A Finding of a Frozen Mummy of a Lemming (Rodentia, Cricetidae, Lemmus) in the Upper Pleistocene of Yakutia.

The external morphological, X-ray, and tomographic study of a frozen rodent mummy from the Upper Pleistocene Yedoma deposits on the Tirekhtyakh River (a Semyuelyakh River tributary, Abyi ulus, Republic of Sakha (Yakutia), Russia) showed its belonging to Lemmus sp. The radiocarbon age of the finding is 41 305-41 885 cal B.P. This is the first Pleistocene discovery of a frozen mummy of a genus Lemmus representative. In terms of the body and skull sizes, coat color, the lower incisor length, and the molar structure, the specimen studied is similar to the recent Lemmus sibiricus (Kerr, 1792). Comparison of the mitochondrial COB gene sequence with the DNA sequences presented in the GenBank database also testified to the maximum similarity with the Recent Siberian brown lemming.

[Expression of cDNA of the Gene for the Capsid Protein VP2 of German Cockroach Densovirus in the Transgenic Strain of Drosophila melanogaster].

Transgenic strains of Drosophila melanogaster capable of expressing a cDNA fragment corresponding to open reading frame (ORF) of the gene for the German cockroach densonucleosis virus capsid protein VP2 (ORF VP2) in specific tissues and at a certain stage of development depending on the type of chosen driver strains (GAL-UAS system) were obtained. The ORF VP2 transcription was examined at the imago stage after crossing the obtained transgenic Drosophila with the driver line expressing the inducer protein (GAL4) under control of actin promoter (the ORF VP2 expression is induced in all tissues of the first-generation Drosophila). It was demonstrated that the greater part of transcribed foreign RNA was represented by three spliced variants in which RNA fragments either between nucleotides 137 and 353 or between nucleotides 609 and 1925 were excised; the third spliced variant was represented by RNA lacking both introns. Using the next-generation sequencing (NGS) technique, the proportion of unspliced form relative to spliced variants of the analyzed RNA was assessed. It was shown that the ratio of unspliced form to the identified spliced variants of the analyzed RNA was approximately 1:6. It is suggested that splicing of viral RNA foreign to Drosophila can be a sort of defense mechanism preventing the large-scale production of the capsid protein, potentially hazardous to the host organism.

[Mammalian cell culture as a model for studying the intracellular traffic of densovirus proteins].

The intracellular localization of the fusion protein composed of green fluorescent protein (GFP) and one of the capsid proteins (namely VP1) of the German cockroach densovirus BgDV1 was investigated using the HeLa human cell culture. The intracellular localization of GFP was analyzed in a series of control experiments. Histochemical analysis with GFP antibodies showed that the fusion protein is localized exclusively inside the nucleus of cells because of the transitory expression of the corresponding vector constructions, whereas the GFP is located both in the nucleus and the cytoplasm. We can conclude that the signal of the nuclear localization of the capsid protein of the German cockroach densovirus is functionally active, not only within the cells of this insect but within the human cell culture as well. This observation extends the experimental possibilities for studying the genetic control of intracellular traffic of densovirus proteins.

[The intracellular localization of the regulatory proteins of the densovirus of German cockroach, Blattella germanica].

The intracellular localization of the regulatory proteins encoded by the genome of the densovirus of German cockroach was analyzed using western-blotting of nuclear and cytoplasmic extracts of the densovirus-infected passaging cells tissue culture BGE-2. Two of the three regulatory proteins, NS1 and NS3, were shown to possess mainly nuclear localization, while NS2 protein was distributed between the nucleus and cytoplasm. Data obtained provide new information necessary for prediction of the functions of densovirus regulatory proteins. Intracellular localization of NS3 protein was described for the densoviruses for the first time.

[Comparative characteristics of intergenic spacers of ribosomal RNA gene cluster in mosquitoes of the genus Culex (Diptera: Culicidae)].

Nucleotide sequences of intergenic spacer of ribosomal RNA gene cluster (rIGS) were identified in mosquitoes Culex modestus, Culex torrentium and Culex pipiens pallens. The level of interpopulation variability of the rIGS in the subspecies C. pipiens pipiens (form pipiens--mosquitoes that inhabit the open waters, and form molestus--mosquitoes that inhabit basements) living in Russia was estimated. No extensive repetitive sequences characteristic of the rIGS of all previously described species of mosquitoes were found within the rIGS of Culex mosquitoes. At the same time, evolutionarily conserved motifs and relatively short degenerate sequences of different classes of transposable elements, as well as multiple blocks of variable microsatellite repeats were identified. Our data demonstrated that the rIGS of Culex mosquitoes can be considered as a promising molecular marker for the analysis of population and phylogenetic relationships within this group of insects.

Molecular modeling of human lanosterol 14α-demethylase complexes with substrates and their derivatives.

Lanosterol 14α-demethylase (CYP51A1) is a key enzyme in sterol biosynthesis. In humans, this enzyme is involved in the cholesterol biosynthesis pathway. The majority of antifungal drugs are aimed at the inhibition of CYP51 in fungi. To elucidate the molecular mechanisms of highly specific protein-ligand recognition, we have developed a full-atomic model of human CYP51A1 and performed docking of natural substrates and their derivatives to the active site of the enzyme. The parameters of the binding enthalpy of substrates, intermediates, and final products of the reaction of 14α-demethylation were estimated using the MMPB(GB)SA algorithm. Dynamic properties and conformational changes of the protein globule upon binding of the ligand near the active site have been investigated by the molecular dynamics method. Our studies reveal that hydroxylated intermediate reaction products have a greater affinity than the initial substrates, which facilitates the multistage reaction without accumulation of intermediate products. The contribution to the free energy of steroid ligand binding of 30 amino acids forming the substrate-binding region of CYP51A1, as well as the influence of their substitutions to alanine on the stability of the protein molecule, has been clarified using alanine scanning modeling. We demonstrate that the most serious weakening of the binding is observed in the case of substitutions Y137A, F145A, V149A, I383A, and R388A. The results of molecular modeling are in agreement with the data obtained by analysis of primary sequences of representatives of the CYP51 family.

[First open reading frame protein (ORF1p) of the Blattella germanica R1 retroposon and phylogenetically close GAG-like proteins of insects and fungi contain RRM domains].

The rDNA locus of insects and other arthropods contains non-LTR retrotransposons (retroposons) that are specifically inserted into 28S rRNA genes. The most frequent retroposons are R1 and R2, but the mechanism of insertion and the functions of these mobile elements have not been studied in detail. A clone containing a full-length R1 retroposon copy was islated from the cosmid library of Blattella germanica genes and sequenced. The amino acid sequences encoded by ORF1 of the R1 retroposon were subjected to bioinformatic analysis. It was found that ORF1 of this mobile element encodes a protein (ORF1p) belonging to the superfamily of zinc finger (CCHC) retroviral nucleocapsid proteins and contains two conserved RRM domains (RNA-recognizing motifs) identified on the basis of analysis of the secondary structure of this protein. The discovery of RRM domains in ORF1p of R1 retroposons can contribute to the understanding of the mechanisms of their retrotransposition. We revealed a coiled-coil motif in the N-terminal region of R1 ORF1p, which is similar to the coiled-coil domain involved in homo- or heteromultimerization of proteins and in protein-protein interactions. The domain organization of homologous Gag-like proteins of retroposons in some insects and fungi was found to be similar to the structure established by us for R1 ORF1p of B. germanica.

[Analysis of the inheritance patterns of 5'-truncated copies of the German cockroach R2 retroposons in individual crosses].

The inheritance patterns of the 5'-truncated copies of R2 retroposons were analyzed in individual crosses of the German cockroach. The recombination level within the cluster of ribosomal RNA genes was determined. It was demonstrated that only the frequencies of individual variants of 5'-truncated retroposon copies are appropriate for population analysis rather than the patterns characterizing individual X chromosomes. The methodical approach used in the work is convenient for studying the genetic variation in ribosomal DNA multigene families.

[Domain organization of the ORF2 C-terminal region of the German cockroach retroposon R1].

Using cosmid vector, a gene library of German cockroach Blattella germanica was constructed. From this library, clones containing full-length copies of two subfamilies of R1 retroposons were selected. Retroposons R1 of German cockroach belonging to different subfamilies were shown to be different in domain organization of the ORF2 C-terminal region. For the first time, retroposons transmembrane domains were identified in the sequences of R1. It was demonstrated that two retroposon R1 subfamilies of German cockroach arose as a result of intragenomic divergence rather than via horizontal transfer of alien mobile element into cockroach genome. The differences in domain organization appeared not as a result of saltatory recombination processes, but as a consequence of gradual mutation accumulation, which led to either degeneration, or to domain formation.

[R1 and R2 retrotransposons of German cockroach Blattella germanica: comparative analysis of 5' truncated copies integrated into genome].

This is the first report providing results on identification, cloning, and sequencing of extended fragments (5'-truncated copies) of R1 and R2 retrotransposons integrated into Blattella germanica genome. Comparative structural analysis of the received clones revealed two distinct subfamilies of R1 elements. However, all B. germanica R1 clones have two common features: poly(T) tails and similar target site duplications. Nucleotide structure and organization of five sequenced R2 fragments was similar. Analysis of R2 nucleotide sequences revealed typical deletions at the 3'end of target sites and lack of homopolynucleotides tails.

[Length polymorphism of integrated copies of R1 and R2 retrotransposons in the German cockroach (Blattella germanica) as a potential marker for population and phylogenetic studies].

Using polymerase chain reaction technique with primers flanking target sites of retrotransposons R1 and R2, integrated copies of these transposable elements were amplified in various cockroach species (Blattodea). It was shown that each species has a unique pattern of "5'-undertranscripts" with the definite set of amplified fragments of different lengths. Intraspecies polymorphism was revealed in analysis of German cockroach specimens obtained upon individual mating. This is the first report providing results of identifying, cloning, and sequencing extended fragments (5'-truncated copies) of Blatella germanica R1 and R2 retrotransposons. It may be assumed that patterns of 5'-truncated copies of R1 and R2 elements can be used as markers in population and phylogenetic studies. Moreover, cloned and sequenced fragments will be employed in our further studies for screening of the German cockroach genomic library in order to detect full-length copies in this class transposable elements.

[Nuclear rDNA variability in laboratory strains of the German cockroach Blattella germanica L. (Blattellidae)].

The polymorphism of nuclear ribosomal DNA has been studied in male German cockroaches. The RFLP analysis has been used to identify seven types of HindIII fragments, the variation of which may serve as the basis for the differentiation of laboratory strains with respect to some population indices, including the average number of fragments per animal (N), the proportion of polymorphic loci (P), and the average pairwise similarity (APS). The interpopulation variation (FST) calculated for nine haplotypes is 53.85%.

[Protein-protein interactions of cytochromes P450 3A4 and 3A5 with their intermediate redox partners cytochromes b5].

Molecular interactions between proteins redox partners (cytochromes Р450 3А4, 3А5 and cytochrome b5) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes Р450 3А4 and 3А5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b5: the microsomal (b5mc) and mitochondrial (b5om) forms of this protein, as well as with 2 "chimeric" proteins, b5(om-mc), b5(mc-om). Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om. Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om, b5(om-mc), and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435  -0.350 V (vs. Ag/AgCl). Cytochrome b5mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 in the presence of its substrate testosterone.

[Detection and analysis of Tetrahymena pyriformis 26S ribosomal DNA domain sequences, differing in degree of evolutionary conservation]. / Vyiavlenie i analyz domenov posledovatel'nosti 26S ribosomnoi DNK Tetrahymena pyriformis, razlichaiushchikhsia po stepeni évoliutsionnogo konservatizma.

Domains of different evolutionary conservatism were defined in the 26S rDNA sequence of T. pyriformis. The fragment of studied DNA (1212 bp) showing high evolutionary conservatism was cloned. It was shown this fragment of DNA may be used to a probe for blot-hybridization analysis of the structure of rDNA from various taxa, protists to mammals. Superconservative and hypervariable domains were defined. The first are good for the primers for PCR analysis of rDNA from various taxa, the second--for species specific primers.

[A densovirus of German cockroach Blatella germanica: detection, nucleotide sequence and genome organization]. / Densovirus ryzhego tarakana Blatella germanica: obnaruzhenie, nukleotidnaia posledovatel'nost' i organizatsiia genoma.

A new Blattella germanica densovirus (BgDNV, Parvoviridae: Densovirinae, Densovirus) was found. Virus DNA and cockroach tissues infected with BgDNV were examined by electron microscopy. Virus particles about 20 nm in diameter were observed both in the nucleus and in the cytoplasm of infected cells. Virus DNA proved to be a linear molecule sized about 1.2 microns. The complete BgDNV genome was sequenced and analyzed. Five ORF were detected: two coded for structural capsid proteins and were on one DNA strand, and three coded for regulatory proteins and were on the other strand. Potential promoters and polyadenylation signals were identified. Structural analysis was performed for terminal inverted repeats containing extended palindromes. The genome structure of BgDNV was compared with that of other Parvoviridae.

[Cloning an extended palindromic sequence of Tetrahymena pyriformis ribosomal DNA]. / Klonirovanie protiazhennoi palindromnoi posledovatel'nosti ribosomnoi DNK Tetrahymena pyriformis.

Simple and effective method for isolation of native copies of ribosomal DNA (rDNA) of T. pyriformis by pulse field gel electrophoresis is suggested. Cloning of long palindrome sequence of rDNA from T. pyriformis in plasmid is described.

[Detection of highly conservative domains in the sequence of 17S ribosomal DNA from Tetrahymena pyriformis]. / Vyiavlenie vysokokonservativnykh domenov v posledovatel'nosti 17S ribosomal'noi DNK Tetrahymena pyriformis.

Domains differing in the degree of evolutionary conservatism were revealed within the 17S ribosomal DNA (rDNA) sequence from Tetrahymena pyriformis by means of computer analysis. The method proposed allows evolution of rDNA structure to be studied in eukaryotes. Superconservative regions revealed within the DNA sequence tested can be used to construct universal primers for studying rDNA structure in various taxa by means of polymerase chain reaction (PCR).

[Genetic variation of nuclear ribosomal DNA of the insect order of cockroaches (Blattaria): phylogenetic analysis of restriction fragment length polymorphism]. / Geneticheskaia izmenchivost' iadernoi ribosomnoi DNK nasekomykh otriada tarakany (Blattaria): filogeneticheskii analiz polimorfizma dlin restriktsionnykh fragmentov.

Species-specific characteristics of nuclear ribosomal DNA (rDNA) have been determined for the first time for six insect species of order Blattaria (Insecta, Dictyoptera) with the use of restriction analysis and Southern blotting. Probes containing highly conserved fragments of 18 S- and 28 S-like genes of the ribosomal RNA (rRNA) of Tetrahymena pyriformis were tested in this study. The genetic similarity tree constructed for the species studied agrees with evidence from classical taxonomy based on morphological, ecological, anatomical, and physiological characters.

[Analysis of expression of foreign genetic material under the control of heterogeneous promoters in the process of early development of the loach (Misgurnus fossilis L.)]. / Analiz ékspressii chuzherodnogo geneticheskogo materiala pod kontrolem geterogennykh promotorov v protsesse rannego razvitiia v'iuna (Misgurnus fossilis L.).

Promoters normally active in the nuclei of evolutionarily distant from fishes taxa-bacteria, insects, and Protozoa-are shown to be able to regulate heterologous genes during early development of the loach. It is shown that particular DNA sequences can drastically influence the function of eukaryotic promoters, enhancing the expression level.

[Intraspecies polymorphism for the structural organization of ribosomal gene cluster in Blattella germanica]. / Vnutrividovoi polimorfizm strukturnoi organizatsii klastera ribosomnykh genov Blattella germanica.

Intraspecific HindIII restriction fragment length polymorphism (RFLP) for the structural organization of the ribosomal gene cluster was described in Blattella germanica. In this species, homologous chromosomes were shown to contain ribosomal DNA (rDNA) repeats that differ in structure. The pattern of inheritance was determined for various structural variants of rDNA.