[Identification of the proteins interacting with neuroprotective peptide humanin in a yeast two-hybrid system].

Humanine is a human neuroprotective peptide with a wide action spectrum. To analyze molecular mechanisms of humanin functioning, a search for proteins interacting with this peptide was conducted using yeast two-hybrid system. Screening of human fetal brain cDNA library identified seven proteins with different functions that specifically interacted with humanin.

[Nucleotide sequences of long terminal repeats of the human endogenous retrovirus (LTR HERV-K) on the short arm of chromosome 7: identification, analysis and evaluation of transcriptional activity]. / Posledovatel'nosti dlinnykh kontsevykh povtorov éndogennogo retrovirusa (LTR HERV-K) korotkogo plecha khromosomy 7 cheloveka: poisk, analiz i opredelenie transkriptsionnoi aktivnosti.

Six clones containing long terminal repeat (LTR) sequences of human endogenous retrovirus of the HERV-K family were found in the YAC library (1200 kb) of the short arm of human chromosome 7. The sequence sizes of the three clones corresponded to the full-length LTR (969 bp). The LTR localization was determined using FISH and verified by comparison with the GenBank database. All three DNA fragments containing solitary LTRs were transcribed in normal germline cells (testicular parenchyma tissue). The differences in the expression of these clones in the germline tumor cells (seminoma) were observed.

[The SRM12/ADA1 gene acts as a cis-active factor when inserted into recombinant plasmids and makes them more stable in Saccharomyces]. / Gen SRM12/ADA1 kak tsis-aktivnyi faktor podderzhaniia rekombinantnykh plazmid v kletkakh drozhzhei sakharomitsetov.

A DNA fragment containing the SRM12/ADA1 gene sequence inserted into a recombinant circular plasmid improves its maintenance in budding yeast (Saccharomyces cerevisiae) cells. Plasmid stabilization caused by the integrated SRM12 sequence does not require the SRM12 function complementing the srm12 mutation and depends on the orientation of the inserted fragment in the vector. This stabilization is mainly due to a decrease in spontaneous plasmid underreplication/copy loss rather than an increase in the fidelity of mitotic plasmid segregation.

A reporter system for prokaryotic and eukaryotic cells based on the thermostable lichenase from Clostridium thermocellum.

The coding region of the licB gene from Clostridium thermocellum was truncated at the 3' end. The modified lichenase encoded by the construct (LicBM2) retained the most important properties of the enzyme - its high activity and thermostability. LicBM2 consists of the catalytic domain and part of the Pro-Thr-box. We demonstrated the application of the licBM2 gene as a reporter system for prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and mammalian) cells by expressing it either as a transcriptional fusion with selected promoters or as a translational fusion with the E. coli uidA gene. The assays available for LicB activity are sensitive, accurate and simple, and can be used for the analysis of various gene fusion systems or for screening of transformants.

Identification of paralogous HERV-K LTRs on human chromosomes 3, 4, 7 and 11 in regions containing clusters of olfactory receptor genes.

A locus harboring a human endogenous retroviral LTR (long terminal repeat) was mapped on the short arm of human chromosome 7 (7p22), and its evolutionary history was investigated. Sequences of two human genome fragments that were homologous to the LTR-flanking sequences were found in human genome databases: (1) an LTR-containing DNA fragment from region 3p13 of the human genome, which includes clusters of olfactory receptor genes and pseudogenes; and (2) a fragment of region 21q22.1 lacking LTR sequences. PCR analysis demonstrated that LTRs with highly homologous flanking sequences could be found in the genomes of human, chimp, gorilla, and orangutan, but were absent from the genomes of gibbon and New World monkeys. A PCR assay with a primer set corresponding to the sequence from human Chr 3 allowed us to detect LTR-containing paralogous sequences on human chromosomes 3, 4, 7, and 11. The divergence times for the LTR-flanking sequences on chromosomes 3 and 7, and the paralogous sequence on chromosome 21, were evaluated and used to reconstruct the order of duplication events and retroviral insertions. (1) An initial duplication event that occurred 14-17 Mya and before LTR insertion - produced two loci, one corresponding to that located on Chr 21, while the second was the ancestor of the loci on chromosomes 3 and 7. (2) Insertion of the LTR (most probably as a provirus) into this ancestral locus took place 13 Mya. (3) Duplication of the LTR-containing ancestral locus occurred 11 Mya, forming the paralogous modern loci on Chr 3 and 7.

[ARS-activity of a DNA fragment from Bombyx mori in Saccharomyces cerevisiae cells]. / ARS-aktivnost' fragmenta DNK Bombyx mor v kletkakh drozhzhei Saccharomyces cerevisiae.

A previously cloned autonomous transgene (pr8a) of silkworm Bombyx mori inherited without changes in the structure was used to clarify the activity of its ARS in yeast cells. ARS of pr8a was also shown to maintain autonomous replication of hybrid plasmids in yeast cells. The same was true for its central 2.4-kb fragment devoid of flanking sequences.

[TAR-cloning of the short arm of human chromosome 7 in yeast and search for terminal sequences]. / TAR-klonirovanie korotkogo plecha khromosomy 7 cheloveka v drozhzhakh ipoisk terminal'nykh posledovatel'nostei.

A partial clone library of the short arm of human chromosome 7 was created in yeast artificial chromosomes (YAC) using TAR-cloning. The DNA of monochromosome somatic hybrid cells (mouse/human) RuRag 14-4-7-44 containing short arm human chromosome 7 was used for cloning. The clone library was screened for YACs with the human DNA; the mitotic stability of these YACs, the sizes of cloned fragments, and an independent clonal distribution in the chromosome were determined. Human YACs were tested for the presence of chromosome 7p telomeric sequences.

[Radiosensitivity of Saccharomyces yeasts and srm genes: effects of srm1 and srm5 mutations]. / Radiochuvstvitel'nost' drozhzhei sakharomitsetov i geny srm: éffekty mutatsii srm1 i srm5.

The presence in the cell genotype of srm1 and srm5 (cdc28-srm) mutations decreasing the spontaneous rho- mutability was shown to have no effect on the rates of spontaneous nuclear gene mutations and gamma-ray-induced mitotic recombination. Mutation cdc28-srm exerts a marked effect on cell sensitivity to the lethal action of ionizing radiation and on the appearance of homoplasmic segregants generated from heteroplasmic diploids. Additive interactions between mutations cdc28-srm and each of the rad6 and rad52 mutations were revealed by an analysis of double mutants with respect to their sensitivity to radiation. Mutation rad9 was epistatic with mutation cdc28-srm. These data agree with the idea that the p34CDC28 gene product is a target for the RAD9-dependent feedback control operating at the cell cycle checkpoints (checkpoint control) and ensuring an additional amount of time for premitotic repair of chromosomal DNA damage.

Mutations of the CDC28 gene and the radiation sensitivity of Saccharomyces cerevisiae.

cdc28-srm, a non-temperature-sensitive (ts) mutation in the CDC28 gene of Saccharomyces cerevisiae that affects fidelity of mitotic transmission of both mitochondrial and nuclear genetic structures (Devin et al., 1990), also affected cell growth and sensitivity to lethal effects of ionizing radiation. At 30 degrees C cdc28-13, a ts mutation, was without appreciable effects on spontaneous mitochondrial rho(-)-mutagenesis, cell growth and radiation sensitivity, whereas all three cell characteristics mentioned were affected (although to a lesser degree than by cdc28-srm) by cdc28-1, another ts mutation. cdc28-srm was without any significant effect on the rates of spontaneous nuclear gene mutations and gamma-ray-induced mitotic recombination. An analysis of double mutants as regards their radiation sensitivity has revealed additive or even synergistic interactions between the cdc28-srm mutation and every one of the rad6-1 and rad52-1 mutations. The rad9 delta allele was found to be epistatic to cdc28-srm. These data suggest that the p34CDC28 protein is involved in the RAD9-dependent feedback control of DNA integrity operating at the cell cycle checkpoints.

[Analysis of maintenance of redundant genetic structures in the yeast Saccharomyces cerevisiae: effect of mutations cdc28-srm and srm1]. / Analiz podderzhaniia izbytochnykh geneticheskikh struktur u drozhzhei Saccharomyces cerevisiae: éffekty mutatsii cdc28-srm i srm1.

The effects of nuclear gene mutations cdc28-srm and srm1 on the maintenance of various recombinant facultative genetic structures (FGSs) in Saccharomyces cerevisiae were studied. These structures are ARS1 TRP1 mini-coils, noncentromeric circular plasmids containing various ARS elements, and extended linear yeast artificial chromosomes (YAC). These mutations led to an increase in the mitotic stability of some of the FGS tested and the disturbed maintenance of the others. Mutation srm1 imposed a stabilizing effect on the maintenance of various recombinant FGSs with ARS chromosomal elements. Mutation cdc28-srm destabilized the maintenance of only those recombinant FGS that shared full or detectable homology with sequences of the nuclear genome of the yeast cell.

[Analysis of maintenance of redundant genetic structures in the yeast Saccharomyces cerevisiae: disomy and spontaneous mitochondrial rho(-)-mutability]. / Analiz podderzhaniia izbytochnykh geneticheskikh struktur u drozhzhei Saccharomyces cerevisiae: disomiia i spontannaia mitokhondrial'naia rho(-)-mutabil'nost'.

With the postmeiotic progeny of triploids used as initial material, n + 1 disomics at chromosomes II, III, VII, VIII, and X were isolated. Disomy at the chromosomes listed (as well as for chromosomes IV and XIV, as demonstrated previously) is associated with decreased spontaneous rho- mitochondrial mutability. This suggests that a disturbance of the chromosome balance itself as such can lead to considerable changes in the spontaneous variability of the mitochondrial genome. From crosses between n + 1 disomics at chromosome IV and for each of the remaining above-mentioned six chromosomes, double n + 2 disomics were isolated, carrying nonchromologous pairs of extra chromosomes. Analysis of mitotic stability of the chromosome IV and spontaneous rho- mutability in double disomics shows that the effect of disomy on spontaneous rho- mutability most probably cannot be explained by direct competition between different genetic structures maintained in Saccharomyces cerevisiae cells. Disturbance of the chromosome balance in disomy is accompanied by essential qualitative changes in processes mediating the maintenance of genetic structures in yeast cells.

[Isolation and characterization of new nuclear srm gene mutation causing coordinated changes in the maintenance of nuclear and mitochondrial genetic structures in the yeast Saccharomyces]. / Poluchenie i kharakteristiki novykh iadernykh gennykh mutatsii srm, vyzyvaiushchikh koordinirovannye izmeneniia podderzhaniia iadernykh i mitokhondrial'nykh geneticheskikh struktur u drozhzhei sakharomitsetov.

From grown cultures of UV-irradiated Saccharomyces cerevisiae cells with disomy at chromosome IV, clones with nuclear gene mutations were isolated, each of which was suggested to change both mitochondrial spontaneous rho- mutability and the mitotic stability of extra natural chromosomes. Four such nonallelic mutations (srm8, srm12, srm15, and srm17) were isolated, and their phenotypic expression characterized. All four mutations are associated with decreased spontaneous rho- mutability and virtually block sporulation in homozygous mutant diploids. Mutation srm8 is temperature-sensitive and, most probably, involves an essential gene. Double mutants of genotypes srm8 cdc28-srm and srm8 srm12 are nonviable. Mutation srm12 increases the rate of spontaneous loss of extra chromosome XIV by disomics by a factor of about 30. Mutation srm15 induces a small (about twofold) but statistically significant decrease of this rate. Mutations srm8 and srm17 drastically decelerate reproduction of cells with disomy, which prevents quantitative estimations of rates of loss of extra chromosomes.

[Variability of rDNA genes, detected as a result of analyzing a pseudogene nucleotide sequence in Drosophila melanogaster]. / Izmenchivost' genov rDNK, obnaruzhivaemaia v rezul'tate analiza nukleotidnoi posledovatel'nosti psevdogena u Drosophila melanogaster.

A pseudogene bearing the bulk of the 18S RNA gene was detected outside the rDNA cluster. It comprised irregularly distributed nucleotide substitutions as well as short insertions and deletions. No sequence alterations were observed in the 5' region of the pseudogene, whereas the frequency of substitutions and alterations per nucleotide number in the 3' region and in the middle of the sequence was 7.6% and 1.8%, respectively. The observed sharp irregularity in distribution of substitutions and alterations was considered the result of successive recombinations between the functional 18S rRNA gene and its diverged or damaged variants. This phenomenon provides experimental evidence that recombinations between the pseudogene and functioning repeats of rDNA are implicated in the mechanism of rDNA sequence correction. A segment of the pseudogene sequence was shown to contain substitutions primarily in regions coding for single-strand parts of the RNA molecule. The same segment contained a deletion and an insertion of a nucleotide, approximating it to the most of the studied eukaryotic 18S rRNA sequences. These observations allowed us to supposed that a structural rDNA variant, a fragment of which appears in the pseudogene sequence, is present in the genome. The data obtained suggest both the presence of 18S rDNA variants, and recombination between them, determining the concerted evolution of rRNA genes.

[A replication enhancer of viral origin increases the mitotic stability of yeast transformants]. / Enkhanser replikatsii virusnogo proiskhozhdeniia povyshaet mitoticheskuiu stabil'nost' transformantov drozhzhei.

It is shown in this paper that a DNA fragment of Hepatitis B virus possessing structural features of yeast replication enhancer increases the mitotic stability of yeast transformants containing hybrid plasmids of episomal and replicative types. The mitotic stability of transformants with plasmid of the replicative type and with the replication enhancer increases only in [cir+] cells. Comparison of primary sequences of HBV DNA of different subtypes revealed that only DNA has unique structural features of the yeast enhancer of replication.

[Cloning of segments of the Drosophila melanogaster genome using artificial chromosomes of the yeast Saccharomyces cerevisiae]. / Klonirovanie uchastkov genoma Drosophila melanogaster v iskusstvennykh khromosomakh drozhzhei Saccharomyces cerevisiae.

A partial genomic library from the Batumi L stock of Drosophila melanogaster was constructed using yeast artificial chromosomes as vectors. The DNA was restricted by Not1 and large fragments were inserted into the YAC5 vector. The size of cloned DNA varied from 90 to 500 kb. 48 random clones were characterized by in situ hybridization to the Batumi L polytene salivary gland chromosome. Single euchromatic sites of hybridization were detected for 27 clones; 11 clones revealed the main euchromatic hybridization site and several additional sites scattered along the chromosomes; 8 clones carried repeats which hybridized to chromocenter and other chromosomal sites; clones with 500 and 90 kb inserts originated from the Y chromosomes and nucleolus, respectively. The library is enriched by the repeated sequences related to the b-heterochromatin.

Region of hepatitis B virus DNA with a homology to the yeast ARS replication enhancer.

A computer analysis of the primary sequence of hepatitis B virus (HBV) subtype ayw DNA, cloned within the pVG2 recombinant plasmid, which raises its stability in Saccharomyces cerevisiae transformants, was performed. This revealed that the structure of the HBV DNA has: two bends in the termination regions of the HBs and HBc genes, and multiple sequences with a high degree of homology to the ARS (autonomously replicating sequence) core consensus in this region of the HBs gene. DNA fragments from the HBs region (330 bp) and from the HBc region (378 bp) have an abnormal electrophoretic mobility in 8% polyacrylamide gels. The similarity of the structural motifs in the stop-region of HBs gene with the B-domain of the S. cerevisiae ARS element is discussed.