[Interaction of proteins from general transcription complex RNA polymerase II with oligoribonucleotides].

We have analyzed an interaction of the general transcription complex RNA polymerase II proteins (RNA polymerase II, factors TBP, TFIIB, TFIIF, TFIIE and TFIIH) S. cerevisiae with the oligoribonucleotides. With the help of method EMSA was shown that labeled 32P labeled oligoribonucleotide 5'-ACUCUCUUCCGCAUCGC-3' (r-17) binds with the proteins and generates three species of the complexes with the three major shifts. All the three species of the complexes are RNA specific because a total RNA S. cerevisiae was a competitor for all three species but the TATA-containing oligodeoxyribonucleotide (500-fold molar excess) was not a competitor for its. Complexes 32P-r-17 with the proteins belonging to the middle shift are the sequence specific because unlabeled r-17 was a competitor for its binding (100-fold molar excess) but unlabeled UA-rich oligoribonucleotide (5'-AUAUUAUGUUCAAAA-3) was not a competitor for this shift (500-fold molar excess). Complexes belonging to the upper shift are RNA specific probably. We think 32P-r-17 interaction with the proteins belonging to the under shift is nonspecific corresponding to a sorbtion of 32P-r-17 on a protein. The data presented demonstrate that oligoribonucleotide and oligodeoxyribonucleotide don't compete for the binding sites on a basal transcription complex proteins.

[Interaction of Escherichia coli RNA polymerase with eukaryotic TATA-binding protein]. / Vzaimodeistvie RNK-polimerazy Escherichia coli s TATA-sviazyvaiushchim belkom éukariot.

Interaction with eukaryotic TATA-binding protein (TBP) was analyzed for natural Escherichia coli RNA polymerase or the recombinant holoenzyme, minimal enzyme, or its sigma subunit. Upon preincubation of full-sized RNA polymerase with TBP and further incubation with a constant amount of 32P-labeled phosphamide derivative of a TATA-containing oligodeoxyribonucleotide, the yield of the holoenzyme-oligonucleotide covalent complex decreased with increasing TBP concentration. This was considered as indirect evidence for complexing of RNA polymerase with TBP. In gel retardation assays, the holoenzyme, but neither minimal enzyme nor the sigma subunit, interacted with TPB, since the labeled probe formed complexes with both proteins in the reaction mixture combining TBP with the minimal enzyme or the sigma subunit. It was assumed that E. coli RNA polymerase is functionally similar to eukaryotic RNA polymerase II, and that the complete ensemble of all subunits is essential for the specific function of the holoenzyme.

Affinity labeling of RNA-polymerase II in the transcriptionally active complex by a phosphorylating analog of the initiation substrate.

Affinity modification of RNA-polymerase II by a phosphorylating analog of the initiation substrate carrying a zwitterionic 5;-terminal phosphate group with a 4-N,N-dimethylaminopyridine residue (DMAP-pA) was studied during specific transcription initiation controlled by the late adenoviral promotor. Super-selective affinity labeling and standard conditions of affinity modification resulted in labeling a polypeptide with molecular weight corresponding to that of the third subunit of the enzyme, RPB3 (45 kD). The initiation substrate (ATP) protects RNA-polymerase II from modification. The third subunit may be involved in the formation of the substrate-binding site of the enzyme.

[Interaction of Escherichia coli RNA polymerase with oligoribonucleotides, homologous to "10"- and "35"- segments of the SPC promotor of bacterial genes]. / Vzaimodeistvie RNK-polimerazy Escherichia coli s oligoribonukleotidami, gomologichnymi "10;- i "35"- uchastkam SPC-promotora bakterial'nykh genov.

It was shown previously that E. coli RNA polymerase in a highly selective manner recognizes and binds 11-14-mere oligodeoxyribonucleotides related to the "-10" region of the nontranscribed DNA strand of bacterial gene promoters. The oligodeoxyribonucleotides cover the Pribnow box with flanking nucleotides up to the transcription start. These affinity oligodeoxyribonucleotides inhibit competitively the transcription of bacterial DNA carried out by E. coli RNA polymerase. The present work has demonstrated that E. coli RNA polymerase is not capable of binding the oligoribonucleotides homologous to the affinity oligodeoxyribonucleotides related to the "-10" area of the spc promoter, but binds the oligoribonucleotides which are complementary to the latter. The oligoribonucleotides with a high affinity for the E. coli RNA polymerase strongly inhibit transcription of the bacterial DNA. Attachment of alkylating groups to the 5'-ends of the affinity oligodeoxy- and oligoribonucleotides provides their covalent binding to the E. coli RNA polymerase subunits. It was shown that the modified affinity 32P-labelled oligodeoxyribonucleotide is covalently bound to the sigma-subunit while the modified affinity 32P-labelled oligoribonucleotide is covalently bound to the beta'beta-subunits of the E. coli RNA polymerase. It is suggested that the affinity oligoribonucleotides can be transcribed from the non-transcribed DNA strand in the region of the open complex and functions presumably as a primer which is splitted later from the nascent RNA or as a regulator of transcription.

[Immunoenzyme analysis of antigen-specific determination of HBsAG-containing circulating immune complexes (CIC HBsAG/IgM and CIC HBsAG/IgG) in blood serum of patients with HBV-infection]. / Immunofermentnyi analiz antigenspetsificheskoe opredelenie HBsAG-soderzhashchikh tsirkuliruiushchikh immunnykh kompleksov (TSIK HBsAg/IgM i TSIK HBsAg/IgG) v syvorotke krovi bol'nykh pri HBV-infektsii.

A complex enzyme immunoassay (ELISA) has been designed for antigen-specific determination of HBsAg-containing circulating immune complexes (CIC HBsAg/IgM and CIC HBsAg/IgG) in human blood sera in parallel with registration of free HBsAg and specific antibodies to viruses of hepatitis A, B and D. It is shown that effective formation of HBsAg-containing CIC serologically is registered predominantly as a mutually incompatible marker with detection of free HBsAg (in 70-85% of the cases). CIC HBsAg/IgM and CIC HBsAg/IgG may be registered both in parallel and as mutually exclusive markers. Effective formation of HBsAg-containing CIC in the presence of anti-HBsAg occurs in case of a mild course of viral hepatitis of epidemic and sporadic type, while in severe forms of VH-free HBsAg is predominantly detected thus pointing either to ineffective formation of HBsAg-containing CIC or to their continuous registration with demonstration of the effect of delay of witching of anti-HBsM over to anti-HBsG (or CIC HBsAg/IgM to CIC HBsAg/IgG). It was also found that in case of epidemic VH in Tajik SSR (1987) serologically marked as VH both A and B convalescent phase was characterized by parallel disappearance (or lowering of the titer levels) of HBsAg-containing CIC and class M antibodies to both hepatitis A (anti-HAV M) and B (anti-HBcM, anti-HBsM) along with the containing parallel registration of relevant G-antibodies (anti-HAV G/anti-HBcG). This observation requires further studies both in terms of close association of viruses of hepatitides A and B and with regards to possible antigenic mimicry.

[Identification of the glucocorticoid receptor binding site at the 5'-flanking region of mouse metallothionein I gene: the effect of base substitutions on binding efficiency]. / Identifikatsiia uchastka sviazyvaniia gliukokortikoid-retseptornogo kompleksa v 5'-flankiruiushchem raione gena metallotioneina I myshi: vliianie nukleotidnykh zamen na éffektivnost' sviazyvaniia.

Interaction of highly purified glucocorticoid receptor complex (GIRC) with synthetic DNA-fragment of mouse metallotionein 1 gene promoter from -209 to -252 b.p. (MTwt) was investigated. By means of nitrocellulose filter binding assay this fragment was shown to contain specific GIRC-binding site. In order to analyse the fine structure of the site, two variants of this DNA-fragment were synthesized and used in gel retardation assay. GIRC specific binding was shown to retain throughout interaction with the fragment in which all base pairs in the surroundings of generally accepted GIRC-binding site consensus G--ACA---TGTTCT C--TGT---ACAAGA were substituted by means of transitions, but it was weaker than the GIRC-binding with MTwt, where the mentioned consensus was situated in the natural surroundings. Complete loss of the GIRC-binding ability was observed when five CG pairs were substituted by AT ones. Two of the CG pairs belonged to the mentioned consensus. Comparison of the data obtained with results of computer analysis allows to consider the consensus as a "core" of GIRC-binding site, flanked with additional elements, interacting with GIRC.

[Study of the antigenic structure of hepatitis B virus proteins. I. Synthesis of pre-S fragments of hepatitis B virus proteins and their immunogenicity]. / Izuchenie antigennoi struktury belkov virusa gepatitaa B. I. Sintez fragmentov pre-S belkov virusa gepatita B i izuchenie ikh immunogennosti.

Fragments of hepatitis B virus envelope proteins corresponding to the parts of the pre-S domain were synthesised and immobilized on the carriers with low own immunogenicity. The highest stimulation of the antibody production was observed for the antigens immobilized on microspherical carriers or gelatin modified by H-Gly-Tyr-OH. Among peptides used for immunization, pre-S fragment 134-144, conjugated with microspherical carrier, proved to be the most active.

[Differentiation of virulent strains of Shigella and entero-invasive Escherichia from their avirulent variants using modified indirect immunoenzyme analysis]. / Differentsiatsiia virulentnykh shtammov shigell i énteroinvazivnykh ésherikhii ot ikh avirulentnykh variantov pri pomoshchi modifitsirovannogo nepriamogo immunofermentnogo analiza.

The data obtained in this investigation confirm that the modified indirect enzyme immunoassay (EIA) permits the differentiation of virulent bacteria of the genus Shigella and enteroinvasive Escherichia (group 1), regularly containing virulence plasmids with a molecular weight of 120-140 MD, from their avirulent variants which have lost these plasmids (group 2). The ratio of the optic density (OD) values of the positive control samples (the OD of group 1) to the OD values of the negative ones (the OD of group 2) is significantly higher than 2.1. As revealed by EIA, the differences between groups 1 and 3 (avirulent Shigella strains and E. coli smooth strain O124, retaining high-molecular plasmids with a molecular weight of 120-140 MD or their fragments) are statistically insignificant. The ratio of the OD of group 1 to the OD of group 3 is significantly less than 2.0. Analysis of outer membrane protein (OMP) preparations isolated from S. flexneri virulent strain 2a and its isogenic avirulent plasmid-containing variant has revealed significant differences in their EIA results. The ratio of the OP of OMP preparations isolated from the virulent strain to the OD of OMP preparations from the avirulent strain exceeds 2.1.

[Immunoenzyme analysis for determining class G and M antibodies to HBsAg]. / Immunofermentnyi analiz dlia opredeleniia antitel klassov G i M k HBsAg.

A scheme of the purification of hepatitis B virus surface antigen (HBsAg) as applied to the enzyme immunoassay (EIA) for the detection of antibodies to HBsAg is described. An indirect EIA technique for the detection of IgG and IgM antibodies to HBsAg has been developed and the diagnostic assay system based on the use of immunoreagents and solid-phase carriers produced in the USSR has been obtained. The sensitivity of the indirect EIA technique in the detection of IgG antibodies to HBsAg exceeds that of double immunodiffusion in gel used for this purpose 2,500- to 5,000-fold. The study has shown the possibility of using the indirect EIA technique for the detection of antibodies to HBsAg, both free and bound in immune complexes, of detecting antibodies to HBsAg in patients with acute and chronic viral hepatitis B, as well as of simultaneous detection of IgG and IgM antibodies to HBsAg without pseudonegative results.

[Choice of the suitability criteria of polystyrene-based solid-phase carriers for performing immunoenzyme analyses]. / Vybor kriteriev prigodnosti tverdofaznykh nositelei na osnove polistirola dlia provedeniia immunofermentnogo analiza.

The authors discuss a tentative approach to the choice of criteria indicating the optimal suitability of different solid-phase carriers made of polystyrene for use in the enzyme immunoassay (EIA), viz. the dependence of specificity, sensitivity, reproducibility and reliability of EIA results on the adsorption properties, transparency expressed in percent and transparency variations of the plates under test. The evaluation of the carriers by four parameters is proposed with the use of assay plates manufactured by Nunc A/S (Denmark) for control. To ensure the objective evaluation of the suitability of polystyrene plates for use in EIA, the choice of uniform criteria is necessary.

[Two-layer rocket-electrophoresis on cellulose-acetate membranes]. / Dvukh"iarusnyi roket-elektroforez na atsetiltselliuloznykh plenkakh.

Two-floor rocket-electrophoresis on gelatinated acetate cellulose membrane "Cellogel" has been developed. The method is based on electroimmunodiffusion detection of the antigen on the acetate-cellulose membranes, containing monospecific antiserum of the test-system. The procedure is followed by the detection of precipitation bands ("rockets") by staining, if the reaction is conducted in the visible zone or by the further treatment of the acetate-cellulose strips, containing invisible precipitates with antiglobulin antibodies, the complement or their combination. An increase in the method sensitivity up to 30-60 ng/ml in the visible zone of the reaction is achieved by simultaneous reduction in the antibody concentration and the growth of the absolute quantity of the antigen, subjected to electrophoresis, up to 50-100 microliters. The method has been applied to human alpha-fetoprotein.

[Highly sensitive electroimmunodiffusion method of detecting antigens on cellulose acetate film]. / Vysokuchuvstivitel'nyi élektroimmunodiffuzionnyi metod opredeleniia antigenov na atsetat-tselliuloznykh plenkakh.

A highly sensitive electro-immunodiffusion test suggested by the authors for antigen detection on cellulose-acetate films consists of three stages: antigen concentration in a discontinuous buffer system on cellulose-acetate films; antigen detection on the same films by immunodiffusion using standard test system; to detect the precipitation bands the washed films are stained with protein dyes in case the reaction takes place in the zone of vision, or subject to further treatment by means of "plating" the precipitates with antiglobulin antibody or by radioautography. The method permits one to reveal the nanogram levels of alfa-fetoprotein and it may be applied for detection of antigens with different molecular weights and electrophoretic mobility.

[Use of bifunctional reagents in the study of histones topography in chromatin]. / Ispol'zovanie bifunktsional'nykh reagentov dlia izucheniia vzaimnogo raspolozheniia gistonov v khromatine

The ability of bifunctional reagents--diimidoesters of sebacinic acid and tetranitromethane--to cross-link the histones in rat thymus chromatin was studied. The histone polymers formed were extracted with 0,8 N H2SO4 and analyzed by polyacrylamide gel electrophoresis. The results obtained suggest that in chromatin some histone fractions are localized in immediate proximity to each other. This phenomenon may probably account for the formation of chromatin structure and its functioning in the cell.

[Detection of the base level of alpha-fetoprotein in the sera of donors by the electrophoresis-precipitation of polyacrylamide gel method]. / Vyiavlenie fonovogo urovnia al'fa-fetoproteina v syvorotkakh donorov metodom élektroforez-pretsipitatsii v poliakrilamidnom gele

Alpha-fetoprotein (AFP) was detected in the sera of adult healthy persons by the method of electrophoresis-precipitation in polyacrylamide gel; it was distinctly determined in about 50% of cases, and its concentration was not over 3ng/ml. AFP was determined all the 20 cases suffering from cirrhosis of the liver examined by the mentioned method.