Biomedical subjects
Publications and source records attributed to N P Mertvetsov.
Introns of human tyrosine aminotransferase (TAT) gene were sequenced. Combined with the literature data about the exon-intron structure of the gene and the sequence of the TAT mRNA, the obtained nucleotide sequences yielded on uninterrupted segment 10989 b. p. long of the human TAT gene.
The polymerase chain reaction technique was employed to isolate cDNA encoding S26 human ribosomal protein. Based on the known sequence of rat S26 ribosomal protein we have designed primers and amplified the corresponding sequence of human cDNA from total placenta cDNA. The fragment of S26 cDNA was cloned in a plasmid vector and sequenced by the Sanger method. Extremely high homology (87.7%) between coding regions of S26 mRNAs in rat liver and human placenta was revealed. The only amino acid substitution (Ser-->Val) at position 38 was observed. Results of blot hybridization with partially digested human genomic DNA suggest the presence of more than 6 copies of the S26 gene.
The treatment of Krebs-2 ascitic tumor cells (TC) with total RNA from the liver of Wistar rats (2 mg/ml) altered their antigenicity. As a result, the growth of treated TC in contrast with untreated TC was inhibited when transplanted i. m. to mice preimmunized with rat liver homogenate. Investigations of poly(A+)mRNA, rRNA and tRNA isolated from the same tissue established that the alteration of antigenicity is due to mRNA (8-24 micrograms/ml). In the cytotoxicity assay with antisera against rat Wistar antigens, the expression of rat antigens in TC treated mRNA was observed in the next cell generations.
EcoRI-fragment No. 5 of the rat tyrosine aminotransferase gene containing exons K, L, intron 11, and a part of the 3'-nontranslatable region was digested with several restriction endonucleases (BspRI, Sau3A, BamHI, Ecl136II, AluI), the subfragments obtained were cloned into M13mp19 and sequenced using the Sanger technique with dye-labelled primers on the automated sequencer "Applied Biosystems", model 370A. The sequences were combined by means of a PC-GENE package and original programs to yield the primary structure (1064 b. p.) of the above fragment No. 5, adjacent to the previously sequenced EcoRI-fragment No. 4 (3677 b.p.).
Restriction fragments' length polymorphism in the region of apolipoprotein A-I (apo A-I) gene was investigated in Novosibirsk (Siberia, USSR) population. Correlation between PstI apo A-I alleles (2,2 kb-P1; 3,3 kb-P2) and total cholesterol, triglycerides, high density polyproteins cholesterol, and apolipoprotein A-I level was analysed. A tendency to increase in cholesterol index of atherogenicity and to decrease in high density lipoproteins cholesterol as well as apolipoprotein A-I level was shown to occur for P1P2 genotype patients.
Expression of pro-opiomelanocortin (POMC) gene in pituitary of rats from newly developed hypertensive strain (ISIAH strain) was studied by dot hybridization. The pUC8 plasmid containing 900 base pair (bp) segment or the human POMC gene corresponding to the major portion of the 3'-nontranslated mRNA region and 60 bp coding for the signal peptide, was used as a probe for hybridization. It was found that the expression of the POMC in pituitary of the hypertensive JSJAH rats was more than 3-fold gene lower as compared to normotensive Wistar strain. The latter is the original strain from which the ISIAH rats were bred. The mechanism of this phenomenon and its possible relation to the arterial hypertension are discussed.
cDNA for proopiomelanocortin (POMC) of mink has been cloned and sequenced. A comparative analysis of the primary structures of mRNAs coding for proopiomelanocortins of eight animal species and man has been performed. The analysis has revealed conserved and variable POMC mRNA regions. High variability of some of the regions is suggested to be due to the peculiarities of their structural organization. A putative mechanism responsible for the mutations in variable regions is proposed. A tree of the evolutionary relations of POMC has been developed.
Genes encoding luminescence of Photobacterium leiognathi have been cloned in Escherichia coli. The luminescent clones were readily apparent. Among them, a clone containing a recombinant plasmid with a 13.5-kb insertion was identified. This DNA fragment contained all of the luminescence-encoding genes. The luciferase-encoding genes (lux) in this DNA fragment were localized. We have sequenced a part of the cloned lux region and identified the luxA, luxB and luxG genes encoding the alpha and beta subunits of luciferase and a gamma protein with an Mr of 26,180, respectively. The analysis of deduced amino acid sequences and comparison with known luciferase sequences from Vibrio harveyi, indicate the common origin of these proteins.
The sequence of a 3677 nucleotide EcoRI fragment was determined that codes for part of the rat liver tyrosine aminotransferase gene. The sequence was compared with the previously determined cDNA sequence and the intron and exon boundaries were deduced.
Tyrosine aminotransferase (TAT) displays a higher activity in rat liver than in cardiac, renal and cerebral tissues. In the latter three tissues the enzyme is not activated by hydrocortisone. In order to investigate the nature of TAT tissue specificity and to understand how its activity is regulated by hormones in different tissues, the content of TAT mRNA in rat liver, kidney, heart and brain was measured by dot-hybridization using TAT cRNA. TAT mRNA was found in all tissues under study, although in the liver its amount was 10 times as high as in kidney, heart and brain, which is consistent with different levels of TAT enzymatic activity. Upon hydrocortisone administration the amount of TAT mRNA in the liver showed a constant increase reaching a maximum (7-fold) thereafter. The TAT mRNA levels in cardiac and cerebral tissues remained unchanged. These data suggest that the tissue-specific expression and regulation of TAT activity are effectuated by changes in the mRNA contents which may be due to the level of TAT gene transcription.
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Treatment of Wistar rats with a single dose of hydrocortisone acetate resulted in a transient induction of tyrosine aminotransferase (TAT) in the liver. TAT activity and the level of TAT mRNA increased 4-6 h after addition of hydrocortisone acetate (induction phase) and declined thereafter (deinduction phase). TAT activity and TAT mRNA failed to respond to readdition of fresh hydrocortisone acetate during the deinduction period, but cycloheximide treatment at this period increased the level of TAT mRNA. Hydrocortisone acetate alone and cycloheximide alone or after hydrocortisone acetate treatment stimulated the rate of TAT gene transcription. TAT displayed a low activity in the rat heart, brain and kidney, and was not induced by hydrocortisone acetate in these tissues. In addition to liver, TAT mRNA was found in heart, brain and kidney, but in the latter three tissues its amount was about one tenth only as in the liver, which was consistent with different levels of TAT activity. Upon hydrocortisone acetate treatment the TAT mRNA levels in brain and heart remained unchanged.
The data concerning the cycloheximide influence on individual mRNA content, transcription rate and chromatin structure in mammalian cells are reviewed. The putative mechanism of cycloheximide effect is explained as being due to the existence of short-living regulatory proteins. It is concluded that cycloheximide is a helpful tool in the study of regulation of gene expression in mammalian cells.
A synthetic gene coding for human angiogenin was cloned in pUR290 plasmid in frame with beta-galactosidase, both parts of the resultant fused protein being joined through an Asp-Pro sequence. The fused protein, synthesised in E. coli cells upon IPTG induction and isolated as inclusion bodies, possessed angiogenic activity on the chick chorioallantois membrane and was cleaved upon acid treatment to yield free angiogenin.
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A cDNA library from the mink pituitary was screened using as probe a synthetic oligodeoxyribonucleotide, 5'-TTCATGACCTCCGA-3', corresponding to the endorphin region of bovine proopiomelanocortin (POMC) cDNA. As a result, several clones containing inserts complementary to POMC mRNA were identified. The sequence of one of the fragments (585 bp, 65% of the total length of mRNA) was determined. A high degree of homology (over 80%) among the primary structures of sequences from mink, man, and bovine cDNA POMC was established. With the cloned mink cDNA fragment as probe, the DNAs from mink-Chinese hamster hybrid clones were studied. The results of segregation analysis of mink POMC sequences and mink chromosomes in the mink-Chinese hamster panel allowed us to assign the POMC gene to mink chromosome 11.
Nucleotide sequence of the Photobacterium leiognathi DNA containing genes of alpha and beta subunits of luciferase has been determined. We also deduced amino acid sequence and molecular mass of luciferase and localized luciferase genes in the sequenced DNA fragment.
The poly(A)-containing mRNA from human pituitary and prolactinoma have been purified and translated in the cell-free system from rabbit reticulocytes. mRNA from prolactinoma was shown to be enriched with specific prolactin mRNA. DNA complementary to the prolactin mRNA from human pituitary was obtained and cloned. Sequencing of the 900 bp insert by the Maxam-Gilbert technique suggested the cDNA cloned to cole for the previously published amino acid sequence, mismatches with mRNA from prolactinoma occurring at the third positions of codons and thus not causing amino acid substitutions.