[Effect of hyperhybridization of nucleic acids]. / Ob zffekte gipergibridizatsii nukleionovykh kislot.

The reasons for the effect of hyperhybridization (HH) of nucleic acids, when the degree of binding of labelled fragments in the heterologous reaction is higher than in the homologous one, are discussed. The object of investigation was DNA of salmon. HH is most demonstrative in hybridization of a DNA fraction which forms thermostable duplexes (Tm greater than or equal to 75 degrees). HH is accounted for by the fact that closely related species and intraspecies forms differ in the percentage of this fraction; therefore if a species with a small content of this fraction in the genome is chosen as a reference species, hybridization of its DNA with that of another species with a high content of the thermostable fraction, exceeds 100% with respect to the homologous fraction). A correction coefficient is suggested allowing comparison between experiments with different reference species. It appears that the genomes of less specialized, as compared with highly specialyzed species, contain more DNA forming thermostable duplexes. It is therefore recommended to use as reference DNA of species, the structure of karyotype and morphologt of which have more in common with the ancestral form.

[A comparative characteristic of DNA pyrimidine sequences of shark, protopterus and perch]. / Sravnitel'naia kharakteristika pirimidinovykh posledovatel'nostei DNK akuly, protopterusa i okunia.

Pyrimidine sequences of DNA from three fishes: shark, protopterus and perch have been studied. These data together with the evidence from the literature were used to support earlier conclusions that dipnoi and cartilagenous fishes should be distinguished as independent classes. The clustering index, beta, and the total molar percentage of long pyrimidine oligonucleotides (Z) containing greater than or equal to 8 nucleotides--a new parameter offered by us--have been used in comparative investigation of DNAs. The new parameter has permitted to obtain a higher resolution in the analysis of our own and literature data on DNA pyrimidine clusters in fishes. Investigation of pyrimidine clusters and of the base composition of individual isoplits of these clusters using statistical analysis showed that DNA from shark, protopterus, sturgeon and perch significantly differ by many features. Significant differences between these DNAs were found also in the base composition. Thus, new evidence for distinguishing cartilagenous fishes and dipnoi as independent classes have been received.

[Analysis of the thermostability of the hybrid DNA molecules of microorganisms as a means of increasing the resolution of the molecular hybridization technic]. / Analiz termostabil'nosti gibridnykh molekul DNK mikroorganizmov kak sredstvo povysheniia razreshaiushchei sposobnosti metoda molekuliarnoi gibridizatsii.

A comparative study of termostability of microorganisms DNA was performed in order to increase the resolution of the method of molecular hybridization. Molecular hybridization was carried out and the curve of hybrid DNA duplexes distribution, acccording to termostability of two groups of microorganisms, related to strains Echerichia coli B1 and Vibrio cholerae eltor 18647, were obtained. It was determined that the form of the curves is specie specific for the microorganisms investigated but there exists a similarity between the closely related strains which can not be distinguished by the percent of homology.

[Kinetic analysis of interaction of human immunodeficiency virus reverse transcriptase with alkaloids]. / Kineticheskii analiz vzaimodeistviia obratnoi transkriptazy virusa immunodefitsita cheloveka s alkaloidami.

The interactions of HIV-I reverse transcriptase with some alkaloids were studied. Among nine compounds tested three--berberine, palmatine and sanguiritrine--inhibited RT. The dependence of the inhibition on the type of template-primer was also demonstrated. The kinetic analysis as well as circular dichroism experiments suggest the complex mechanism of RT inhibition by alkaloids. This mechanism includes both enzyme-alkaloid and alkaloid-template interactions; the latter effect also results in RT inhibition.

[DNA metabolism in the process of the maturation of male sex cells. I. Amplification of ribosomal genes in the spermatogenesis of rats]. / Metabolizm DNK v protsesse sozrevaniia muzhskikh polovykh kletok. Soobshchenie I. Amplifikatsiia ribosomnykh genov v spermatogeneze krys.

It has been shown that amplification of ribosomal genes takes place in prophase I of rat spermatogenesis. At the pachytene I step, a 2-fold excess of rDNA was found. Amplification of ribosomal genes starts in the premeiotic interphase on the chromosomal DNA. At the initial stage of rDNA amplification, a rDNA-RNA hybrid was detected. Our results allow the suggestion to be made that RNA plays the role of a primer in synthesis of the excess rDNA. It is possible that AT-rich prenucleolar DNA is involved in amplification of rDNA.

[Intra- and interpopulation differentiation of human genomes by molecular DNA hybridization]. / Izuchenie vnutri- i mezhpopuliatsionnoi differentsirovki genomov cheloveka metodom molekuliarnoi gibridizatsii DNK.

The DNA-DNA hybridization method was used to compare the repetitive sequences with a low degree of intragenomic divergence in various etno-territorial groups (Russians, Bouriats and Paleoasiats). Values of intergenomic divergence within groups and between them were estimated by a decrease in melting temperature of hybrid duplexes in relation to homologous 3H-labeled thermostable fraction reassociates of DNA of a Russian. Statistically valid differences in melting temperature were revealed when Russian, Bouriat and Paleoasiatic groups were compared. No such differences were found within each of the groups. Though the thermostability profiles had much in common in each case, some quantitative differences in melting temperature allowed to differentiate local groups in humans.

[Study of polymorphism and divergence of genomic DNA at the species and population levels (using DNA of domestic sheep and wild rams as an example)]. / Issledovanie polimorfizma i divergentsii genomnoi DNK na vidovom i populiatsionnom urovniakh (na primere DNK porod domashnikh obets i dikikh baranov).

Genetic divergence in repetitive sequences of nuclear DNA of wild and domestic sheep was studied by general restriction endonuclease mapping (i.e., the taxonoprint method). The PCR RAPD method with one and two arbitrary primers was also used to analyze the nuclear DNA polymorphism in some other regions. The taxonoprint method, performed using six endonucleases, showed specificity and virtually complete similarity in the patterns of repetitive DNA sequences of two wild forms, argali and mouflon, and five domestic sheep breeds. Central Asian breeds, Kazakh fine-fleeced, karakul, ghissar, and eadeelbay, and an English breed, Lincoln, were examined. The results confirm the opinion that wild and domestic sheep may be considered one polytypic species. The PCR-RAPD method, both with one and two arbitrary primers, revealed a closer similarity of all the sheep breeds examined when argali, rather than with mouflon, was used. These results indicate that the domestication area of sheep was much broader than was earlier presumed. Otherwise, hybridizations of domestic and wild forms could occasionally occur in the area of their coexistence. The amplification patterns of PCR-RAPD products are the most promising population genetic markers.

[Comparison of repeat DNA sequences in the Erinaceidae mammal family by restriction analysis]. / Sravnenie povtoriaiushchikhsia posledovatel'nostei DNK mlekopitaiushchikh semeistva Erinaceidae metodom restriktaznogo analiza.

Hedgehog (Erinaceidae) DNA was digested with teh Sau 96 I, Bsp 143 I, Csp 6 I, Taq I, Hinf I, Msp I, Eco 130I, Bcn I, BsuR I restriction endonucleases. The obtained products were end-labeled and electrophoretically separated in polyacrylamide gel. DNA fragments consisting of highly repetitive genomic sequences were detected as a set of bands corresponding to fragments between 30 and 500 bp in length. Comparison of DNA restriction patterns of the species analyzed revealed the presence of species-specific bands as well as common bands. Phylogenetic trees were constructed by means of the maximum parsimony method and the bootstrap procedure. Our data suggest that hedgehog species from arid areas are clearly distinguished from forest species.

[DNA synthesis in the process of rat spermatogenesis]. / Sintez DNK v protsesse spermatogeneza u krys.

The rat spermatogonia were fractionated in STA-PUT system within 24 hrs following 3H-thymidine injection. DNA isolated from the cells at certain stages of spermatogenesis (spermatogonia, leptotene-zygotene, pachytene, spermatids-spermatozoa) was studied by the method of reassociation kinetics and ultracentrofugation in CsCl gradient. A selective 3H-thymidine incorporation in the region of midrepetitious nucleotide sequences was found at all stages under study, except spermatids-spermatozoa. DNA synthesized during spermatogenesis forms in CsCl density gradient several "satellite" peaks in the "heavy" and "light" gradient zones.

[The detection of provirus in lymphocyte DNA. The monitoring of HIV infection at the genetic level]. / Detektsiia provirusa v DNK limfotsitov. Monitoring VICh-infektsii na geneticheskom urovne.

The presence of HIV provirus in the cell culture and in the patients' blood was studied by polymerase chain reaction followed by hybridization in solution. It was shown that: (i) the hybridized product could be detected both by gel electrophoresis and by binding on hydroxyapatite; (ii) the detection level achieved was no more than 10 infected lymphocytes per million; (iii) the hybridization signal and sensitivity of detection could be enhanced by the transcription of PCR product by the phage T7 RNA polymerase. The observed lack of complete correlation between the amount of provirus and of the p24 antigen in the patients' blood possibly reflects the peculiarities of HIV infection.

[Whitefish: a new mechanism of reproductive isolation]. / Sigovye ryby: novyi mekhanizm reproduktivnoi izoliatsii.

Taxonoprint (a modification of DNA restrictase analysis) allows to distinguish sympatric species, that do not mate or produce hybrid offspring that are sterile or not viable. It is shown that taxonoprints of whitefish are very similar of identical. Sympatric whitefish are continuing to be separate despite they easily mate in experiments and in nature (up to 30% of individuals in nature are hybrids) and hybrids offspring have some features of heterosis. However it appears that hybrids of the second generation are not viable and can exist only because of back crossing with parents. In allows to keep a species independence in the process of gene exchange and to use heterosis of the first generation. Similar isolation mechanism is determined for other fish families (Acipensepidae, Clupeidae, Cyprinidae, Percidae) and some mammals (camels, sheep, bulls).

[The estimation of the degree of genetic divergence in Bovidae by DNA restrictase analysis]. / Otsenka stepeni geneticheskoi divergentsii nekotorykh polorogikh metodom restriktanogo analiza DNK.

Restrictase DNA analysis was performed in seven species of Bovidae family--European (Belovezh) bison, Belovezh-Caucasian bison, European-American bison, mountain Caucasian bison, American bison, domestic bull, and domestic sheep. DNA was digested with MspI and TaqI restriction endonucleases. The products obtained were end-labeled and electrophoretically separated in poliacrilamid gel. DNA fragments consisting of highly repetitive genomic sequences were detected as a set of bands corresponding to fragments between 464 and 67 bp in length. All forms of Bison genus analysed were characterized by the identical sets of bands. The band patterns of bulls is similar to these of the bison, whereas the patterns of sheep is rather different. These data indicate that American bison and European bison geographically isolated forms of the same polytypic species.

Quantitative PCR as a method for monitoring retroviral infection on the gene level.

For monitoring retroviral infection on the gene level, we propose the use of quantitative PCR with two internal standards: one for a fragment of the viral genome and the other for the host cell marker gene. The standards (one for HIV and the other for a human DNA marker gene HLA-DQ alpha) were constructed by PCR-mediated joining of DNA fragments and were found to be effective in quantitative PCR despite rather different structures of amplified fragments in target and standard DNAs. The number of HIV DNA copies was found to be 2-500 per 1000 lymphocytes in blood from HIV-infected patients and up to 5000+ per 1000 cells in chronically infected cell lines. The degree of infection thus measured was found to change over the course of treatment.

Restriction endonuclease analysis of highly repetitive DNA as a phylogenetic tool.

Multiple band patterns of DNA repeats in the 20-500-nucleotide range can be detected by digesting genomic DNA with short-cutting restriction endonucleases, followed by end labeling of the restriction fragments and fractionation in nondenaturing polyacrylamide gels. We call such band patterns obtained from genomic DNA "taxonprints" (Fedorov et al. 1992). Here we show that taxonprints for the taxonomic groups studied (mammals, reptiles, fish, insects-altogether more than 50 species) have the following properties: (1) All individuals from the same species have identical taxonprints. (2) Taxonprint bands can be subdivided into those specific for a single species and those specific for groups of closely related species, genera, and even families. (3) Each restriction endonuclease produces unique band patterns; thus, five to ten restriction enzymes (about 100 bands) may be sufficient for a statistical treatment of phylogenetic relationships based on polymorphisms of restriction endinuclease sites. We demonstrate that taxonprint analysis allows one to distinguish closely related species and to establish the degree of similarity among species and among genera. These characteristics make taxonprint analysis a valuable tool for taxonomic and phylogenetic studies.