[Genotypic Diversity of Wolbachia pipientis in Native and Invasive Harmonia axyridis Pall., 1773 (Coleoptera, Coccinellidae) Populations].

The distribution and variability of reproductive symbiotic Wolbachia pipientis bacteria were studied in seven native and six invasive H. axyridis populations. Wolbachia-infected individuals were found in two invasive and two native populations. We demonstrated for the first time an infection of invasive H. axyridis populations with Wolbachia. Two new molecular forms of Wolbachia were detected by a system of multilocus typing. The supergroup A Wolbachia was found for the first time in H. axyridis. The detected genotypic diversity of Wolbachia indicates repeated and independent infection events in the evolutionary past of H. axyridis.

[Identification of potentially invasive species of black flies [Diptera: Simuliidae] from Armenia based on an analysis of variability in the mtDNA barcode of the cox1 gene and chromosomal polymorphism].

Black flies (Diptera, Simuliidae) are well known for their medical, environmental, and veterinary importance. The simuliid fauna of Armenia includes 53 species. A number of dominant species are of ecological importance. Complex analysis, which involved morphometric, cytogenetic, and molecular genetic approaches, was conducted to characterize the species status of black flies inhabiting the territory of Armenia. It was shown that the predominant simuliid species, Simulium paraequinum and Simulium kiritshenkoi, belong to a group of species with minimal variability of the cox1 gene. The recently discovered species, Simulium noellery and Simulium [B.] erythrocephalum, which are new to Armenia, can be considered as potentially invasive, which is supported by the low level of variability of the cox1 gene.

[Drosophila melanogaster Cell Culture as an Experimental Model to Study Recombination in Wolbachia pipientis].

Wolbachiapipientis is an obligate intracellular endosymbiont that commonly infects arthropods. Comparative genomic studies of Wolbachia reveal traces of numerous events of intergenic and intragenic recombination. The molecular mechanisms of recombination in Wolbachia are not currently known. We conducted experimental verification of the possibility of recombination of two strains of Wolbachia: wMel and wRi, after using these strains for double infection of the Dm2008Wb1 (D. melanogaster) cell culture clone permissive to Wolbachia. We obtained cell culture subclones with double Wolbachia infection and subclones infected only by strain wMel. Dual infection with the Wolbachia strains wMel and wRi has been stably maintained in the subclones for two years. Multilocus sequence typing (MLST) of the obtained subclones revealed the presence of dual infection for all five Wolbachia genes used for MLST Cloning and nucleotide sequence analysis of individual forms of the fbpA gene of Wolbachia from cell clones with dual infection showed intragenic recombination events between strains wMel and wRi, which occurred in the permanent D. melanogaster culture cell culture. The fact that putative recombination sites contain no insertions of nucleotide sequences of phages or IS elements, as well as the asymmetrical character of recombinants, favors the hypothesis that gene conversion is the most probable molecular mechanism of recombination in Wolbachia.

[Transfer of mitochondrial DNA to nuclear genome of cells of passaged cell line of Drosophila virilis].

The variability of the chromosomal fragments of the atp6 mitochondrial gene, which is integrated into chromosomal DNA in the lines of flies of different geographic origins and in the passaged cell lines of D. virilis has been analyzed. We did not reveal any nucleotide variability in this DNA marker among the studied fly lines. This result is consistent with the proposition that the D. virilis species is monomorphic. The new fragments of the atp6 gene that are associated with the insertions of the Tv1 retrotransposon and are absent in the fly genome are revealed in the genome of the passaged cell line of D. virilis. This fact is evidence of the activation of the mitochondrial DNA transfer into the nuclear genome of the cells of passaged cell culture.

[Mitochondrial genome variation in domesticated sable (Martes zibellina)].

The first comparison of mitochondrial variations in sables from captive and natural populations of the Urals, Central Siberia, Yakutia, Kamchatka, and Japan has been performed. The object of comparative analysis was a 427-bp 5' fragment of the mitochondrial control region, including the D-loop. Two main haplogroups of the sable mitochondrial genome have been found, which provides new data for reconstruction of the spread of the sable over its current range. Asymmetry of the haplotype abundances in the captive populations of sables has been detected. The mitochondrial haplotypes characteristic of sable breeds have been identified. The possible role of the frequent mitochondrial haplotypes of the captive population in the sable adaptation to the conditions of captivity is discussed.

[Establishment of a new continuous cell line of Drosophila melanogaster strain infected by the intracellular endosymbiotic bacterium Wolbachia pipientis under natural conditions].

Wolbachia pipientis is an obligately intracellular bacterium infecting a number of arthropod and nematode species. At the body level, Wolbachia infection may cause parthenogenesis, feminization of genetic males, male killing, or cytoplasmic incompatibility; it may also be asymptomatic. Of special interest is DNA transfer from Wolbachia to the host insect genome, which was discovered recently. At the cellular level, the effects caused by Wolbachia have been studied more poorly. Only one of the known insect cell lines has been obtained from an insect species (the mosquito Aedes albopictus) infected by Wolbachia. In this study, a continuous cell line Dm2008Wb1 has been obtained from embryos of Drosophila melanogaster infected under natural conditions. Wolbachia both persists in a primary cell culture and is retained upon its transformation into a continuous culture. The presence of this bacterium in cells in a free form is evidenced by the fact that tetracycline treatment can cure the cells of Wolbachia and by successful transfer of Wolbachia to another cell line (S2), where it has not been detected before.

[Variability of continuous insect cell lines and their identification].

Continuous insect cell lines make a special object of research in biology. Insect cells in the established lines differ in the number of attributes from both normal differentiated, and embryonic cells. The period of genome destabilization necessarily precedes cell line immortalization. Genome destabilization is manifested by changes in genome size, cell karyotype, amplification of some retrotransposone families, and induction of their expression. The existence of significant genetic variability in one line puts a problem of searching for invariant attributes providing culture identification and defining the limits of normal polymorphism of cells in the culture. Using the vast collection of insect continuous cell lines stored at the N. I. Vavilov Institute of General Genetics RAS, nine lines were identified by RFLP method of mitochondrial DNA. Variability of DNA-polymorphisms, cellular karyology, morphology, immunological and biochemical attribute in the culture is discussed.

[Mitochondrial DNA polymorphism in the natural populations of the Drosophila virilis species group]. / Polimorfizm mitokhondrial'noi DNK prirodnykh populiatsii drozofil gruppy virilis.

Restriction fragment length polymorphism (RFLP) analysis has been used to evaluate mitochondrial DNA (mtDNA) variation in 12 sibling species forming the Drosophila virilis species group. The variation thresholds corresponding to the interspecific and interstrain levels have been determined. The results indicate that interspecific hybridization has significantly contributed to the evolutionary history of the virilis species group.

Gypsy group retrotransposon Tv1 from Drosophila virilis.

We have determined the nucleotide sequence of the 6868 bp full-size retrotransposon termed 'Tv1'. Tv1 was isolated from the DNA fraction of extracellular virus-like particles of Drosophila virilis culture cells. Tv1 has the typical structure for a gypsy-group retrotransposon. The Tv1 element was found to be flanked by 453 bp long terminal direct repeats identical to each other. The central part of the element contains three long open reading frames which resemble the gag, pol and env genes of retroviruses. ORF2 includes conservative motifs of protease, reverse transcriptase, RNase H and integrase in the order characteristic for the gypsy-group retrotransposons. Although most copies of Tv1 are located in pericentromeric heterochromatin, the amplification of this family demonstrated in the cell culture and site polymorphism observed in different Drosophila strains suggest functional activity of the Tv1 element.

[Polymorphism of virus-like particles of retrotransposons in Drosophila and yeast cells]. / Polimorfizm virusopodobnykh chastits retrotranspozonov v kletkakh drozofily i drozhzhei.

Data on the molecular arrangement of viruslike particles (VLPs) of yeast and Drosophila retrotransposons are presented. Two methods for identifying VLPs from specific retrotransposon families have been offered. The first method is based on VLPs fractionation by electrophoresis in agarose gel under strictly controlled conditions. VLPs of the Drosophila melanogaster retrotransposon families copia and gypsy and D. virilis retrotransposon Tv1 were identified by this method. The method based on heterologous induction of retrotransposons in cells of the mutant spt3 strain of Saccharomyces cerevisiae was used to identify VLPs of yeast retrotransposon Tyl and D. melanogaster gypsy retrotransposon.

Transient expression assay in a baculovirus system using firefly luciferase gene as a reporter.

Transient gene expression assays were developed to assess the function of the regulatory sequences of baculoviruses Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica nuclear polyhedrosis virus (AcNPV) in insect cells of Bombyx mori and Spodoptera frugiperda, respectively. DNA sequences encoding luciferase (luc) of the firefly Photinus pyralis was successfully employed in the expression assay as a reporter gene. Recombinant plasmids were constructed containing the luc gene under control of baculovirus-specific or heterologous promoters. Cotransfection of Bombyx mori and Spodoptera frugiperda cells with recombinant plasmids carrying virus-specific promoter sequences and BmNPV and AcNPV DNA, respectively, gave rise to efficient synthesis of luciferase (Luc), while heterologous promoters induced a low level of luc expression. We found that flanking sequences of the AcNPV DNA in the transfer plasmid contained an unknown promoter conferring an efficient luc expression. The activity of this promoter was modulated by the polh promoter sequences. The assay allows one to conduct highly sensitive monitoring of the transient expression of foreign genes from the transfecting plasmids prior to construction of recombinant viruses.

[Phenotypic manifestations of reverse transcriptase activity in yeast cells]. / Fenotipicheskoe proiavlenie aktivnosti obratnoi transkriptazy v drozhzhevykh kletkakh.

The yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe transformed by plasmids containing retrotransposon from yeast or Drosophila under the control of a strong promoter show the remarkable reverse transcriptase activity. The activity results in the impaired yeast growth and decreased mitotic stability of the plasmids. The phenotypic expression of the reverse transcriptase activity is observed within 30 days.

[Expression of homologous and heterologous retrotransposons in a model system of yeast cells]. / Ekspressiia gomologichnykh i geterologichnykh retrotranspozonov v model'noi sisteme drozhzhevykh kletok.

Yeast cells Saccharomyces cerevisiae were transformed by the recombinant plasmids containing the Yeast retrotransposon Ty and the Drosophila mobile element gypsy under the control of a strong Yeast promoter. The exogenous Ty-element induces the complete cycle of Ty-retrotransposition including the TyRNA synthesis, formation of virus-like particles, synthesis of all reverse transcriptase intermediates in the virus-like particles with the subsequent circles formation and transposition. The Drosophila mobile element gypsy is capable of inducing the formation of the virus-like particles containing RNA, DNA and proteins of the Ty-retrotransposon only. The Ty-circles and induction of transposition were not observed. The obtained data demonstrates the existence of the multistep repression system for Ty-transposition cycle. The possibility and efficiency of using the model to study the mechanism for retrotransposon transposition is discussed.

Retrotransposon transposition intermediates are encapsidated into virus-like particles.

Virus-like particles (VLPs) possessing reverse transcriptase activity are persistently present in Drosophila melanogaster cultured cells and are formed in yeast induced for transposition. Different retrotransposon transposition intermediates consistent with those expected from the model of reverse transcription pathway of retrotransposon transposition have been detected during the analysis of nucleic acids isolated from VLPs. These data indicate that the act of reverse transcription takes place in VLPs which may be considered as functional intermediates of transposition.

[Expression of Drosophila retrotransposon in yeast]. / Ekspressiia retrotranspozona drozofily v drozhzhakh.

The recombinant plasmids pPTX and pPGX were constructed, containing the sequences of yeast Ty retrotransposon and Drosophila element mdg4, correspondingly. Transformation of yeast by these plasmids lead to induction of reverse transcriptase activity associated with virus-like particles, containing only the sequences of Ty. The data obtained show that mdg4 is capable of expression in yeast and the products of its expression are used to form the yeast virus-like particles. The system described may be used to study the expression of different retrotransposons from various cells in yeast.

[Structure of long terminal repeats of transcriptionally active and inactive copies of the mobile dispersed gene MDG3 in Drosophila melanogaster]. / Struktura dlinnykh kontsevykh povtorov transkriptsionno aktivnykh i neaktivnykh kopii mobil'nogo dispergirovannogo gena MDG3 Drosophila melanogaster.

The nucleotide sequences of long terminal repeats (LTRs) and adjacent regions are determined in the transcribed and non-transcribed variants of a mobile dispersed genetic element MDG3. MDG3 is similar to other mdg elements. A 4 bp duplication of the host DNA is generated upon its integration. MDG3 is flanked by a 5 bp inverted repeat. The length of the LTRs varies in different MDG3 variants, the difference being connected mainly with duplications of certain sequences in U3 and R regions. The copies with 267 bp LTR are the most abundant ones and perfectly conservative in their primary structure. They are transcribed in 67J25D cell culture and are not transcribed in Kc cell line, where another variant with LTR length 293 bp is transcriptionally active. S1-mapping of transcription initiation and termination sites has demonstrated that in both MDG3 variants they are situated in the same positions, and the LTR itself may be subdivided into U3, R and U5 regions, like retroviral LTRs. Possible factors involved in the regulation of mdg transcription are discussed.

[Fine structure of long terminal repeats and stages of reverse transcription of mobile dispersed genes in Drosophila]. / Tonkaia struktura dlinnykh kontsevykh povtorov i étapy obratnoi transkriptsii mobil'nykh dispergirovannykh genov drozofily.

In Drosophila melanogaster cultured cells, RNA reverse transcription intermediate forms connected with initiation of minus and plus DNA strand synthesis (minus and plus strong-stop DNA) are detected for mobile dispersed genetic elements MDG1, MDG3 and MDG4 (gypsy). A comparative analysis of intermediate forms has proved that mdg elements pass the same stages of reverse transcription as retroviruses, revealing a complete similarity between intermediate products. It has also been established that these three mdg elements possess a common mechanism of reverse transcription, despite their structural differences. The length of the minus strong-stop DNA, that gives the position of the RNA start site, coincides with the data obtained from SI nuclease analysis of transcription initiation. SI mapping has also revealed that mdg RNA carries a repeated sequence R on its ends, similar to retroviral RNA molecules, and that mdg LTRs have a U3-R-U5 structure analogous to that of proretroviral LTRs. Transcription of mdg1, mdg3 and mdg4 is initiated within or immediately after the same sequence TCAGTPy. Neither TATA box nor CAAT box can be found in their characteristic positions upstream of the 5' ends of mRNA.