[Archaeal diversity in permafrost deposits of Bunger Hills Oasis and King George Island (Antarctica) according to the 16S rRNA gene sequencing].

Archaeal communities of permafrost deposits of King George Island and Bunger Hills Oasis (Antarctica) differing in the content of biogenic methane were analyzed using clone libraries of two 16S rRNA gene regions. Phylotypes belonging to methanogenic archaea were identified in all horizons.

Evolutionary relationships within 'pygmaeus' group microphallids using genetic analysis and scanning electron microscopy.

There are four species of 'pygmaeus' microphallids, namely Microphallus pygmaeus, M. piriformes, M. pseudopygmaeus and M. triangulatus (Trematoda: Microphallidae) which are parasites of marine birds and their sporocysts give rise to transmissible metacercariae inside littoral gastropods (mostly littorines). Universally primed polymerase chain reaction (UP-PCR) showed no apparent pattern between genetic diversity of the metacercariae as estimated by genomic banding profiles and their geographic region or molluscan host species. At the same time UP-PCR product cross-hybridization showed that M. pseudopygmaeus and M. triangulatus are genetically very similar, indicating that these taxa represent one species complex. In contrast, M. pygmaeus and M. piriformes are genetically well separated from each other and also from the pseudopygmaeus-triangulatus complex. Scanning electron microscopy of ventral spines, and analyses of spine angles and the number of teeth per spine, showed that all species differed significantly from one another. It was concluded that M. piriformes represents the original western member of the 'pygmaeus' group. Microphallus pygmaeus probably diverged from M. piriformes as it progressively specialized for sea duck final hosts. Microphallus pseudopygmaeus and M. triangulatus diverged from each other and the piriformes-pygmaeus ancestral line relatively recently. Microphallus pseudopygmaeus specialized for adoption of a wide range of gastropod host species and M. triangulatus developed morpho-functional specialization associated with final host exploitation.

Phylogenetic relationship of Fusarium langsethiae to Fusarium poae and Fusarium sporotrichioides as inferred by IGS, ITS, beta-tubulin sequences and UP-PCR hybridization analysis.

Fusarium langsethiae was recently described to accommodate "powdery" isolates of Fusarium poae, which morphologically resemble F. poae, but whose metabolite profile is similar to that of Fusarium sporotrichioides. In order to investigate the phylogenetic relationship of F. langsethiae to closely related species, we sequenced the internal transcribed spacer (ITS) regions 1 and 2 and part of the intergenic spacer (IGS) region of the rDNA cluster and part of the beta-tubulin gene from 109 strains of F. poae, F. sporotrichioides, F. langsethiae and Fusarium kyushuense from different geographic origin. Sequence analysis of ITS1 and 2 was unable to separate all F. sporotrichioides strains from F. langsethiae strains. Sequence analysis of beta-tubulin distinguished all four species, but it did not resolve the phylogenetic relationship between these two species. Sequence analysis of the IGS region distinguished the four species and led to a higher number of subgroups of the individual species, of which that of F. sporotrichioides var. minus isolates was even better supported than that of F. poae and F. langsethiae. Neighbor-joining and POY analyses of all combined sequences reliably separated all species studied, including F. langsethiae, clearly from F. sporotrichioides. The high intraspecific variability of the IGS sequences were found useful to group isolates according to their geographic origin. These results are in accordance with the results of the UP-PCR hybridization analysis. In summary, our data offer molecular support for the description of F. langsethiae as a new species in section Sporotrichiella.

Differentiation of six sibling species in the Saccharomyces sensu stricto complex by multilocus enzyme electrophoresis and UP-PCR analysis.

UP-PCR analysis and multilocus enzyme electrophoresis were used to characterize 37 strains of the sibling species Saccharomyces cerevisiae, S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae and S. paradoxus. The results demonstrate that both molecular approaches are useful for discriminating between these phenotypically indistinguishable Saccharomyces species. The data obtained are in excellent agreement with previously reported genetic analyses, sequencing of the 18S rRNA and ITS regions, and DNA-DNA reassociation data.

[Glass slide smears and imprints from infected organs for identification of Leishmania major by polymerase chain reaction methods]. / Vozmozhnost' ispol'zovaniia mazkov i otpechatkov na stekle ot porazhennykh organov dlia identifikatsii Leishmaniia major metodami PTSR analiza.

The paper presents data showing that the DNA isolated from the smears and imprints of L. major-infected hamsters is suitable for use in the polymerase chain reaction (PCR) to detect the causative agent of leishmaniasis. The most solid data have been obtained with the smears unexposed to staining and examination by using immersion oil and to benzene treatment. The DNA isolated from these smears infected may preserve for at least 1.5 months in a domestic refrigerator. The immersion oil-treated smears may be also used to identify leishmanias, but DNA should be isolated from the infected specimens of these smears just before PCR. The original primer pair L-unit/L-mail that has shown itself well in the experiments on cultured promastigotes may be, if required, used to differentiate L. major and L. turanica in the infected material collected from infected rodents.

Identification of a universally primed-PCR-derived sequence-characterized amplified region marker for an antagonistic strain of Clonostachys rosea and development of a strain-specific PCR detection assay.

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.

Intraspecific variation in gamma-radiation resistance and genomic structure in the filamentous fungus Alternaria alternata: a case study of strains inhabiting Chernobyl reactor no. 4.

This is probably the first report on intraspecific variation in radiation resistance for filamentous fungi. It was revealed that natural ("field") strains of the filamentous fungus Alternaria alternata are extremely variable in response to gamma-irradiation ranging from supersensitive to highly resistant to radiation. At the same time nearly all strains originating from the highly radiation-polluted reactor of the Chernobyl (Ukraine) Nuclear Power Plant possessed high radiation resistance. The genome structure of strains studied by universally primed polymerase chain reaction (UP-PCR) was found to be well conserved in "reactor" but not in "control" strains. The "reactor" strains appear to be genetically adapted to this high radiation habitat by means of selection, thus providing a natural source of genetically homogeneous fungal lineages.

[A polymorphism study of the genomic DNA of trematodes at different phases of development]. / Issledovanie polimorfizma genomnoi DNK trematod v raznykh fazakh razvitiia.

Polymorphism of the genome DNA in different development stages of trematodes by means of the polymerase chain reaction (PCR) with universal primers was investigated. Interspecific variation in Trichobilharzia ocellata (cercaria) was shown in samples from the Moscow area and some regions in Byelorussia (Naroch Lake). Population groupments of T. ocellata are correlated with migration routes of subpopulation groupments of city populations of the mallard duck (Anas platyrhynchus). The cercariae of T. ocellata from rather distant geographical isolates (Moscow and Naroch Lake) differ by PCR pattern in greater extent than groupments of cercariae within Moscow. These distant isolates are considered to be members of separate populations. The cercariae of T. ocellata emitted from infected snails belonging to the different species (Lymnaea auricularia, L. ovata, L. pusilla pusilla) and during definite period (6 days with 24 h analysis) do not differ by the PCR patterns. T. ocellata cercariae emitted from L. auricularia in autumn period (from molluscs of the new generation in "new" summer infection) are found to be different significantly from the "spring" cercariae of the same year and the "autumn" ones of the previous year. The study of the experimentally reproduced life cycle of Opisthorchis felineus revealed an essential difference of cercariae genome. DNA from that in other development stages. This indicates, that genetic reorganisation in the genome of products of parthenogenetic development took place in the parthenita stage.

Comparison of some molecular-genetic techniques for identification of Leishmania circulating in natural foci of zoonotic cutaneous leishmaniasis in the central Asia region.

Different molecular-genetic methods were used to identify a cohort of Leishmania strains from natural foci of zoonotic cutaneous leishmaniasis located in Central Asia, on the former USSR territory. The results obtained using isoenzymes, PCR, restriction fragment length polymorphisms of kDNA and molecular hybridization techniques are discussed in terms of their applicability, discrimination power and feasibility for answering questions related to molecular epidemiological research and for detecting mixed Leishmania infections.

[Genetic structure of soil population of the fungus Fusarium oxysporum Schlechtend.: Fr.: molecular reidentification of the species and genetic differentiation of isolates using polymerase chain reaction with universal primers (UP-PCR)]. / Geneticheskaia struktura pochvennoi populiatsii griba Fusarium oxysporum Schlechtend.: Fr.: molekuliarnaia reidentifikatsiia vida i geneticheskaia differentsiatsiia izoliatov metodom polimeraznoi tsepnoi reaktsii s universal'nymi praimerami (UP-PTsR)]

The genetic structure of three soil populations of fungus Fusarium oxysporum was analyzed using polymerase chain reaction with universal primers (UP-PCR). Distinct UP-PCR variants revealed by means of cross-dot hybridization of amplified DNA and restriction analysis of nuclear ribosomal DNA represent subspecies or sibling species of F. oxysporum. The remaining isolates of F. oxysporum showed moderate UP-PCR polymorphism characterized by numerous types, whose relatedness was analyzed by computer treatment of the UP-PCR patterns. The genetic distance trees based on the UP-PCR patterns, which were obtained with different universal primers, demonstrated similar topology. This suggests that evolutionarily important genome rearrangements correlatively occur within the entire genome. Isolates representing different UP-PCR polymorphisms were encountered in all populations, being distributed asymmetrically in two of these. In general, soil populations of F. oxysporum were represented by numerous genetically isolated groups with a similar genome structure. The genetic heterogeneity of the isolates within these groups is likely to be caused by the parasexual process. The usefulness of the UP-PCR technique for population studies of F. oxysporum was demonstrated.

[Amplification of DNA for 15-30 minutes in single-use pipette tips]. / Amplifikatsiia DNK za 15-30 minut v odnorazovykh nakonechnikakh dlia mikrodozatorov.

Amplification of 200-1000-bp DNA fragments was performed in 15-30 min using a rapid thermal cycler based on the commercial instrument TC-1000-1 (IRLEN, St. Petersburg, Russia). Plastic pipette tips were used as thin walled, high surface to volume-ratio tubes, to increase the rate of heating (cooling) of 20 microliters samples, which allowed the time required for DNA amplification to be considerably reduced (5-10 times).

[The production of clones of "man x Chinese hamster" hybrid cells containing different parts of the human genome]. / Poluchenie klonov gibridnykh kletok "chelovek x kitaiskii khomiachok", soderzhashchikh raznye chasti genoma cheloveka.

Some approach has been described to create hybrid cell lines (human x Chinese hamster) which contain different parts of human genome, and then efficiently to reveal and isolate the human DNA from these. This method involves the introduction of a selective marker in different sites of the human cell genome, by transfecting them with plasmid SV2neo, and the use of flow cytometry and DNA polymerase chain reaction with primers specific only for human DNA.

[The detection of human DNA in cell hybrids by the polymerase chain reaction with universal primers: the species specificity of the amplified DNA]. / Vyiavlenie DNK cheloveka v kletochnykh gibridakh metodom polimeraznoi tsepnoi reaktsii s universal'nymi praimerami: vidospetsifichnost' amplifitsirovannoi DNK.

The human DNA detection method is developed on the basis of DNA cross-hydridization of the amplification products obtained by the universally primed polymerase chain reaction (UP-PCR) technique. These PCR products are characterized by species-specificity in hybridization assay. Two somatic cell hybrids "human x Chinese hamster" supposed to contain the human DNA, according to selection procedure, were analysed by this method. As a result, the presence of human DNA, unable to be tested by cytological techniques, have been proven. The amplified human DNA can be mapped by this method.

Comparative analysis of spontaneous mitotic recombination in [cir0] and [cir+] strains of the yeast Saccharomyces cerevisiae.

The influence of the 2 microns plasmid on homologous recombination in the right arm of chromosome XV of the yeast Saccharomyces cerevisiae has been examined. No differences between spontaneous mitotic recombination rates in [cir0] and [cir+] derivatives of two yeast diploid tester strains were detected. In the course of analysis an unusually high coincident conversion frequency at ADE2, HIS3, and two RFLP loci adjacent to ADE2, was observed. The character of coincident homozygotization of linked markers argues for a "break-and-replicate" mechanism underlying the coincident conversion events.

[Polymerase chain reaction with universal primers for studying genomes]. / Polimeraznaia tsepnaia reaktsiia s universal'nym praimerami dlia izucheniia genomov.

Universal primer ability of generating conservative and variable UP-PCR (universally primed polymerase chain reaction) species-specific patterns was analysed on bacteria to serve as an example. Also, two important properties of the UP-PCR patterns (species/primer DNA hybridization specificity) are characterized.

[The identification of marker strains of Leishmania major, L. turanica and L. gerbilli by the polymer chain reaction with a universal primer]. / Identifikatsiia markernykh shtammov Leishmania major, L. turanica, L. gerbilli metodom polimeraznoi tsepnoi reaktsii s universal'nym praimerom.

The Leningrad Nuclear Physics Institute, Academy of Science of the USSR, and Martsinovskii Institute of Medical Parasitology and Tropical Medicine, USSR Ministry of Health, developed polymerase chain reaction (PCR) technique with universal primer 3-2 for Leishmania identification. The primers were patented in the USSR (Patent No 4757254, 1989). Reference strains of three Leishmania species were identified: L. major--MRHO/SU/59/Neal P; L. gerbilli--MRHO/CN/60/gerbilli; L. turanica--MRHO/SU/80/Cl 3720 and MRHO/SU/83/KD 051. Each Leishmania species is specific and different in its PCR pattern whereas the two L. turanica strains have identical PCR patterns of the given primer.

[The gene identification of bacterial species and serovariants by the polymerase chain reaction with universal oligonucleotides: the reidentification of earlier isolated strains of Yersinia pseudotuberculosis]. / Genoidentifikatsiia vidov i serovariantov bakterii metodom polimeraznoi tsepnoi reaktsii s universal'nymi oligonukleotidami: reidentifikatsiia ranee vydelennykh shtammov Yersinia pseudotuberculosis.

Genetic analysis of 19 standard strains belonging to 6 Yersinia species (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica, Y. kirstensenii, Y. frederiksenii, Y. intermedia) revealed that gene typing by the method of polymerase chain reaction (PCR) with the use of universal primers permitted the identification of species in bacterial cultures by PCR patterns and the determination of Y. pseudotuberculosis serovars within 4 hours. By this method 23 Y. pseudotuberculosis strains (serovar 1), earlier isolated in different regions of the USSR from humans and rodents, were studied. The study showed that out of 14 strains of human origin only two strains could actually be classified with serovar 1, while the remaining strains were reidentified as belonging to serovar 5. Among 9 strains isolated from rodents those of serovar 1 prevailed (8 strains). The authors suppose that strains of serovar 5 cause outbreaks and sporadic cases of pseudotuberculosis, occurring considerably more often than it is commonly believed in the USSR.

[Genetic study of plasmid integration into yeast chromosomes. VI. Patterns of the destabilization of chimeric chromosomes and their use in gene mapping]. / Geneticheskoe izuchenie integratsii plazmid v drozhzhevye khromosomy. Soobshchenie VI. Zakonomernosti destabilizatsii khimernykh khromosom i ikh ispol'zovanie dlia kartirovaniia genov.

The 2 microns DNA-dependent destabilization of yeast chimeric chromosomes III, IV, V was analysed. The comparison of its peculiarities with the earlier localized sites of episomal plasmid integration allowed to derive genetic regularities of destabilization process. Two destabilization rules that describe patterns of the loss of genetic information in the chromosome were formulated. The usefulness of this for mitotic intrachromosomal gene mapping in yeast was demonstrated using plasmid integration site mapping in chromosome I.