[Functioning of integrative vectors, based on phage phi C 31, in the strain Streptomyces bambergiensis 712]. / Funktsionirovanie integrativnykh vektorov, sozdannykh na osnove faga phiC31, v shtamme Streptomyces bambergiensis 712.

Actinomycete integrative vectors were constructed. The vectors contain the Escherichia coli plasmid ColE1 replicon, the thiostrepton resistance gene used for selection in Streptomyces and a fragment of the phi C31 actinophage genome with integrative functions. The pS133 and PS135 vector DNAs transformed Streptomyces bambergiensis 712, a strain producing the phosphoglycolipid antibiotic moenomycin. Two types of the transformants were detected. The first type was not affected in the ability to produce moenomycin and the vector pS135 DNA was shown to integrate into the S. bambergiensis 712 genome by the site-specific pattern with the psi C31 phage DNA fragment. The second type of the transformants lost the ability to produce moenomycin. The Southern analysis and cloning of the inserted DNA indicated that in this case the vector pS135 and pS133 DNAs also integrated specifically into the genome but the integration took place not within the phage DNA fragment. Its realization was suggested to proceed via homologous recombination.

[Actinomycete plasmid and integrative vectors based on DNA of the temperate Phi C31 actinophage, determining limitation of lytic development of phage Phi C31, not dependent on repressor]. / Aktinomitsetnye plazmidnye i integrativnye vektory na osnove DNK umerennogo aktinofaga Phi C31, opredeliaiushchie organichenie liticheskogo razvitiia faga Phi C 31, ne zavisimoe ot repressora.

Bireplicon plasmids were constructed. The plasmids consist of DNAs of the streptomycete plasmid pIJ702, the Escherichia coli plasmid pUC19 and the phi C31 actinophage genome fragment encoding the function of the site-specific integration into the chromosome. Part of the plasmids transformed Streptomyces lividans TK64 to thiostreptone resistance. The DNA transforming activity depended on the mutual orientations of the blocks used for the construction and probably depended on the structural stability of the plasmids in S. lividans. The integrative vectors consisting of the pUC19 plasmid DNA and the phage genome fragment with the integrative function efficiently transformed S. lividans. No phage plagues were detected with the standard procedure of integrants' infection by phi C31 phage, despite the absence of the phi C31 phage repressor gene in the integrated DNA. The phi C31 phage mutants including clear and virulent ones were not capable of lytic growth on the integrants. The region determining the limitation of the phi C31 phage lytic development was localized by the deletion analysis of the bireplicon plasmids. As a result actinomycete monoreplicon plasmids were formed. The region is the 1.3 kb phage fragment whose right end maps at 0.2 kb preceding the right end of the phi C31 phage genome linear map.

[Physical mapping of Streptomyces coelicolor A3(2) actinophages. VII. Formation of deletions in the region of phi C43 insertion sequence]. / Fizicheskoe kartirovanie akinofagov Streptomyces coelicolor A3(2). VII. Obrazovanie deletsii v oblasti vstavochnoi posledovatel'nosti faga phi C43.

The correlation between the plaque morphology and the presence of the insertion sequence at the phage genomes have been found for different variants of phi C43, obtained by spontaneous induction of strain S. lividans 803. Phages carrying complete insertion have Tum phenotype and they are represented in 4% of induced lysate. Phages carrying the part of insertion have Tu phenotype, their portion in induced lysate is about 26%. Phages without insertion have Stu phenotype and they are the majority of the lysate (70%). All variants phi C43, except wild-type phage, have lost the fragment of phage genome. Phage phi C43ins1, carrying the complete insertion give rise spontaneously at the frequency 10(-2) to deletion derivatives. Heteroduplex experiments show that in the deletion derivatives phi C43ins1 also in the Stu and Tu variants phi C43 one of the termini of the deletions is located within the insertion or at its end. Considerable portion of deletion variants in induced lysate phi C43 also the formation of deletion derivatives phi C43ins1 with high frequency strongly suggest that insertion sequence generates deletions similar to IS of Tn elements.

[Structural peculiarities of the SCP2 plasmid of Streptomyces coelicolor A3(2)]. / Strukturnye osobennosti plazmidy SCP2 Streptomyces coelicolor A3(2).

Structural peculiarities of SCP2 plasmid isolated from different derivatives of Streptomyces coelicolor A3(2) were studied. By means of heteroduplexing 800 b.p. DNA insertion in SCP2 plasmid of S18-1 derivative was detected. The position of this insertion was localised on the SCP2 restriction map. A transposon-like structure with similar characteristics was detected and localised in different variants of SCP2 plasmid. This 600 bp DNA region is flanked by 20 bp inverted repeats and has the single EcoRI cleavage site. It is noted that the stretching of single-strand DNA circles of SCP2 plasmid was significantly less than that of single-strand DNA circles of pBR322 and PAS3; Tn9 plasmids. This may likely depend on the GC content of different plasmids.

[Physical mapping of actinophage Streptomyces coelicolor A3(2). VI. The use of deletion mutants of actinophage phi C31 for construction of phage vectors]. / Fizicheskoe kartirovanie aktinofagov Streptomyces coelicolor A3(2). VI. Ispol'zovanie deletsionnykh mutantov aktinofaga phi C31 dlia konstruirovaniia fagovogo vektora.

Nonessential region responsible for G function has been identified in theta C31 phage genome by means of deletion mutants. The mutant phenotype is expressed upon theta C31 phage propagation in Streptomyces albus G strains differing in functioning of restriction and modification systems. Based on their increased resistance to EDTA, deletions were located in theta C31 delta 10 and theta C31 delta 65 phage mutants. Data are presented on physical mapping of nonessention region of theta C31 phage. The total length of this region is 24.1% of the overall length of DNA molecules. The DNA segment of 19.1% of the whole genome contains overlapped deletions. Theta C31 actinophage is proposed to be used as a cloning vector for Streptomyces. Various deletion mutants obtained, with the capacity of about 3 thousands base pairs may serve as "insertion vectors". The presence of the stretched nonessential genome region allows to use theta C31 phage as a "replacement vector". Then, insertion of foreign DNA to replace the EcoRI--C fragment of theta C31 DNA of 6.4 x 10(3) base pairs is possible. The phages comprising hybrid molecules may be selected for G and Lyg phenotypes.

[Circular permutation of DNA molecules of Streptomyces griseus Kr actinophage Pg2]. / Tsirkuliarnaia permutatsiia molekul DNK aktinofaga Pg2 Streptomyces griseus Kr.

DNA molecules of phage Pg2 have terminal repetitions and are partially circularly permuted. This was established by denaturation self-reannealing experiments and analysis of the distribution of denaturation loops in partially denatured Pg2 DNA molecules. The genome size is 40.8 kb, terminal repetitions are of about 2.2%, the degree of permutation is 35%. On the basis of the data on DNA melting temperature the content of GC pairs was found equal to 65%.

[Physical mapping of Streptomyces coelicolor A3(2). V. Structural modifications of actinophage phiC43 DNA molecules]. / Fizicheskoe kartirovanie aktinofagov Streptomyces coelicolor A3(2). V. Strukturnye modifikatsii molekul DNK aktinofaga phiC43.

As shown by genetical and physical methods, the total preparation of phiC43 phage obtained after spontaneous induction of the prophage from S. lividans 803 strain is a heterogenous population. The wild-type phage (phi C43 wt) is only represented in 5--10% of the population. The majority of phage variants are not able to establish the lysogenic state. The structure of DNA molecules of some phages from the total preparations was characterized by electron microscopy of DNA heteroduplexes. Molecules of phiC43 wt DNA appeared to be completely homologous to those of recently studied phiC62 phage, except for two small regions of approximately 0.3 kb in the central part. Phage variants defective in establishment of the lysogenic state were distributed to two groups. One of them consists of deletion variants, the other--deletion/insertion variants. Deletions in DNA molecule of all nonlysogenizing phage overlap. The region of overlapping seems to be responsible for establishment of the lysogenic state. In the same region, deletion of DNA molecules of mutant phiC311 yg2 has been located. Three deletion/insertion variants contain homologous foreign sequences of various length. It is likely that these insertions are fragments of the host chromosomal DNA.

[Plasmids of Bacillus thuringiensis var. galleriae strain 612]. / Plazmidy Bacillus thuringiensis var. galleriae, shtamm 612.

We studied Bacillus thuringiensis var galleriae, strain 612 plasmids. B. thuringiensis cells contain double-stranded plasmid DNA molecules (ranging of about 12% from total DNA content) with buoyant density 1.59 g/cm3. Plasmid DNA content was constant during the exponential and stationary phases of bacterial growth. The plasmid fractions consist of DNA molecules with molecular weights of 5.9 x 10(6), 10.0 x 10(6), and 110.9 x 10(6) daltons (pVD1, pVD2 pVD3, respectively). Endonuclease EcoRI cuts the plasmids pVD2 and pVD3 into two and four fragments, respectivelyy, but pVDI seemed to be resistent to EcoRI treatment. We found that pVD2 and pVD3 plasmids contain a common DNA fragment with the molecular weight of 6.7 x 10(6) dalton as it was shown by restriction analysis. In contrast, the same plasmids contain the common fragment with molecular weight of 7.5 x 10(6) dalton as shown by heteroduplex analysis. Plasmid pVD3 has a transposon-like structure.

[Physical mapping of Streptomyces coelicolar A3(3) actinophages. III. Restriction analysis]. / Fizicheskoe kartirovanie aktinofagov Streptomyces coelicolar A3(2). III. Restriktsionnyi analiz.

It has been shown that endonucleases HindII, HindIII, SalI and BsuI treatment of phiC62, or phiC43 and phiC31 DNAs forms more than 20 fragments. EcoRI cleaves phiC62, phiC31, phiC31c5 and phiC31c28 into seven fragments, but phiC311yg33 into six fragments. Comparison of molecular weights of DNA restricts obtained after hydrolysis of phage DNAs containing deletions by endonuclease EcoRI made it possible to determine the location of four fragments on restriction map and to orientate this map in relation to the molecule's ends. BamHI cleaves phiC43 DNA into two fragments. By heteroduplexing BamHi site was mapped within the phiC43 insertion sequence.

[Physical mapping of Streptomyces coelicolor Ae(2) actinophages. IV. Partial denaturation maps]. / Fizicheskoe kartirovanie aktinofagov Streptomyces coelicolor A3(2). IV. Karty chastichnoi denaturatsii.

By measuring temperature transition profiles evidence was obtained that DNA of actinophage phiC31 has a random base distribution and the content of GC pairs is 63%. The denaturation maps of the DNA molecules of actinophages phiC31c28, phiC43del and phiC62 are similar and characteristic of these random base distributions. Main peaks are located at positions: 0.03, 0.25, 0.33, 0.53 and 0.99 from the left end (in relative units). Positions of the regions deleted in phages phiC31c28 and phiC43del were established in respect to phage phiC62 genome and shown not to coincide with the position of the denaturation peaks on the phiC62 DNA map. It seems unlikely that AT rich regions are preferrable ones for deletions in actinophage DNAs.

[Physical mapping of Streptomyces coelicolor A3(2) actinophage DNA. II. Transposon-like structure in DNA molecules of phiC43 phage DNA, localization of region responsible for establishment of the lysogenic state]. / Fizicheskoe kartirovanie DNK aktinofagov Streptomyces coelicolor A3(2). II. Transpozonopodobnaia struktura v molekuliakh DNK faga phiC43, lokalizatsiia oblasti, otvetstvennoi za ustanovlenie lizogennogo sostoianiia.

Structural properties of DNA molecules of phages phi C43, phi C43del and mutant phage phi C311yg33 were studied. Actinophages phi C43del and phi C311yg33 have been isolated and shown to have a phenotype characteristic of phages defective in integration, i. e. turbid plaques and inability to establish the lysogenic state. By means of heteroduplexing, deletions were mapped in the genomes of these phages. DNA molecules of phi C43del and phi C11yg33 are devoid of the common fragment, which suggests that the mutant phenotypic character is associated with structural alterations in DNA molecules. The presence of transposon-like structure in phi C43 DNA molecules has been inferred from the analysis of phi C43/phi C43del heteroduplexes and phi C43 homoduplexes. Also, a deletion in phi C43 genome has been detected covering the same region where deletions in phi C43del and phi C11yg33 DNAs were located.

[Physical mapping of Streptomyces coelicolor A3(2) actinophage DNA. I. Location of the c-region of actinophage phiC31]. / Fizicheskoe kartirovanie DNK aktinofagov Streptomyces coelicolog A3(2). I. Lokalizatsiia c-oblasti aktinofaga phiC31.

Mutants of temperate actinophage phi C31 Streptomyces coelicolor A3(2) having an increased resistance to chelating agents (sodium EDTA and sodium citrate) were isolated. Most of these mutants were not able to lysogenize sensitive cultures (c-mutants). DNA molecules of four c-mutants resistant to chelating agents were shown to be deleted by electron microscopy of DNA heteroduplexes. The four deletions were located in the central region of phi C31 DNA molecule. The deleted segment of 1000 base pairs common for molecules of all c-mutants is located in a region 47,2--49,9% and indicates the position of c-region on the physical map of phi C31 actinophage. The size of the region containing delections of all analyzed c-mutants is 1700 base pairs. The c-region on the heteroduplex map was oriented in relation to the known genetic map of phi C31.

[Genetic characteristics and genome structure of Streptomyces coelicolor actinophages]. / Geneticheskaia kharakteristika i struktura genomov aktinofagov Streptomyces coelicolor.

Actinophage phi C31 of Streptomyces coelicolor A3 (2) and two novel temperate actinophages phi C43 and phi C62 isolated from strains of blue actinomycetes group are homoimmune, serologically and functionally related. DNA molecules of phages phi C31, phi C43 and phi C62 have cohesive ends; sizes of DNAs of these phages and some mutants have been determined. The extent of homology between the DNAs of three phages is 93-96% as shown by heteroduplex analysis. The regions of non-homology are of a deletion-insertion type and of approximately 1500 base pairs in the length. Location of deletions in DNAs of mutant phages phi C31 vd and phi C31 c5 has been shown. Structural modifications in phage dnas have been found only to occur in the right part of molecules. Heteroduplex maps have been constructed for all phages studied.