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A I Gurevich

Publications and source records attributed to A I Gurevich.

At least 19 recordsLinked to original sources
PubMed1999

[The dependence of the E.coli gene expression level on the structure of the translation initiation region (TIR). IV. Distal complementary TIR interactions with the mRNA coding region].

To evaluate the effect on translation of distal regions of the encoding mRNA part capable of the complementary binding to the ribosome binding site (RBS), a series of plasmids were constructed containing fragments inserted into the il3 gene and determining secondary interactions in mRNA. A comparison of the levels of the in vivo gene expression showed that the complementary interactions of the translation initiation region (TIR) with distal regions of the mRNA encoding part affect translation. The effectiveness of these interactions decreased with an increase in the distance between the RBS and the complementary mRNA region, whereas the secondary structure formed by the TIR and the adjacent mRNA region was more stable despite the presence of regions in mRNA capable of forming energetically more favorable structures involving these elements.

Codon, Initiator↗
PubMed1997

[Recombinant proteins containing amino acid sequences of two ectatomin chains].

Artificial genes for chains A and B of ectatomin, an Ectatomma tuberculatum ant toxin, were obtained by chemical and enzymic synthesis and cloned into new plasmid vectors. Expression plasmids with the genes of hybrid proteins were constructed containing human interleukin-3 or its terminal 63-mer fragment as well as chains A and B of ectatomin, which are linked via a region containing the cleavage site of specific protease, enterokinase (hybrid proteins IL3ETOXA, IL3ETOXB, ILETOXA, and ILETOXB). Escherichia coli producer strains providing a high yield of IL3ETOXA and IL3ETOXB proteins as inclusion bodies were obtained.

Amino Acid Sequence↗
PubMed1997

[Relation between the level of E. coli gene expression and the structure of translation initiation region (TIR). III. Sites of complementary interaction of TIR with 16S rRNA].

Potential sites of the complementary interaction of the translation initiation region (TIR) with 16S rRNA are revealed, and the role of these sites in the gene expression level is studied. The high expression level of a gene depends not only on the complementary interaction of TIR with 16S rRNA in sites proximal to the start codon [anti-Shine-Dalgarno (ASD) (delta G > -8 to -10 kcal/mol) and downstream box (DB)] and located at the -15 to +20 mRNA region but also on complementary interactions in distal sites of the untranslated branch of TIR (mTIR). Among them, the UB (upsteam box) 1 site, complementarily interacting with the exposed 452-490 segment of the 440-490 loop of 16S rRNA, may be located in the -15 to -50 mTIR segment. In the -50 to -70 mTIR segment may be located UB2 and UB3 sites, which interact with the exposed segment 478-488 of the 440-490 loop and segment 520-532 of the 520-540 loop of 16S rRNA, the UB3 site being much more efficacious. The high expression level requires that the total free energy of complementary interactions of UB1, UB2, and UB3 sites with 16S rRNA exceeds -20 kcal/mol.

Base Sequence↗
PubMed1996

[Recombinant proteins containing oxytocin oligomeric sequences].

Expression plasmids were constructed with genes encoding the ILOX3, ILOX6, and ILOX9 recombinant proteins, which contain the C-terminal fragments of trimer, hexamer, or nonamer of oxytocinoyl-Lys. Upon expression in E. coli, all three genes yielded inclusion bodies containing protein products of similar length and heterogeneous in the C-terminal region. It is likely that in the case of the ilox3 gene, the obtained protein mixture includes the full-length product of translation with the C-terminal lysine. In the case of the ilox6 and ilox9 genes, the protein products are formed as the result of a site-specific proteolysis in the regions between the second and the fourth oxytocin units.

Amino Acid Sequence↗
PubMed1996

[Mechanism of action of the plant hormone jasmonate. 1. Jasomonate-interacting proteins that regulate transcription of the p. pinII potato gene].

A fragment containing the regulatory region of the p. pinII gene was isolated from potato DNA by polymerase chain reaction. Interactions of this DNA region with jasmonate determines the transcriptional activation. The isolated DNA fragment was cloned into the pTE2pb plasmid, which was used for preparing an affinity sorbent. Using this sorbent, four proteins were isolated from the total protein capable of desorption at physiological concentration of jasmonate. These proteins are likely to be subunits of two transcription repressors, whereas jasmonate serves as an inducer. Three sequences of the regulatory regions (boxes G, I, and III) are binding sites for repressors; similar sequences were found in various plant genes activated by jasmonate.

Base Sequence↗
PubMed1995

[Dependence of the level of gene expression in E. coli on the structure of the translation initiation segment (TIR). I. Primary structure of TIR].

The comparison of expression levels of two genes-interleukin-3 (il3) and epidermal growth factor connected to leader peptide of OmpF (lompegf)-was carried out using specially constructed plasmids contained various structures of translational enhancers. It was shown that besides already known binding sites from mRNA translation initiation region (TIR) to 16S rRNA (SD, UB1 and DB), there is additional binding site of TIR disposed from -30 to -60 nt upstream of start codon AUG (UB2) having significant increasing effect on translation initiation and correspondingly on expression level.

Bacterial Outer Membrane Proteins↗
PubMed1995

[Dependence of the level of gene expression in E. coli on the structure of the translation initiation segment (TIR)].

The expression levels of genes that are transcribed to give mRNAs with identical leader sequences and even with identical extended coding regions may differ considerably. In order to determine the mechanism of this phenomenon, secondary structures of some mRNAs synthesized from a series of expression plasmids were studied. It was shown that the effect of the mRNA secondary structure in the translation initiation region on the initiation efficiency is due not only to the hairpin formation in this region but also to long-range interactions. When complementary structures tighter than those resulted from the interaction of regions SD, UB1, UB2, and DB with 16S rRNA are formed, the efficiency of the translation initiation and, consequently, the expression level decrease.

Base Sequence↗
PubMed1993

[Cloning the repeat sequence of the oxytocin gene].

Cloning of a synthetic gene of the oxytocin trimer is accompanied by deletions, caused by nicks in the plasmid DNA. Use of covalently closed circular double-stranded DNA greatly reduces the number of the deletions.

Amino Acid Sequence↗
PubMed1992

[Recombinant interleukin-3 expression system in E. coli].

Plasmid pTOTE2IL3 (III) has been constructed, expressing an artificial human interleukin-3 (hIL3) gene under conditions of the induced protein biosynthesis. Levels of the recombinant protein synthesis have been compared in several E. coli strains containing expression plasmids pTE2IL3 (I) (constitutive biosynthesis) and (III) (induced biosynthesis). Optimal combinations of the expression plasmids and the bacterial strains are of importance. A simple and effective method has been elaborated for isolation, purification and renaturation of the recombinant protein accumulated in inclusion bodies.

Base Sequence↗
PubMed1991

[Chemical-enzymatic synthesis of the amplifier of the T7 phage gene 10 and function of its structural elements].

The translational enhancer (TREN) sequence of the phage T7 gene 10 (in full and also its proximal or distal parts) have been obtained by chemical-enzymatic synthesis and cloned into the plasmids immediately before the human interleukin 3 (hIL3) artificial gene. Expression levels of the hIL3 gene in E. coli in these constructions show that the region controlling the specific activity is placed in distal part of TREN more than 40 nucleotides upstream from the initiation codon.

Amino Acid Sequence↗
PubMed1991

[Isolation of recombinant interleukin-3 produced by E. coli].

A synthetic gene coding for human interleukin-3 (hIL3) was cloned in the plasmid pTE2IL3, the gene expression being controlled by the phage fd PVIII promotor and the phage T7 gene 10 translational enhancer. Under constitutive biosynthesis conditions in E. coli, the accumulation of recombinant hIL3 (in the inclusion bodies) was up to 30-40% of the total cell protein. An effective procedure of the hIL3 isolation is suggested. The hIL3 was solubilized in 5 M guanidinium chloride, renaturated and purified to homogeneity by a single chromatographic step. The protein's yield was 34 mg/g wet cells. The isolated hIL3 showed a specific biological activity.

Chromatography, Gel↗
PubMed1989

[Recombinant plasmids containing hybrid protein genes with antigenic determinants of the foot and mouth virus].

Plasmids have been constructed which contain genes coding for fused proteins including beta-galactosidase or human leukocyte interferon alpha 2 and monomeric or pentameric form of the main antigenic determinant of the foot-and-mouth disease virus (FMDV) serotype 01K. Expression of the hybrid genes has been studied. It is shown that fused proteins, containing beta-galactosidase and the antigenic determinant (monomer or pentamer), interact specifically with anti-FMDV anti-sera and with antibodies against peptide 141-160 of FMDV VP1 coat protein.

Amino Acid Sequence↗
PubMed1988

[Recombinations during DNA cloning].

RecA-independent recombinations accompanying the processes of plasmids preparation and cloning into Escherichia coli cells are induced within the short direct and inverted repeats of several types.

Base Sequence↗
PubMed1987

[The role of structural elements of ColE1-related plasmids in replication control].

Effect of deletions downstream from the replication origin on copy number of ColE1 related (pBR322 derived) plasmids has been studied. Along with main control elements (RNAI of defined secondary structure, the repressor protein, effectiveness of promoter upstream of gene of RNA primer) replication is influenced by transcription level propagated into the replicon region from promoters even in distal parts of plasmid. Structure of the region adjacent to the ori is important for startpoint recognition specificity, so that deletion of this region reduces the effectiveness of replication.

DNA Replication↗