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Biomedical subjects

A V Itkes

Publications and source records attributed to A V Itkes.

At least 19 recordsLinked to original sources
PubMed2004

Selection of E. coli strains for stable transformation with recombinant plasmids containing full-length genome of clinical HIV-1 isolates.

Strain chi6007 obtained from the parent E. coli strain chi5097 is a result of ptsH5 mutation, which allowed cells to grow without common components of the phosphoenolpyruvate-dependent phosphotransferase system. Segregants of strain chi6007 retaining the Pol+ gene responsible for inability to grow at 37 degrees C, but gaining rifampicin resistance (RifR) were used for cloning of cointegrate plasmids. Pre-integration complexes of HIV-1 were co-integrated with the pBR-322 plasmid and transformed strain chi6018. Sequencing showed that the pPIC91 hybrid plasmid contains full-length genome of HIV-1 with shortened 5-terminal LTR and full-length copy of pBR322. Elimination of the pPIC91 plasmid from chi6018 cells was followed by the appearance of auxotrophic insertion mutants. Sequencing of the insert region showed that chromosome DNA of the host cell includes integrated genomes of pBR-322 and HIV-1.

Base Sequence↗
PubMed2001

Type D retrovirus specific sequences in lymphocytes of the children with Burkitt-type lymphoma and their parents.

Type D retroviruses cause immunodeficiency in monkey. Earlier we have revealed genetical and serological markers of type D retroviruses in children with Burkitt-type lymphoma. Using PCR/Southern blotting assay we have found sequences related to MPMV in PBMC's DNA from children with Burkitt-type lymphoma and from their parents. Moreover, the data on sequencing of virus specific sequences from one ill child and from his mother have been presented.

Blotting, Southern↗
PubMed1997

The role of phosphorylation of DNA-binding proteins in regulation of transcription of the human c-myc gene.

The goal of the present study was to identify transcriptional factors, to determine their specificity toward nucleotide sequences of binding sites, and to elucidate their regulatory effects on transcription of the human c-myc oncogene. Three novel phosphorylated proteins (transcriptional factors) participating in the regulation of transcription of the c-myc gene have been identified, and the following data have been obtained. A) The binding of the regulatory phosphoproteins activates in vitro transcription of the human c-myc gene. B) The recognition sites for the three discovered DNA-binding proteins, i.e., p70, p35, and p23, have been mapped in the c-myc promoter. C) The site-specific mutagenesis of p70 binding sites of the c-myc gene has been performed and the recognition sequence has been identified. The mutagenesis was carried out using the PCR technique using oligonucleotide primers with mismatches in the p70 binding site. The wild-type sequence GGGAAAAGAAAAAA showed the highest affinity toward p70, while the mutant sequence GAAAAtGAcAct exhibited a lack of affinity for the protein. The participation of p70 and other phosphorylated transcriptional factors in regulation of the human c-myc gene expression is discussed.

Binding Sites↗
PubMed1995

Enhancement of in vitro transcription of human c-myc correlates with the binding of phosphorylated protein factors from HeLa cell extract.

Hela cell extract overphosphorylated with endogenous protein kinases was used for in vitro transcription of human c-myc DNA. The activation of transcription initiated from P1, P1a and P2 cap-sites was observed. Three phosphoproteins of 70, 35 and 23 kDa are found, whose binding to DNA correlates with activation of c-myc transcription. Sites of binding of these proteins to DNA are located.

Base Sequence↗
PubMed1994

Cloning of a cDNA encoding a human protein which binds a sequence in the c-myc gene similar to the interferon-stimulated response element.

A human cDNA clone encoding a c-myc promoter-binding protein (IRLB) was selected by screening a human fibroblast lambda gt11 phage library with the hexamer oligodeoxyribonucleotide (oligo) 5'-GGCGGGAAAAAGAACGGA, corresponding to the protein-binding element of human c-myc similar to the interferon-stimulated response element (ISRE). The lambda gt11 phage clone, encoding a fusion protein which bound the probe oligo, was used to create an strain of Escherichia coli. The deduced amino-acid sequence of the cloned protein contains a putative alpha-helix which is expected to act as the DNA-binding domain. DNase footprinting analysis and oligo-binding specificity assays showed that the cloned factor recognizes the ISRE-like element of the P2 promoter region of human c-myc.

Amino Acid Sequence↗
PubMed1994

Oligoadenylate and cyclic AMP: interrelation and mutual regulation.

The data obtained are in good agreement with the hypothesis that cAMP is involved in the control of 2-5A metabolism, including the mediation of the regulation of 2-5A by IFNs; 2-5A, in turn, affects the intracellular cAMP level. The general question originating from the data is that of a biochemical mechanism connecting the activation of the cAMP/2-5A system and the effect of depression of cell division. In my opinion, this universal effect is the result of the action of the known 2-5A-dependent mechanism, namely, RNase L (see review by Pestka et al. 1987), rather than by any new 2-5A-stimulating enzyme. The RNase L activated by 2-5A decreases the total level of protein synthesis and accelerates the degradation of cellular RNA, resulting in the inhibition of cell growth. It should be mentioned that such activation of RNA turnover is generally characteristic for nondividing cells, especially for cells in the resting state (Epifanova et al. 1983). Thus, the regulatory system of cAMP/2-5A is involved evidently in the antiproliferative mechanism characteristic for the resting cells, controlling the variations in the levels of RNA turnover and protein synthesis.

2',5'-Oligoadenylate Synthetase↗
PubMed1993

Unexpected effect of an anti-human immunodeficiency virus intermolecular triplex-forming oligonucleotide in an in vitro transcription system due to RNase H-induced cleavage of the RNA transcript.

A 16-mer oligodeoxynucleotide (ODN) which specifically recognizes the polypurine tract (PPT) located upstream of the 3' long terminal repeat (LTR) of human immunodeficiency virus (HIV) proviral DNA via triplex formation is shown to have a dramatic effect on in vitro transcription from the HIV-LTR promoter. In the presence of HeLa cell extracts, a shorter RNA transcript is obtained in the presence of the 16-mer ODN. This truncated RNA lacks about 200 nucleotides from its 3' region. The PPT sequence is not responsible for this effect. Instead, this process involves a purine-rich sequence in the gag mRNA located around position +400. The imperfect hybrid formed between the 16-mer ODN and mRNA is precisely cleaved by RNase H contained in HeLa cell extracts. These data show that sophisticated control experiments must be designed before any conclusion can be drawn on the effect of oligonucleotides used in vitro and in cell cultures.

Base Sequence↗
PubMed1991

Binding of proteins of HeLa S3 cell extract to oligonucleotides containing the consensus interferon-response sequence (IRS) and to the IRS-containing fragment of the human c-myc gene.

The human c-myc proto-oncogene was recently found to contain a regulatory sequence similar to the consensus interferon-response sequence (IRS) of interferon-activating genes. Binding of regulatory protein(s) to this sequence of cloned fragment of c-myc, lacking the main part of 5'-nontranscribing region, regulates in vitro transcription from I1/I2 initiation sites located in the first intron of the gene. Here, we have shown that HeLa S3 nuclear extract contains different protein factors, at least two, that bind preferentially to the IRS sequence of either the c-myc gene or the interferon-dependent 6-16 gene. Moreover, each of these factors 'cross-binds' to the region of the other gene, although affinity of this interaction is lower. Binding constants of these proteins to oligonucleotide fragments of c-myc and 6-16 genes were determined. In vitro transcription of the human full-length c-myc gene (i.e. the gene containing the complete 5'-noncoding region) initiated from I1/I2 sites, that is controlled by the IRS region, was demonstrated to be blocked. A possible physiological role for the mechanisms described is discussed.

Base Sequence↗
PubMed1990

Human c-myc gene contains a regulatory site similar to consensus of interferon response sequence (IRS).

Expression of c-myc proto oncogene is regulated by multiple mechanisms. Here, we report that the consensus of the regulatory region of interferon-dependent genes, GGAAAN1-3 GAAA, was found after computer search in the 5'-terminal flank of human c-myc gene in position (-76:-67). In vitro transcription of c-myc gene fragments showed that the consensus region competes with oligonucleotide GGGAAAATGAAACT for binding to specific protein(s). This oligonucleotide was shown to bind selectively the interferon-dependent positive transcription factor. Transcription of c-myc fragments lacking 5'-terminal region up to positions -101 or +71 was initiated at two sites located in the first intron. These sites did not coincide with P1 in vivo RNA cap-site. Binding of the protein factor(s) to the regulatory region of c-myc gene -76:-67 blocked the in vitro transcription initiated in the first intron.

Base Sequence↗
PubMed1990

Expression of c-myc gene in human ovary carcinoma cells treated with vanadate.

The widely accepted hypothesis of vanadate action on cells postulates that this ion inhibits protein phosphatase(s) that dephosphorylates protein phosphotyrosine residues. This inhibition causes tyrosine hyperphosphorylation of cell proteins followed by changes in physiological action of phosphoproteins resulting in stimulation of cell proliferation, expression of protooncogenes, and transient cell transformation. We have found that treatment of human ovary carcinoma (CaOv) cells with vanadate causes the increase in total protein phosphorylation from 1.5- to 2.0-fold whereas the ratio between phosphoserine, phosphothreonine, and phosphotyrosine content remains unchanged. At the same time, enhancement of c-myc gene expression (not c-fos) was observed. Hence, the increase in the ratio of phosphotyrosine to phosphoserine and phosphothreonine is not an obligatory intermediate stage before vanadate-dependent activation of c-myc expression.

Cell Transformation, Neoplastic↗
PubMed1988

A route to 2',5'-oligoadenylates with increased stability towards phosphodiesterases.

The rates of enzymatic hydrolysis of 2',5'-oligoadenylates and their synthetic analogs have been measured. These compounds were treated with either NIH 3T3 cell lysates, mouse liver homogenates or snake venom phosphodiesterase. All analogs with 3'-terminal acyclic nucleoside residues demonstrated greater stability compared with the natural compound adenylyl(2'-5')adenylyl(2'-5')adenosine.

Adenine Nucleotides↗
PubMed1988

[Effect of modulation of the production of interferon by L-929 cells treated with theophylline].

The specific inhibitor of cAMP phosphodiesterase theophylline has been shown to evoke in L929 cells 2.3-fold induction of 2-5A-synthetase activity and 3.5-fold superinduction of the same enzyme activity while acting in combination with actinomycin D. It has been shown also that temporal coincidence of 2-5A-synthetase induction with the active period of interferon production resulted in 8-16 times decrease in the level of interferon production. The result was supported by the experiments of superinduced cells (containing the high stable level of 2-5A-synthetase) fusion with monolayer of poly(I).poly(C)-induced L929 cells (taken at the start of interferon production). In this case the production of interferon was dramatically decreased in comparison with the control. Possible role of 2-5A-synthetase in regulation of interferon production is discussed.

2',5'-Oligoadenylate Synthetase↗
PubMed1988

[Induction of interferon-specific enzymes in cultures of cells sensitive and resistant to interferon].

Induction of 2'-5'-oligoadenylatesynthetase (2-5A synthetase) by interferons and theophylline by means of activation of cAMP-system in interferon susceptible and resistant cell lines were studied. In interferon resistant cell lines the basal activity of 2-5A synthetase exceeded the level of the same enzyme in interferon susceptible cell lines. Activity of 2-5A synthetase is increased in interferon susceptible cell lines by interferon treatment, but the activity of the enzyme is not altered in interferon resistant cell lines. Among the studied cell lines the induction of 2-5A synthetase by theophylline was possible only in L929 cell line. The common mechanism for the absence of 2-5A synthetase induction by interferon and theophylline in interferon resistant cells is discussed.

2',5'-Oligoadenylate Synthetase↗
PubMed1988

[Effect of interferon on the cAMP system in cells].

The increase in cAMP concentration in CaOv cells affected by alpha-interferon has been found to have a two wave character with the maximums at 4 and 24 h after the effect. The waves are due to the increase in adenylate cyclase activity and to the decrease in the activity of cAMP phosphodiesterase. The described changes were characteristic of the native and partially purified interferon and depended on the concentration of interferon used (optimal effect at 1200 IU/ml-1). It suffices to notice that the described effects were more largely expressed when the preparations of the native alpha-interferon were used. The correlation was noticed between the increase in adenylate cyclase activity, the decrease in cAMP phosphodiesterase and the concentration of the cyclic nucleotide as well as the expression of antiproliferative effect. The correlation was less significant for antiviral effect.

3',5'-Cyclic-AMP Phosphodiesterases↗