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The investigation of the microbial community of Lake Baikal by the methods of general and molecular microbiology showed that culturable bacterial strains were represented by various known genera. The lake water contains a great number of bacterial morphotypes, as revealed by electron microscopy, and a great diversity of nonculturable microorganisms belonging to different phylogenetic groups, as revealed by 16S rRNA gene fragment sequencing. The inference is made that the microbial community of Lake Baikal contains not only the known species but also new, possibly endemic to the lake, bacterial species.

A new method for the determination of lipopolysaccharide (LPS) admixtures in protein solutions has been developed. The method includes the periodate oxidation of LPS, biotinylation with biotin hydraside, immobilization on a nitrocellulose membrane and the development of biotinylated LPS in the streptavidin--alkaline phosphatase system. Proteins are previously removed from the solution by treatment with hot phenol. Development with the use of 5-bromoinodyl phosphate and nitrotetrazolium blue makes it possible to detect about 30 pg of LPS immobilized on the nitrocellulose membrane.

Phylogenetic analysis of the bacterial community inhabiting the water of Lake Baikal was performed on the basis of 16S rRNA sequencing. The composition of the bacterial community was shown to vary significantly with depth. Cyanobacteria were dominant species at the surface of the lake. At a moderate depth (400 m), actinomycete relatives were most abundant. At a great depth and near the bottom, the community was composed mainly of proteobacteria and cyanobacteria (the latter are probably brought from the surface layers by vertical near-shore water fluxes). Most of the bacterial 16S rRNA sequences detected exhibited low similarity to those known and formed separate clusters in the phylogenetic tree, which may indicate the endemic nature of the corresponding bacteria.

Bme 361 I, a new site-specific type II deoxyribonuclease, was purified from Bacillus megaterium 361 by chromatography on phosphocellulose P 11 and hydroxylapatite. The enzyme recognizes and cleaves the nucleotide sequence 5'-GG decreases CC-3' in double-strand DNA. Thus it is a true isoschizomer of deoxyribonucleases Hae III and BspR I.

A new approach to synthesis of oligoribonucleotides is suggested, based on transcription by E. coli RNA polymerase of synthetic immobilized DNA-templates with AUG as primer. The approach has been experimentally verified by synthesis of two oligonucleotides, viz., a RNA fragment of the fr phage (16 nucleotides long) and a RNA fragment of the tickborne encephalitis virus (18 nucleotides long). Fraction of the synthesized RNA fragments in the whole nucleotide material is about 20%. The templates can be used repeatedly. Sequences of the oligoribonucleotides were confirmed. Advantages of this approach and its usefulness for SP6 DNA-dependent RNA polymerase are discussed.

The decadeoxynucleotide d(pCCACGAAACC) transcription by E. coli RNA-polymerase was studied. The transcript was shown to be several times longer than the template. The oligonucleotide GpGpGpGpUp complementary to the "contact" of two neighbouring template molecules was found in the pancreatic RNase digest of the RNA-product. This fact is consistent with our hypothesis reported recently. Pentanucleotide d(pGGTTT) may funtion as a primer in the decadeoxynucleotide transcription being incorporated into RNA.

The ColEI plasmid DNA transcription in E. coli RNA-polymerase system in vitro was studied. Three RNA types: of 110-140 residues long, 1700 residues long and of the length similar to that of DNA-template were synthesized in the reaction, containing 10 mM MgCl2, 50 mM KCl and 15% glycerol. The transcription was shown to be asymmetric, the DNA H-chain being transcribed. The ColEI DNA region transcribed in vitro was located by the blotting techniques. This region comprised most of the ColEI genome except the colicin EI gene. The addition of mitomycin C to the reaction mixture caused only a little stimulation of colicin EI gene transcription. Four fragments of HaeIII digest of ColEI DNA were shown to have affinity to RNA-polymerase in the absence of NTPs and two fragments to have it in the presence of 3 NTPs (in the RNA initiation conditions). These two fragments seem to contain two strong promoters. One of them is in the HaeIIIA fragment, where the colicin EI gene origin is situated, and another one is the HaeIIIB fragment (in the immunity region).

The plasmid pBR322 transcription in the isolated E. coli DNA-dependent RNA-polymerase system was studied. Transcription regions as well as transcripts orientation were defined using both the technique of "criss-cross" hybridization and the annealing of RNA-products with L- and H-strands of plasmid. Summing up obtained data together with available data on promoter localization a circular transcription map of plasmid pBR322 was constructed. Effects of heparin, ion strength and E. coli S-30 system on in vitro transcripts were also studied. The 110-long RNA transcript synthesized in Ori region of pBR322 was found to be the most sensitive to all these factors. RNA-transcripts obtained in in vitro system are able to direct protein synthesis in cell-free S-30 system.

The decadeoxynucleotides d(pTTC)3 and d(CCATCTTTT) transcription by E. coli RNA-polymerase was studied. The nucleotide composition of d(pTTC)3 transcript was shown to be consistent with that of the template, but RNA-product was several times longer than the template. With nonanucleotide d(CCATCTTTT) the poly(A) synthesis was observed. This fact may be attributed to the reiteration on the TTTT-cluster. When using the oligonucleotide primers d(pGGA) and r(pAAAA) complementary to the templates d(pTTC)3 and d(CCATCTTTT), accordingly, the nucleosidetriphosphates concentration being reduced and the RNA-polymerase "holo" being replaced by "core", the considerable decrease in the unhomogeneity of the transcript length and in the length itself was found. With d(pTTC)3 as a template the length of RNA-product was found to be of 24-25 nucleotides and with d(CCATCTTTT) it was of 18-19 nucleotides. The sequence of RNA transcribed from both templates in the presence of primers was in accordance with the structure of templates.

The efficiency of hybrid promotor Ptac', comprising a synthetic trp-promoter and lacUV5 "-10" sequence, was studied. By means of electrophoresis and hybridization of RNA-products obtained in vitro and in minicells, with promotor-containing plasmids; the hybrid promotor was found to be 6 times and 3-4 times as efficient as trp and lacUV5 promoters, respectively.

The behavior of nucleoside triphosphates (NTP) in the RNA-polymerase system from E. coli was studied. The conversion of NTP to the corresponding nucleoside diphosphate (NDP) was observed. This phenomenon was accounted for a contaminating enzyme activity in the RNA-polymerase preparations. The possibility to remove such a contamination was demonstrated, the best technique being the DNA-cellulose affinity chromatography. The purified enzyme does not catalyze the intermediate formation of NDP's from NTP's during RNA synthesis with poly(U) and poly(dG)-poly(dC) as templates.

The poly(A) synthesis in the E. coli RNA-polymerase system on the oligothymidilates, oligouridilates and oligonucleotides d(5'-OH-CCATCTTTT-3'-OH) and d(5'-HO-TCTTTT-3'-OH) as templates was studied. The reaction was shown to proceed more intensively on the oligothymidilates compared to oligouridilates. The insertion of phosphate residues into 3' end of oligouridilates deteriorates properties of their templates though the length of the synthesized poly(A) is not changed in this case. The synthesis of poly(A) on the nonanucleotide d(CCATCTTTT) proceeds intensively, but there is no AMP incorporation on the hexanucleotide of similar structure. The author's data combined with certain literature results suppose that the template activity of the oligonucleotides containing homopolymer sequences is determined in the RNA-polymerase system in reiterative regime by two factors: by the total amount of oligonucleotide links and by the homopolymer block size. The total amount of links seems to be important for the oligonucleotide enzyme bind stability and must comprise not less than 6 charges of the phosphate groups. The homopolymer block size must be no less than of 4 links.

A recombinant DNA was constructed by inserting polynucleotide (dA).(dT) of 80-100 base pairs long into EcoRI site of the pBR325 plasmid DNA. Transcription of this DNA was studied in E. coli RNA polymerase system in vitro. Some transcripts obtained with the recombinant plasmid were shown to have poly(U) clusters at the 3'-ends. Obtained data indicate that poly(dA).poly(dT) sequence acts as a terminator of RNA synthesis. Orientation of this sequence in recombinant DNA was also established.

The 99 base pair poly(dA).poly(dT) stretch was inserted into EcoRI site of pBR plasmid. The effect of this insertion on tet-gene expression from P2 promoter, and bla-gene expression from P1 and P3 promoters was studied. The insertion results in a slight enhancement of the resistance of the cells to tetracycline and a decreased sensitivity to ampicillin. It is suggested that these findings reflect the effect of insertion on the transcription of the corresponding genes.