[Microbial biodiversity in the Lake Baikal water]. / Kharacteristika bioraznoobraziia mikrobnogo soobshchestva vodnoi tolshchi ozera Baikal.

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.

Comparative 16S rRNA analysis of lake bacterioplankton reveals globally distributed phylogenetic clusters including an abundant group of actinobacteria.

In a search for cosmopolitan phylogenetic clusters of freshwater bacteria, we recovered a total of 190 full and partial 16S ribosomal DNA (rDNA) sequences from three different lakes (Lake Gossenköllesee, Austria; Lake Fuchskuhle, Germany; and Lake Baikal, Russia). The phylogenetic comparison with the currently available rDNA data set showed that our sequences fall into 16 clusters, which otherwise include bacterial rDNA sequences of primarily freshwater and soil, but not marine, origin. Six of the clusters were affiliated with the alpha, four were affiliated with the beta, and one was affiliated with the gamma subclass of the Proteobacteria; four were affiliated with the Cytophaga-Flavobacterium-Bacteroides group; and one was affiliated with the class Actinobacteria (formerly known as the high-G+C gram-positive bacteria). The latter cluster (hgcI) is monophyletic and so far includes only sequences directly retrieved from aquatic environments. Fluorescence in situ hybridization (FISH) with probes specific for the hgcI cluster showed abundances of up to 1.7 x 10(5) cells ml(-1) in Lake Gossenköllesee, with strong seasonal fluctuations, and high abundances in the two other lakes investigated. Cell size measurements revealed that Actinobacteria in Lake Gossenköllesee can account for up to 63% of the bacterioplankton biomass. A combination of phylogenetic analysis and FISH was used to reveal 16 globally distributed sequence clusters and to confirm the broad distribution, abundance, and high biomass of members of the class Actinobacteria in freshwater ecosystems.

[Diversity of bacteria at various depths in the southern part of lake Baikal as detected by 16S rRNA sequencing]. / Bioraznoobrazie bakterii na razlichnykh glubinakh iuzhnoi kotloviny ozera Baikal, vyiavlennoe po posledovatel'nostiam 16S rRNK.

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.

[Nucleotide sequence of a fragment of the Cottocomephorus grewingki phosphofructokinase gene]. / Nukleotidnaia posledovatel'nost' fragmenta gena fosfofruktokinazy Cottocomephorus grewingki.

The nucleotide sequence of a 208-bp fragment of the pfk gene encoding phosphofructokinase from Baikalian fish Cottocomephorus grewingki (Cottidae family) was determined. The fragment shows the codes of a sequence of 38 amino acid residues and contains a 94-bp intron. The nucleotide sequence of the C. grewingki phosphofructokinase gene and the corresponding amino acid sequence display the maximum homology to phosphofructokinases from rabbit muscles and a rat liver.

[Topography of bacteriophage T7 RNA polymerase using monoclonal antibodies]. / Izuchenie topografii RNK-polimerazy bakteriofaga T7 s pomoshchiu monokklonal'nykh antitel.

Four clones producing monoclonal antibodies inhibiting enzymatic activity were used to localize the functionally important antigenic determinants of T7 RNA polymerase. All antibodies were shown to bind to C-terminal fragment of the protein (residues 589-883). The competition studies showed the specificity of the three antibodies toward one epitope and the fourth antibody to another one. By means of limited cleavage of the RNA polymerase with cyanogen bromide with subsequent electrophoretic separation and immunoblotting the peptides containing antigenic determinants were localized. These are Met861-Ala883 for antibody 4H8 and Met750-Met832 for antibodies 9B2, 3H11 and 2A2.

[Highly selective affinity labeling of DNA-dependent RNA-polymerase II from human placenta]. / Vysokoselektivnoe affinnoe mechenie DNK-zavisimoi RNK-polimerazy II iz platsenty cheloveka.

RNA polymerase II from human placenta was affinity labelled in crude preparation using two-step technique, which includes treatment of the enzyme with an aldehyde-containing reactive analogue of ATP, ADP or AMP in the presence of poly[d(A-T)] followed (after borohydride reduction) by the elongation of the attached label with [alpha-32P]UTP. A polypeptide of the molecular mass ca. 140 kDa proved to be the labelling target. No labelling was observed in the absence of poly[d(A-T)] or the reagent or in the presence of alpha-amanitin. All the results suggest the attachment of the affinity reagents to the second-largest subunit of the human RNA polymerase II, which therefore takes part in the initiation substrate's binding.

[Highly selective labeling of the E. coli RNA-polymerase promoter complex with reactive derivatives of oligonucleotide primers of various specificity]. / Vysokoselektivnoe mechenie kompleksa RNK-polimerazy E. coli s promotorom reaktsionnosposobnymi proizvodnymi oligonukleotidov- zatravok razlichnoi spetsifichnosti.

A technique of highly selective affinity labelling, which includes covalent modification of the enzyme-T7A2 promoter complex with reactive oligonucleotide derivatives and subsequent elongation of the attached oligonucleotide residue with a radioactive substrate was used to study the product-binding site of E. coli RNA polymerase. Different oligonucleotides complementary to the T7A2 promoter (with lengths ranging from 2 to 8 residues) containing 5'-terminal phosphorylating, alkylating or aldehyde groups were used for the labelling. The procedure resulted in labelling DNA and beta-, beta'- or sigma-subunits of the enzyme, which are therefore believed to contact with growing RNA in the course of initiation. Consideration of the labelling patterns as a functions of the oligonucleotide's length as well as of the structure and chemical specificity of the reactive groups led to a tentative topographic scheme of the RNA polymerase product-binding region.

[Replication complex of tick-borne encephalitis complex. I. Identification of a nuclear fraction protein responsible for the initiation of RNA synthesis using affinity labeling]. / Replikativnyi kompleks virusa kleshchevogo éntsefalita. I. Vyiavleniebelka iadernoi fraktsii infitsirovannykh kletok, otvetstvennykh za initsiatsiiu sinteza RNK, s pomoshch'iu affinnogo mecheniia.

The pig embryo kidney cells infected by tick-borne encephalitis virus were fractionated into nuclear-associated, cytoplasmic and membrane fractions. The main part of the virus replicase activity was associated with the nuclei. The replication complex is able to synthesize full-length viral RNAs in vitro. To identify proteins involved in the initiation of the replication at the late stages of the infection, the highly specific affinity labelling technique was used. It was shown that with aldehyde-containing derivatives of ATP, ADP and AMP and [alpha-32P]GTP the target of labelling is a polypeptide having molecular weight about 69 kDa. The same protein is immunostained with TBE virus specific antibodies after blotting onto nitrocellulose. The conclusion is made that nonstructural protein NS3 takes part in virus replication at the late stage of the infection.

[Covalent labelling of the Klenow fragment of DNA-polymerase I from E. coli]. / Kovalentnoe mechenie fragmenta Klenova DNK-polimerazy I E. coli.

Incubation of the Klenow fragment of E. coli DNA polymerase I with [alpha-32P] dNTP (or NTP) results in the covalent radiolabelling of the enzyme, the bond being stable in acid (pH 2) and alkaline (pH 12) conditions and nucleophiles, such as beta-mercaptoethylamine, efficiently inhibiting the labelling. It is suggested that radiolabelling of the enzyme is the result of formation of chemically active products of the radiolysis of [alpha-32P]NTP (which are likely to be radicals). Non-radioactive NTP hinder the labelling, whereas Mg2+ and polynucleotide do not affect it. Cleavage of the enzyme by hydroxylamine and cyanogen bromide and analysis of gel-electrophoretic patterns of the cleavage products led to conclusion that 32P-label is located between Gly-544 and Met-647.

[Localization of a lysine residue near the site of initiating substrate binding of T7 bacteriophage RNA polymerase]. / Lokaliatsiia ostatka lizina v oblasti sviazyvaniia initsiruiushchego substrata RNK-polimerazy bakteriofaga T7.

A highly selective affinity label was introduced into the T7 phage RNA polymerase by means of GMP ortho-formylphenyl ester and [alpha-32P]UTP nearby the enzyme's active site, which was located using limited cleavage technique. Hydroxylamine, bromine, N-chlorosuccinimide, and cyanogen bromide were employed as the reagents. Analysis of gel-electrophoretic patterns of the cleavage products led to a conclusion that Lys631 is the target of labelling. The region nearby this residue has a high degree of sequence homology with regions of RNA polymerases from T3 and SP6 phages and yeast mitochondria.

[Immune electron microscope localization of RNA-polymerase on the Escherichia coli chromosome using monoclonal antibodies against the beta-subunit]. / Immuno-élektronno-mikroskopicheskaia lokalizatsiia RNK-polimerazy na khromosome Escherichia coli pri pomoshchi monoklonal'nykh antitel protiv beta-sub''edinitsy.

Possibility of the immunoelectron microscopic visualization of RNA polymerase on the Escherichia coli chromosome with monoclonal antibodies against the beta-subunit labelled by [protein A.gold] complex was demonstrated. Using this method RNA polymerase molecules were revealed within nucleoid as well as on the membrane-free chromosome.

[Elongation of oligonucleotides covalently bound to the active center of RNA polymerase]. / Izuchenie élongatsii oligonukleotidov, kovalentno fiksirovannykh v aktivnom tsentre RNK-polimerazy.

Elongation (mediated by RNA polymerase and NTPs) of the primer oligonucleotide residues, covalently fixed near the active centre of RNA polymerase, has been studied. Hepta- and octanucleotide residues covalently attached to beta-subunit could not be elongated (evidently, because the translocation step is prevented), whereas the same residues attached to sigma-subunit were easily elongated. It was concluded that ease of the oligonucleotide residue elongation is due to the dissociation of sigma-subunit from the transcription complex. The mechanism of this dissociation is discussed.

[Addressed modification of the promoter region of DNA in a complex with RNA polymerase by alkylating oligonucleotide derivatives]. / Adresovannaia modifikatsiia promotornogo uchastka DNK v komplekse c RNK-polimerazoi alkiliruiushchimi proizvodnymi oligonukleotidov.

Addressed chemical modification of double-stranded DNA unwound with the specific protein has been demonstrated. 4(N-2-chloroethyl-N-methylamino)benzyl-5'-phosphoamides of oligonucleotides d(CG)rC, d(ATCG)rC, d(AATCG)rC, which can serve as primers in the RNA polymerase-catalyzed transcription, alkylated A2 promoter region of pSK-A2 plasmid in its "open" complex with E. coli RNA polymerase.

[Highly selective affinity labeling of DNA-dependent RNA-polymerase of bacteriophage T7]. / Vysokoselektivnoe affinnoe mechenie DNK-zavisimoi RNK-polimerazy bakteriofaga T7.

T7 phage RNA polymerase was affinity labelled in the presence of its promoter by treatment with an ATP gamma-derivative (a phosphoamide obtained from 4-(N-chloroethyl, N-methyl)aminobenzylamine, or one of esters obtained from 2-methoxy-4-formylphenol, 4-formylphenol, and 2[N-(4-formylphenyl), N-methyl]-aminoethanol) followed by addition of [alpha-32P]GTP. The most efficient labelling took place with the alkylating phosphoamide reagent.

[Affinity modification of E. coli RNA-polymerase in a complex with the promoter by phosphorylating derivatives of primer oligonucleotides]. / Affinnaia modifikatsiia RNK-polimerazy E. coli v komplekse s promotorom fosforiliruiushchimi proizvodnymi oligonukleotidov-zatravok.

Oligonucleotides 2 to 7 nucleotide residues long, complementary to the codogenic strand of T7 promoter A2, have been synthesized; all of them contained a ribo-unit at the 3'-end. They were converted into 5'-(N-methyl)phosphoimidazolides, and the affinity reagents obtained were allowed to bind covalently to RNA polymerase in the presence of a promoter. Some of the nucleotide residues covalently attached occupied proper positions relative to the active centre of the phosphodiester bond synthesis and on addition of [alpha-32P]UTP were elongated, so that highly selective affinity labelling occurred. With oligonucleotides of various lengths, different distribution of the label between beta, beta' and sigma subunits of RNA polymerase took place. Most efficient was labelling of beta-subunit by the residue--pCpGpCpU, and of sigma-subunit by the residue--pApApApTp-CpGpCpU (p--radioactive phosphorus atom). In both cases, the amino acid residues labelled were histidines.

[Gamma-anilides of nucleoside-5'triphosphates as substrates of DNA-dependent RNA-polymerase of Escherichia coli]. / Gamma-anilicy nukleozid-5'-trifosfatov kak substraty DNK-zavisimoi RNK polimerazy Escherichia coli.

Characteristics of NTP gamma-anilidates (AnpppN) as substrates of E. coli RNA-polymerase have been studied. Michaelis constants for AnpppN are about an order of magnitude greater than those for the usual substrates, whereas the maximum rates differ but unsignificantly. AnpppA is almost completely transformed to polyA and Anppi when synthesis of polyA takes place with AnpppA as the only substrate with denatured DNA as template (reiteration system). The AnpppA-residue is present at the 5'-terminus of RNA synthesized from AnpppA, CTP, UTP and GTP on T7 DNA template by the holo-enzyme.