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T I Tikhonenko

Publications and source records attributed to T I Tikhonenko.

At least 19 recordsLinked to original sources
PubMed1994

[Genetic engineering in the bacterial synthesis of somatostatin].

Series of recombinant plasmids for expression of the synthetic gene somatostatin-14 (SST) as a fusion protein were obtained. The somatostatin gene was fused to chloramphenicol acetyltransferase (cat) or its deleted variant genes. Both parts of the resultant fusion protein were joined through a Met residue. The hybrid gene was expressed under the control of the cat gene promoter (Pcat), the tryptophan operon promoter (Ptrp) or the promoter of bacteriophage T5 (PT5). These fusions gave insoluble polypeptide products amounting from 5-10% of the total cellular protein under constitutive biosynthetic conditions (Pcat) to 5-30% upon induction (Ptrp, PT5). A correlation between the efficiency of expression and the length of cat, the power of the promoter used and the absence or presence of transcription terminators, was studied. The scheme for SST isolation from bacterial cells was developed. SST was liberated from the fused polypeptide by treatment with cyanogen bromide and purified to homogenity by a combination of chromatographic steps: gel filtration, ion-exchange and rpHPLC. The renaturated recombinant SST showed specific biological and immunological activities and had 98% purity. The yield was 1 mg of the purified cyclic SST/1 culture of E.coli.

Chloramphenicol O-Acetyltransferase↗
PubMed1993

[Synthesis of somatostatin in Escherichia coli cells: isolation and characteristics].

A synthetic gene coding for somatostatin-14 (SST) was cloned in plasmid expression vectors in frame with the chloramphenicol acetyl transferase (CAT) gene, both genes being divided by a Met residue. The hybrid gene was expressed under the control of the CAT gene promoter (Pcat) or the tryptophan operon promoter (Ptrp). Them fused genes gave insoluble polypeptide products amounting from 5% of the total cellular protein under constitutive biosynthesis conditions (Pcat) to 30% upon induction (Ptrp). SST was liberated from the fused polypeptide by treatment with cyanogen bromide, purified to homogeneity by gel-filtration and reverse phase HPLC, and finally refolded by dilution and air oxidation. The renaturated recombinant SST showed the specific biological and immunological activities of the native peptide.

Amino Acid Sequence↗
PubMed1992

An investigation of the effect of antisense RNA gene on bovine leukaemia virus reproduction in cell culture.

A possible approach to control of bovine lymphoproliferative disease caused by bovine leukaemia virus (BLV) may be the development of an "antiviral information immunity" based on the effect of anti-sense RNA (asRNA). A numbers of constructs were obtained, under control of various promotors (herpesvirus thymidine kinase, T-antigen SV40 promoter), carrying as DNA against gene X, the expression product of which is a transactivator of viral transcription from the BLV LTR promotor. As a model system for the analysis of antiviral activity of constructs developed, cloned continuous cell lines of BLV-producing FLK cells were used. The level of BLV expression in cells transfected with the constructs was determined by various parameters. Differences were detected in different clones obtained from non-transfected cells, as well as variation between transfected clones, as measured by reverse transcriptase, competitive radio-immunoassay for BLV p24, the viral particle count on agar membrane, and the tumorigenicity for nude mice. The differences in inhibition of expression of BLV genes and their products may be explained in terms of the site of integration of asDNA and the number of integrated copies.

Animals↗
PubMed1990

[Purification and properties of the hexon antigen of canine adenovirus serotype CAV-1].

The main antigen and immunogen of canine adenovirus type 1 (CAV-1) has been purified to near homogeneity from cultural fluid of a CAV-1-infected primary cell culture by hydrophobic and anion-exchange chromatography. The hexon native form (trimer) was shown to be resistant against denaturation by SDS under conditions of SDS-PAGE performed without heating the samples. The monomer chain of the CAV-1 hexon was apparently identical in terms of electrophoretic mobility with that of the previously sequenced BAV-3 hexon polypeptide (103 kDA). In blot enzyme immunoassay only native trimers of CAV-1 hexon were detected by cross-specific polyclonal and monoclonal anti-hexon antibodies.

Adenoviridae↗
PubMed1990

Inhibition of adenovirus replication by the E1A antisense transcript initiated from hsp70 and VA-1 promoters.

The E1A region of the adenoviral genome, important for initiation of virus infection and activation of other viral genes, was chosen as a target for engineering antisense RNA (asRNA) to inhibit adenovirus 5 (Ad5) replication in COS-1 cell culture in vitro. The hsp70 promoter, taken from the appropriate heat-shock-protein gene of Drosophila melanogaster, and the VA-1 RNA promoter, derived from the Ad5 gene coding for low-molecular-mass VA-1 RNA and recognized by RNA polymerase III were used as regulatory elements of transcription. The two types of recombinant constructs contained E1A fragments of 710 bp (hsp70 constructs) or 380 or 740 bp (VA-1 RNA constructs) in reverse orientation relative to the promoter position, as well as a transcription termination signal, the SV40 ori, and the gene controlling Geneticin (antibiotic G418) resistance (G418R). After selection of transfected COS-1 cells in the presence of G418, a number of stable G418R cell lines were raised which expressed engineered asRNAs. Plating of Ad5 suspensions of known titre on monolayers of transfected COS-1 cells clearly showed strong inhibition of adenovirus replication by asRNAs: 75% with the hsp70 promoter and 90% with the VA-1 RNA promoter.

Adenoviridae↗
PubMed1990

[Human hepatitis B virus and hepatocellular carcinoma].

Hepatitis B virus (HBV) is the causative agent of hepatocellular carcinoma (HCC) in man. The HBV genome is a circular partially double-stranded DNA molecule of about 3.2 kb. The HBV genome contains four structural genes coding for the HBV envelope (HBsAg) and core (HBcAg/HBeAg) proteins, endogenous DNA-polymerase with the additional enzymatic activity of a reverse transcriptase and polypeptide X functioning as a trans-activator of cellular and viral genes. HBV DNA integration in the genomes of HCCs and hepatocytes of HBV carriers is an important evidence establishing a relationship between the HBV infection and the development of HCC. The mechanism of HBV DNA integration into the cellular genome and the possible role of integrated HBV DNA sequences in the malignant transformation of hepatocytes are discussed.

Carcinoma, Hepatocellular↗
PubMed1990

[New shuttle-type vectors for cloning in Escherichia coli and Agrobacterium tumefaciens].

The vectors capable of replication in Escherichia coli and Agrobacterium tumefaciens have been constructed on the basis of the plasmid pUB5502. The constructed vectors pVA12, pVA12-2, pVA12-4 contain the mini-replicon and trimethoprim resistance gene (Tp) of a broad host-range plasmid R388 (IncW). The pVA12 vector (8.8 kb) has been constructed by insertion of a kanamycin resistance gene (Km) from the plasmid pUC-4K into a Psti site. It possesses 7 unique restriction sites for XhoI, SmaI, PvuI, PvuII, HindIII, EcoRI, BamHI and the markers for kanamycin and trimethoprim resistance (Km and Tp). The pVA12-2 and pVA12-4 vectors were obtained as a result of changing of the PvuII-EcoI fragment of pVA12 carrying the Tp gene for the PvuII-EcoRI fragment of pBR322 carrying the Tc gene. These plasmids have the same size of 9.7 kb and 8 unique sites for restriction endonucleases XhoI, SmaI, PvuI, PvuII, EcoRI, EcoRV, SalI, BalI and Km and Tc genes. No difference has been registered between the two plasmids by restriction analysis, but pVA12-4 has the dramatically increased copy number in Escherichia coli cells. All three vectors are transferable to Agrobacterium tumefaciens with the same frequencies by transformation or conjugation and do not affect the oncogenicity of pTi.

Cloning, Molecular↗
PubMed1990

[Biotinylated pH-sensitive liposomes--containers for guided administration of biologically active substances to the cells].

The technique is described for obtaining pH-sensitive biotinylated liposomes. The liposomes included phosphatidyl ethanolamine, oleic acid, cholesterol, biotin amidocaproyl phosphatidyl ethanolamine, with a molar ratio of 7:3:3:0.1 or 4:2:4:0.1. The fluorescein stain calcein at ++self-limited concentration, 32-P-dTTP, or DNA was incorporated into the internal liposome medium. The liposomes were stable in neutral or mildly alkaline medium but became leaky and showed agglutination at pH 5.7. Liposomes were agglutinated in the presence of avidin and bound specifically to the layer of avidin on the surface of immunological plates. The apparent liposome affinity to the avidin layer was up to 10(-11) M. The avidin-biotin system allows for specific binding of liposomes to human lymphocytes via biotinylated anti-CD5-antibodies. A simple, convenient, and rapid procedure has been elaborated for the binding of water-soluble substances (plasmid DNA included) to liposomes without DNA destruction.

Avidin↗
PubMed1989

[Polymerization of DNA fragments coding epitopes of the surface antigen of hepatitis B in Escherichia coli cells].

Synthetic oligonucleotides coding for the amino acid residues 135-151 (A, B) and 93-109 (C) of the hepatitis B surface antigen (HBsAg) have been polymerized. The obtained polymers (AC)n and (BCAC)n were inserted into the beginning of the lacZ gene under the control of the chloramphenicol promoter or the right promoter PR of bacteriophage lambda. Stability of the obtained plasmids was investigated during transformation, cell growth and gene expression.

Amino Acid Sequence↗
PubMed1989

[A phased poly-linker for cloning and expression of genes].

15 oligodeoxynucleotides were synthesized using the phosphotriether technique which were subsequently enzymatically ligated in polylinker with subsequent phasing of left and right sides. Based on the phased polylinker a series of vehicles for the gene cloning and expression was constructed. The vectors of pRK series contain all three variants of polylinker with the frame shift of the reading frame for 1, 2, or 3 nucleotides in both chains. The obtained polylinkers do not effect the enzymatic activity of the beta-galactosidase alpha-peptide. Structure of the phased polylinker was confirmed by Maxam and Gilbert's sequencing method.

Amino Acid Sequence↗
PubMed1989

[Immunoenzyme analysis of adenovirus hexon. Determination of antibodies from hyperimmunized chickens].

We have elaborated three systems of enzyme-linked immunosorbent assay (ELISA) for detection of chicken IgG antibodies specific for hexon antigens of three immunologically distinct adenovirus groups: those of mammalian adenoviruses (Mastadenovira), typical avian adenoviruses (Aviadenovira) and of egg-drop syndrome-76 (EDS-76) virus. In each system the antibodies against respective hexons were specifically detected. In mammalian adenovirus hexons the ELISA detects primarily the type-specific (epsilon) and genus-specific (alpha) antigenic determinants. The time course of anti-hexon antibodies content was followed during immunization. The level of anti-hexon antibodies in egg yolk reflects adequately their content in blood serum. The technique is suitable for serological diagnosis of chicken adenoviral infections as well as for characterization of egg-yolk antibodies obtained by preparative hyperimmunization of hens.

Adenoviridae↗
PubMed1987

Unstable visible mutations induced in Drosophila melanogaster by injections of oncogenic virus DNA into the polar plasm of early embryos.

When RSV DNA cloned in pBR 322 or DNA of simian adenovirus Sa7 (C8) is injected into the pole plasm of embryos of various Drosophila stocks, the progeny of 1-70% of the surviving flies display visible mutations. The mutagenesis is partially directed: the loci mutating due to retrovirus and adenovirus DNA do not overlap. The majority of resulting mutants are characterised by high instability: reversions and new mutations occur in them, which sometimes spread over the whole population ("explosive" instability). The injected sequences are revealed by dot-hybridization in the DNA of many mutant strains, but only rarely by Southern blotting procedures. The results show that the microinjection of oncovirus DNA into embryos is an approach for obtaining highly unstable strains even from wild-type stable Drosophila stocks without crosses with MR lines or the introduction of P elements. The sets of unstable mutations induced by oncovirus DNA is different from those in hybrid dysgenesis.

Adenoviridae↗