PubMed Health⌕ Search

Biomedical subjects

M A Grachev

Publications and source records attributed to M A Grachev.

At least 37 records · Page 2Linked to original sources
PubMed1986

Highly selective affinity labelling of RNA polymerase B (II) from wheat germ.

DNA-dependent RNA polymerase B (II) from wheat germ was modified by incubation with 4-[N-(beta-hydroxyethyl)-N-methyl]benzaldehyde esters of AMP, ADP or ATP, followed by reduction with NaBH4. Reaction of the modified enzyme with [alpha-32P]UTP in the presence of various DNA templates led to a highly selective affinity labelling of the subunit with Mr 140 000 by covalently linked ApU. Labelling was inhibited by 1 microgram/ml alpha-amanitin.

Affinity Labels↗
PubMed1986

[A simple method of determination of antigenic determinants in proteins with known primary structure].

A simple and rapid method is proposed for the localization of antigenic determinants in proteins of known primary structure exemplified by human myoglobin. The polypeptide chain of myoglobin was cleaved with BrCN (at Met residues) or with bromosuccinimide (at Trp and Tyr residues) under conditions which on average gave less than one scission per myoglobin molecule. The "single-hit" cleavage products were separated by gel electrophoresis and transferred to nitrocellulose by electroblotting. The peptides containing intact antigenic determinants were vizualized by immuno-peroxidase staining with four monoclonal anti-myoglobin antibodies. Comparison of the lengths of the immuno-reactive peptides with the known positions of methionine, tryptophan and tyrosine residues suggested that the four monoclonal antibodies were bound by myoglobin over the region Trp-14 to Met-55. As compared with other methods of localization, the method proposed is much faster and takes much lesser amount of protein.

Amino Acid Sequence↗
PubMed1986

[Affinity modification of E. coli RNA-polymerase in a complex with the promoter by phosphorylating derivatives of primer oligonucleotides].

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.

Binding Sites↗
PubMed1985

[Amino acid sequence of various tryptic peptides of the envelope protein of the tick-borne encephalitis virus].

Protein E of the tick-borne encephalitis virus (TBEV) (strain Sofin) was treated with purified trypsin in 1% Triton X-100, and the peptides thus obtained were separated by micro-column reversed-phase chromatography. Four of the purified peptides were sequenced, their structures being in accordance with the nucleotide sequence of the viral protein E gene. Amino acid sequences of peptides deduced from the cDNA primary structure are: Ser-Val-Leu-Ile-Pro-Ser-His-Ala-Gln-Gly-Asp-Leu-Thr-Gly-Arg (N-terminal peptide of protein E); Thr-Glu-Gly-Ala-Gln-Asn-Trp-Asn-Ala-Glu-Arg: Trp-Leu-Glu-Gly-Asp-Ser-Leu-Arg; Leu-Val-Glu-Phe-Gly-Ala-Pro-His-Ala-Val-Lys.

Amino Acid Sequence↗
PubMed1984

Oligonucleotides complementary to a promoter over the region -8...+2 as transcription primers for E. coli RNA polymerase.

Primer-dependent transcription by E. coli RNA polymerase on T7 promoter A2 has been studied. Synthetic deoxyribonucleotides complementary to the promoter over the region -8...+2 were taken as primers. A ribonucleoside residue was present at the 3'-end of some of these oligonucleotides. The octanucleotide complementary to the region -8...-1 appeared to be an active primer. Oligonucleotides having lengths from 3 to 6 nucleotide residues complementary to the promoter over the region -4...+2 also exhibited primer activity. The latter was some 5-10 times greater in the case of oligonucleotides having a ribonucleoside residue at the 3'-end. Oligonucleotides which on complementary binding do not reach the center of phosphodiester bond synthesis, as well as the decanucleotides (-8...+2) and octanucleotides (-6...+2) of both the ribo- and deoxyribo-series were inactive as primers.

Base Sequence↗
PubMed1984

[Phage T7 RNA-polymerase: gene cloning and its structure].

Gene 1.0 to T7 phage coding for a phage-specific RNA polymerase has been cloned in a plasmid vector. One of the clones obtained, viz. E. coli K12 HB101 (pSK-T7-1.0a), produced an active T7 RNA polymerase as revealed by the fact that its extract stimulated RNA synthesis on the T7 DNA template in the presence of rifampicin. Analysis of gene 1.0 fragments isolated from pSK-T7-1.0a plasmid made it possible to refine the sequence of this gene which has been recently published by the authors of the present paper and, independently, by other workers. Another plasmid, pSK-T7-1.0b, did not differ from pSK-T7-1.0a in the restriction map. However, it failed to induce production of a rifampicin-insensitive RNA polymerase. Sequencing revealed a deletion of one T residue in gene 1.0 present in this plasmid.

Amino Acid Sequence↗
PubMed1983

A new system for ATP-metric immunoanalysis.

Treatment of amino-group-containing antigens with adenosine-5'-trimetaphosphate results in their chemical modification by -pppA residues. An immunoanalytical system is proposed based upon competition of these ATP-labelled antigens with those of the sample for immobilized antibodies. Mild acidic treatment of complexes of ATP-labelled antigens with immobilized antibodies results in quantitative liberation of intact ATP. The latter may be determined by the ultrosenstive bioluminescent techniques based upon emission of light with firefly luciferase. The validity of the system has been studied with two clinically important antigens, thyroxine and myoglobin.

Adenosine Triphosphate↗
PubMed1981

A chemical method to enrich RNA by molecules having 5'-terminal triphosphate groups.

A method is proposed to enrich RNA molecules having a 5'-terminal triphosphate group. The method is based upon selective chemical modification of 5'-triphosphate groups by an antigen-containing amine followed by affinity chromatography on an absorbent loaded with antibodies specific to this antigen. A purification factor up to 37 may be achieved.

Adenosine Triphosphate↗
PubMed1980

A route to RNA with an alkylating group at the 5'-triphosphate residue.

Reaction of ATP with N,N,N'-tris(2-chloroethyl), N' (p-formyl-phenyl)propylenediamine-1,3 (abbreviation C13R) afforded a gamma-ester of ATP (abbreviation C1RpppA) - the product of alkylation by an aliphatic nitrogen mustard residue of C13R. The alkylating activity of the aromatic nitrogen mustard residue of C1RpppA is suppressed by the electron-acceptor effect of the p-formyl group. C1RpppA is a substrate of RNA-polymerase, and affords RNA with C1RpppA-residues at the 5'-termini.

Adenosine Triphosphate↗
PubMed1979

A rightward promoter to the left of the att site of lambda phage DNA: possible participant in site-specific recombination.

The binding has been studied of Escherichia coli RNA-polymerase to the fragments of lambda DNA obtained by digestion with restriction endonucleases BsuI, HindIII, BamHI, EcoRI and HindII + III. There are at least six sites of RNA-polymerase binding in the b2-region. In vitro transcription of those BsuI-fragments of the b2-region which contain six binding sites is rightward. Therefore, the fragments contain promoters rather than mere RNA-polymerase binding sites. One of the promoters of the b2 region named patt was calculated to be about 50 bp to the left of the att site. We postulate that this promoter might correspond to the hef-target which was described as important for the site-specific recombination.

Bacteriophage lambda↗
PubMed1978

Measurement of the differential melting profile of a promoter-containing fragment of T7 DNA by means of a microspectrophotometer.

A double-beam microspectrophotometer with a 5 microliter cell has been used to study denaturation of DNA in an aqueous solution. This instrument enables measurement of high-resolution differential melting profiles simultaneously at several wavelengths with 0.5 to 1 microgram of DNA. Therefore it becomes possible to study nucleic acids which are difficult to obtain in large amounts. The techniques have been employed to measure the differential melting profiles of T7 DNA and of a fragment of this DNA 1000 base pairs long which contains the four early promoters.

Base Sequence↗