bakteriiakh Escherichia coli: zavisimost' fenotipa bakterii ot chisla kopii genoma D3112.

Escherichia coli (RP4 :: D3112) bacteria manifest Tcs phenotype (thirty centigrade sensitivity), i.e. the cells do not divide and form colonies under conditions of lowered temperature (30 degrees C and lower), while cells grow normally at 42 degrees C. In this work it is demonstrated that replication-transposition of D3112 and the Tcs phenotype depend on no recA system of E.coli. Following events lead to the loss of the Tcs phenotype (in E.coli (RP4 :: D3112) cells survived after growing at 30 degrees C): occurrence of mutations in bacterial, phage and plasmid genomes, elimination of DNA of hybrid plasmid or RP4 DNA (a portion of DNA) as well as integration of the hybrid plasmid into bacterial chromosome. In the latter case, the E.coli (D3112) cells acquired the properties shared by the initial bacteria and those with the Tcs phenotype. Such clones are designated tcl (thirty centigrade low sensitivity), they are able to form colonies at 30 degrees C but their growth is more slow, they maintain instability at lowered temperature and continue to produce D3112 phage. The tcl clones in which replication-transposition of D3112 DNA in less effective than in the tcs clones are a suitable object for the study of genetic rearrangements caused by D3112 phage transposon. It is shown that either complete RP4 genome or its portion are comprised between direct repeats of D3112 and are built into various chromosomal sites, i.e. cointegrates are being formed. Two types of deletions are revealed: eliminating sites of RP4 plasmid adjacent to the left end of D3112 genome as well as deletions of the D3112 genome. It is demonstrated that alteration in the growth nature of E.coli, carrying D3112 DNA, at 30 degrees C depends on the copy number of D3112 per bacterial cell.

[2 site-specific endonucleases of the cyanobacterium Nostoc linckia]. / Dve sait-spetsificheskie éndonukleazy tsianobakterii Nostoc linckia.

Two restrictases Nli387/7 I and Nli387/7 II have been isolated from cyanobacterium Nostoc linckia using chromatography on phosphocellulose, "Mono Q" column, and heparin sepharose 4B. The preparations are described by the method of electrophoresis in polyacrylamide gel under denaturing conditions. Catalytic properties of restrictases are determined: optimal pH of the action--9.0--9.5, optimal concentration of Na+--5 mM, that of Mg2+--6 mM, optimal temperature--37 degrees C. The isolated enzymes are isoschizomers of restrictases avaI and AvaII. The point of cutting is determined for enzyme Nli387/7 I. It is shown that restrictase Nli387/7 I is a false isoschizomer Ava I.

[A new site-specific endodeoxyribonuclease EcoHI]. / Novaia saitspetsificheskaia éndodezoksiribonukleaza EcoHI.

A new sitespecific endonuclease of the II class EcoHI has been isolated from Escherichia coli strain and characterized. Restriction endonuclease EcoHI recognises the nucleotide sequence C C (C/G) G G with the cleavage site between the fourth and fifth nucleotide. It is an isoshizomer of the restriction endonuclease CauII. The yield of enzyme is 2500 units of activity per 1 g of biomass. The producing strain Escherichia coli HI is nonpathogenic, easily grown with the antibiotic resistance markers permitting to cultivate the strain under selective conditions.

TGATG vector: a new expression system for cloned foreign genes in Escherichia coli cells.

A TGATG vector system was developed that allows for the construction of hybrid operons with partially overlapping genes, employing the effects of translational coupling to optimize expression of cloned cistrons in Escherichia coli. In this vector system (plasmid pPR-TGATG-1), the coding region of a foreign gene is attached to the ATG codon situated on the vector, to form the hybrid operon transcribed from the phage lambda PR promoter. The cloned gene is the distal cistron of this hybrid operon ('overlappon'). The efficiently translated cro'-cat'-'trpE hybrid cistron is proximal to the promoter. The coding region of this artificial fused cistron [the length of the corresponding open reading frame is about 120 amino acids (aa)] includes the following: the N-terminal portions of phage lambda Cro protein (20 aa), the CAT protein of E. coli (72 aa) and 3' C-terminal codons of the E. coli trpE gene product. At the 3'-end of the cro'-cat'-'trpE fused cistron there is a region for efficient translation reinitiation: a Shine-Dalgarno sequence of the E. coli trpD gene and the overlapping stop and start codons (TGATG). In this sequence, the last G is the first nucleotide of the unique SacI-recognition site (GAGCT decreases C) and so integration of the structural part of the foreign gene into the vector plasmid may be performed using blunt-end DNA linking after the treatment of pPR-TGATG-1 with SacI and E. coli DNA polymerase I or its Klenow fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

[Structure and stability of cointegrating plasmids mediated by the IS1 elements in transposon delta Tn9']. / Struktura i stabil'nost' kointegratnykh plazmid, oposredovannykh élementami IS1 v sostave transpozona delta Tn9'.

In order to elucidate the structural features of the transposon Tn9', representative of the Tn9 family, which define the ability of the transposon to produce unstable cointegrates, we have obtained a derivative of this transposon carrying a deletion in its central region. The deletion in the obtained transposon delta Tn9' covers a DNA segment of about 50 bp in length, occupying the most distal position in relation to the cat gene, at its junction with the right copy of the IS1. The structure and stability of the IS1/delta Tn9'-mediated cointegrates between the plasmids pDK57.1 (pBR322::delta Tn9') and pRP3.1, a deletion derivative of RP1, have been studied. The three types of cointegrates were found. Those of the type I are predominantly formed, due to the left copy of the IS1 which in delta Tn9' occupies proximal position to the promoter of the cat gene. These cointegrates contain three copies of IS1 and are of high stability. The cointegrates of the type II contain two entire copies of delta Tn9' (i.e. four copies of IS1) as well as the structures of the type II, representing the cointegrate equivalent of inverse transposition and also containing four copies of IS1. Cointegrates of the type II and III dissociate efficiently in the rec+ cells but, in contrast to the cointegrates mediated by the original transposon Tn9', are unable to dissociate efficiently in the recA- cells. It was concluded that a DNA segment in the central region of Tn9' may be essential for the expression of the IS1-specific resolvase encoded by the right copy of IS1.

Phasmids as effective and simple tools for construction and analysis of gene libraries.

Phasmid lambda pMYF131, a hybrid of phage lambda vectors and plasmid pUC19, was constructed. The phasmid and its derivatives were shown to be efficient vectors for construction and analysis of gene libraries in Escherichia coli cells. The lambda pMYF131 DNA molecule contains all the genes and regions essential for phage lytic development. The plasmid cannot be packaged either in the monomeric or the oligomeric form due to its specific length. Elongation of the DNA molecule by ligation with fragments of foreign DNA can make it packageable and this is easily detected by plaque formation. Hence, the procedures used to construct genomic libraries can be simplified by selection of only recombinant DNA molecules just at the time and on the basis of their packaging in vitro. The output of recombinant clones per vector molecule was several times higher for vector lambda pMYF131, compared to phage vector lambda L47.1AB, and attained 3 x 10(6) clones per micrograms DNA. Vector and recombinant phasmids can be obtained in large quantities in plasmid form. lambda pMYF131 contains nine unique restriction sites which allow the cloning of DNA fragments with blunt ends and of fragments with various types of cohesive ends, obtained by digestion with 14 prototype restriction enzymes. The maximal size of the cloned DNA fragments is approx. 20 kb for lambda pMYF131. Phasmid vectors were used to construct libraries of bovine, pig and quail genomes, and genomic libraries of 17 species of bacteria. Application of suitable methods allowed the identification 13 individual genes within these libraries.

[Construction and use of phasmid vectors of large capacity]. / Konstruirovanie i ispol'zovanie fazmidnykh vektorov bol'shoi emkosti.

lambda vector phages--pUC19 phasmid hybrids were constructed. The hybrids, phasmids lambda pMYF131 and lambda pSL51, were used as vector molecules for making genomic libraries. Vector phasmids exist as plasmids in vivo. They contain all the genes and specific sites necessary for lytic development, but the DNA molecules are not long enough to be packaged in lambda capsid. Elongation of the molecule due to insertion of a foreign DNA fragment renders phasmid all features of non-defective phage. Output of recombinants is up to 3 X 10(6) per 1 microgram of phasmid vector DNA. The fraction of non-recombinants in libraries is less than 1-0.1%. The capacity of the vectors is 19.6 and 22 kb for lambda pMYE131 and lambda pSL51 accordingly. It is possible to clone DNA fragments with blunt ends and various sticky-end fragments obtained by digestion with 14 prototype restrictases, into nine unique restriction sites of vector lambda pMYF131. The created vectors allowed to construct more than 30 representative genomic libraries of eukaryotic and prokaryotic organisms. 13 individual genes were identified within the libraries.

[Two loci in the genome of the Pseudomonas aeruginosa transposable phage B39 which affect the integration process. II. Mapping of the pdeX and pdeY loci by restriction and heteroduplex analysis]. / Dva lokusa v genome faga-transpozona B39 Pseudomonas aeruginosa, vliiaiushchie na protsess integratsii. Soobshchenie II. Opredelenie polozheniia lokusov pdeX i pdeY metodami restriktsionnogo i geterodupleksnogo analizov.

The coordinate function of two loci - pdeX and pdeY - in the genome of a transposable phage (TP) provides the phage function pde+ (good growth on bacteria with Rms163 plasmid). When these two loci in hybrid phages originate from different TP, some of the hybrid phages have Pde- phenotype. To localize pdeX and pdeY, the structure of hybrid TP genomes with Pde+ and Pde- phenotype obtained in crosses between B39ts+ and PH132 were studied using restriction and heteroduplex analysis. On the basis of data obtained, pdeX and pdeY were mapped in 2.85-6.4 and 6.4-16 kbp regions, respectively.

[Limitation of phage multiplication by microbes of the genus Bordetella]. / Ogranichenie razmnozheniia fagov mikrobami roda Bordetella.

Possible causes limiting the multiplication of Bordetella phages or inducing their restriction, such as the influence of lysogenic immunity and the restriction-modification (R-M) system or the incompatibility of the receptor apparatus, have been studied. The limitation of the multiplication of phages by some B. bronchiseptica and B. pertussis strains has been shown to be due to the presence of the R-M system and lysogenic immunity. In five B. bronchiseptica strains and two B. pertussis strains site-specific endonucleases (restrictases) with Hind III specificity have been detected. One B. bronchiseptica strain without the R-M system has been detected. B. bronchiseptica strains producing site-specific endonucleases are practically nonpathogenic for humans, grow in common culture media and selectively produce only one restrictase, type Hind III, which guarantees from the admixture of other specific endonucleases. The B. parapertussis strains under study (altogether 100 strains) have not been found to limit the multiplication of Bordetella test phages. The absence of site-specific endonucleases has also been confirmed biochemically. These strains are recommended as indicator strains for the multiplication of Bordetella phages.

[Localized mutagenesis of the tetracycline gene in the plasmid pBR322 induced by sodium bisulfite in vitro]. / Lokalizovannyi mutagenez tetratsiklinovogo gena plazmidy pBR322 bisul'fitom natriia in vitro.

The technique of localized in vitro mutagenesis in the cohesive ends of plasmid pBR322 DNA has been elaborated (separately for BamHI and HindIII sites). Plasmid DNA digested by restriction endonucleases has been treated with sodium bisulphite deaminating cytosine to form uracil in single stranded DNA (cohesive ends of the plasmid). The mutagenized plasmid DNA, free of mutagen, has been treated with bacteriophage T4 ligase. E. coli C600 cells were subsequently transformed by the ligated DNA preparation. The clones having tetracycline gene mutagenized represented 4.0-11.1% and 1.2-3.1% among HindIII and BamHI mutants, respectively, selected as TcR----TcS transformants. Selection of mutagenized DNA by the second endonuclease restriction has increased the mutant yields up to 55.6-78.0% and 10.0-75.4%, respectively. The yield of TcS mutations in the control DNA treated at all stages of experiment, except for mutagen treatment, has reached 0.06% and 0.2%, respectively.

[Interaction of the peptide antibiotic bacitracin with DNA and synthetic polynucleotides]. / Issledovanie vzaimodeistviia peptidnogo antibiotika batsitratsina s DNK i sinteticheskimi polinukleotidami.

The aim of the present paper was to study the specific character of interaction of peptide antibiotic bacitracin with DNA and to estimate the interaction constant. The influence of bacitracin on bacteriophage DNA restriction by HindIII and SmaI endonucleases was studied. The possibility of arranging the polynucleotide template by small ligands was shown.

[Characteristics of Bacillus thuringiensis phages with circular permutation in the DNA molecule]. / Kharakteristika fagov Bacillus thuringiensis s tsirkuliarnoi permutatsiei molekul DNK.

Bacteriophages Tm2 and Tg27 of different origins but identical in biological properties have been compared. Physicochemical characteristics of bacteriophages have revealed the existence of end repeats and circular permutation of phage DNA. Phages Tm2 and Tg27 share the same dimensions of incapsulated DNA, differing in the sizes of phage genome and end repeats. Bacteriophage Tm2 genome is 45.2 kb. long with the end repeats containing 5.7%. The genome of Tg27 is 42.53 kb and 11.8% of end repeat. The bacteriophages relation has been confirmed by heteroduplex and restriction analysis. Tm2 and Tg27 share 84% of homology. Two regions of nonhomology are found representing a single-stranded loop and equishouldered vesicle with the sizes 2.19 kb and 5.03 kb, respectively.

[Molecular genetic studies of the DNA promotor regions in Bacillus thuringiensis subsp. galleriae 69-6. I. Structural and functional analysis]. / Molekuliarno-geneticheskie issledovaniia promotornykh raionov DNK Bacillus thuringiensis subsp. galleriae 69-6. Soobshchenie I. Strukturno-funktsional'nyi analiz.

Three recombinant plasmids pPBT9, pPBT10 and pPBT74 carrying promoter-containing regions of DNA of Bacillus thuringiensis which are responsible for the expression of the promoterless tet gene, were studied. In the in vitro experiments, it had been shown that these promoter-active HindIII fragments of bacillar DNA contained RNA polymerase binding sites. The AluI subfragments that specifically bind to Escherichia coli RNA polymerase promote the tet gene expression, similar to the whole HindIII fragments. Sequence analysis revealed that the approximately 220 base pair AluI subfragment of the bacillar insertion of the pPBT10 plasmid contained sites typical for "-10" and "-35" homology regions of promoters specific for sigma 55-RNA polymerase from Bac. subtilis. The 1.45 kb HindII bacillar fragment of the plasmid pPBT9 had three AluI subfragments that bind to E. coli RNA polymerase. Only approximately 400 base pair AluI subfragment among these restored the tet gene expression in vivo. Bireplicon pBP plasmids were constructed that promoted the expression of the enterobacterial antibiotic resistance gene under the control of Bac. thuringiensis promoters in Bac. subtilis cells.

Bacteriophages of methanotrophs isolated from fish.

Bacteriophages of methanotrophic bacteria were isolated from 67 fish. Only two phages isolated from two fish species specifically lysed Methylocystis sp. and Flavobacterium gasotypicum. The phages lysing these species were designated 63-F and CMF-1-F, respectively. The isolated phages differed greatly in the fine structure of the virion, plaque morphology, spectrum of lytic action, serological properties, and UV sensitivity. At the same time, they had identical one-step growth characteristics: their latent period equalled 5 h, lysis time was 3 to 4 h, and burst size was about 240 virions. The phages had guanine- and cytosine-rich double-stranded DNAs consisting of common nitrogen bases. The molecular masses of the DNAs as determined by the sums of restriction endonuclease cleavage fragments were 28 X 10(6) daltons for phage 63-F and 31 X 10(6) daltons for phage CMF-1-F.