Flexibility difference between double-stranded RNA and DNA as revealed by gel electrophoresis

A systematic study of agarose gel electrophoresis of double-stranded RNA in the kilobase range of sizes was performed. The dsRNA to dsDNA relative mobility was found to depend on gel concentration: in low density gels RNA moves slower and in high density gels - faster than DNA of the same molecular size. The electrophoretic differences were interpreted within the reptation theory to be mainly due to the molecular stiffness differences. The dsRNA persistence length was roughly estimated to be about twice as great as that of DNA.     

Genome comparisons of three closely related flax species and their hybrids with chromosomal and molecular markers

Chromosome C-banding patterns were analyzed in three closely related flax species (Linum usitatissimum L., 2n = 30; L. angustifolium Huds., 2n = 30; and L. bienne Mill., 2n = 30) and their hybrids. In each case, the karyotype included metacentrics, submetacentrics, and one or two satellite chromosomes. Chromosomes of the three flax species were similar in morphology, size (1-3 microns), and C-banding pattern and slightly differed in size of heterochromatic regions. In all accessions, a large major site of ribosomal genes was revealed by hybridization in the pericentric region of a large metacentric. A minor 45S rDNA site was observed on a small chromosome in L. usitatissimum and L. bienne and on a medium-sized chromosome in L. angustifolium. Upon silver staining, a nucleolus-organizing region (NOR) was detected on a large chromosome in all species. In L. angustifolium, an Ag-NOR band was sometimes seen on a medium-sized chromosome. In the karyotypes of interspecific hybrids, silver-stained rDNA loci were observed on satellite chromosomes of both parental species. RAPD analysis with 22 primers revealed a high similarity of the three species. The greatest difference was observed between L. angustifolium and the other two species. The RAPD patterns of L. bienne and L. usitatissimum differed in fewer fragments. A dendrogram of genetic similarity was constructed for the three flax species on the basis of their RAPD patterns. Genome analysis with chromosome and molecular markers showed that L. bienne must be considered as a subspecies of L. usitatissimum rather than a separate species. The three species were assumed to originate from a common ancestor, L. angustifolium being closest to it.     

Whole-genome experimental identification of insertion/deletion polymorphisms of interspersed repeats by a new general approach

A new experimental technique for genome-wide detection of integration sites of polymorphic retroelements (REs) is described. The technique allows one to reveal the absence of a retroelement in an individual genome provided that this retroelement is present in at least one of several other genomes under comparison. Since quite a number of genomes are compared simultaneously, the search for polymorphic REs insertions is very efficient. The technique includes two whole-genome selective PCR amplifications of sequences flanking REs: one for a particular genome and another one for a mixture of ten different genomes. A subsequent subtractive hybridization of the obtained amplicons with DNA of a particular genome as driver results in isolation of polymorphic insertions. The technique was successfully applied for identification of 41 new polymorphic human AluYa5/Ya8 insertions. Among them, 18 individual Alu elements first sequenced in this work were not found in the available human genome databases. This result suggests that significant part of polymorphic REs were not identified during genome sequencing and remain to be detected and characterized. The proposed method does not depend on preliminary knowledge of evolutionary history of retroelements and can be applied for identification of insertion/deletion polymorphic markers in genomes of different species.     

Cell line fingerprinting using retroelement insertion polymorphism

Human cell lines are an indispensable tool for functional studies of living entities in their numerous manifestations starting with integral complex systems such as signal pathways and networks, regulation of gene ensembles, epigenetic factors, and finishing with pathological changes and impact of artificially introduced elements, such as various transgenes, on the behavior of the cell. Therefore, it is highly desirable to have reliable cell line identification techniques to make sure that the cell lines to be used in experiments are exactly what is expected. To this end, we developed a set of informative markers based on insertion polymorphism of human retroelements (REs). The set includes 47 pairs of PCR primers corresponding to introns of the human genes with dimorphic LINE1 (L1) and Alu insertions. Using locus-specific PCR assays, we have genotyped 10 human cell lines of various origins. For each of these cell lines, characteristic fingerprints were obtained. An estimated probability that two different cell lines possess the same marker genotype is about 10-18. Therefore, the proposed set of markers provides a reliable tool for cell line identification.     

Ultrafine structure of salivary glands of the chicken mite Dermanyssus gallinae (Acarina, Gamasina, Dermanyssidae)

Using the method of transmission electron microscopy, structure of salivary glands of the chicken mite Dermanyssus gallinae (De Geer, 1778) is studied. Structure of the glands and of their ducts is described. The cellular composition, ultrastructural characteristics of secretion, and peculiarities of its release from cells are revealed.     

Chromosome Localization of 5S and 45S Ribosomal DNA in the Genomes of Linum L. Species of the Section Linum (Syn. Protolinum and Adenolinum)

Fluorescence in situ hybridization (FISH) was for the first time used to study the chromosomal location of the 45S (18S–5.8S–26S) and 5S ribosomal genes in the genomes of five flax species of the section Linum (syn. Protolinum and Adenolinum). In L. usitatissimum L. (2n = 30), L. angustifolium Huds. (2n = 30), and L. bienne Mill. (2n = 30), a major hybridization site of 45S rDNA was observed in the pericentric region of a large metacentric chromosome. A polymorphic minor locus of 45S rDNA was found on one of the small chromosomes. Sites of 5S rDNA were colocalized with those of 45S rDNA, but direct correlation between signal intensities from the 45S and 5S rDNA sites was observed only in some cases. Other 5S rDNA sites mapped to two chromosomes in these flax species. In L. grandiflorum Desf. (2n = 16) and L. austriacum L. (2n = 18), large regions of 45S and 5S rDNA were similarly located on a pair of homologous satellite-bearing chromosomes. An additional large polymorphic site of 45S and 5S rDNA was found in the proximal region of one arm of a small chromosome in the L. usitatissimum, L. angustifolium, and L. bienne karyotypes. The other arm of this chromosome contained a large 5S rDNA cluster. A similar location of the ribosomal genes in the pericentric region of the pair of satellite-bearing metacentrics confirmed the close relationships of the species examined. The difference in chromosomal location of the ribosomal genes between flax species with 2n = 30 and those with 2n = 16 or 18 testified to their assignment to different sections. The use of ribosomal genes as chromosome markers was assumed to be of importance for comparative genomic studies in cultivated flax, a valuable crop species of Russia, and in its wild relatives.     

Effect of the 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole residue on the stability of DNA duplexes and triplexes.

3-Nitropyrrole (M) was introduced as a non-discriminating 'universal' base in nucleic acid duplexes by virtue of small size and a presumed tendency to stack but not hydrogen bond with canonical bases. However, the absence of thermally-induced hyperchromic changes by single-stranded deoxyoligomers in which M alternates with A or C residues shows that M does not stack strongly with A or C nearest neighbors. Yet, the insertion of a centrally located M opposite any canonical base in a duplex is sometimes even less destabilizing than that of some mismatches, and the variation in duplex stability is small. In triplexes, on the other hand, an M residue centrally located in the third strand reduces triplex stability drastically even when the X.Y target base pair is A.T or G. C in a homopurine. homopyrimidine segment. But, when the target duplex opposition is M-T and the third strand residue is T, the presence of M in the test triplet has little effect on triplex stability. Therefore, a lack of hydrogen bonding in an otherwise helix-compatible test triplet cannot be responsible for triplex destabilization when M is the third strand residue. Thus, M is non-discriminating and none-too-destabilizing in a duplex, but in a triplex it is extremely destabilizing when in the third strand.     

Localization of DNA probes for human ribosomal genes on barley chromosomes]

To estimate the possibility of plant genome mapping using human genome probes, the probes fluorescent in situ hybridization (FISH) of human 18S-28S rDNA (clon 22F9 from the LA-13NCO1 library) was carried out on chromosomes of the spring barley Hordeum vulgare L. As a control, wheat rDNA probe (clon pTa71) was taken. Hybridization of the wheat DNA probe revealed two major labelling sites on mitotic barley chromosomes 5I (7H) and 6I (6H), as well as several minor sites. With the human DNA probe, signals were detected in the major sites of the ribosomal genes on chromosomes 5I (7H) and 6I (6H) only when the chromosome preparations were obtained using an optimized technique with obligatory pepsin treatment followed by hybridization. Thus, this study demonstrates that physical mapping of plant chromosomes with human DNA probes that are 60 to 75% homologous to the plant genes is possible. It suggests principal opportunity for the FISH mapping of plant genomes using probes from human genome libraries, obtained in the course of the total sequencing of the human genomes and corresponding to the coding regions of genes with known functions.     

Genetic and epigenetic changes of rDNA in a synthetic allotetraploid, Aegilops sharonensis x Ae. umbellulata.

The synthetic allotetraploid Aegilops sharonensis x Ae. umbellulata (genomic formula S(sh)U) was used to study inheritance and expression of 45S rDNA during early stages of allopolyploid formation. Using silver staining, we revealed suppression of the NORs (nucleolar organizing regions) from the S(sh) genome in response to polyploidization. Most allopolyploid plants of the S(2)-S(4) generations retained the chromosomal location of 45S rDNA typical for the parental species, except for two S(3) plants in which a deletion of the rDNA locus on one of the homologous 6S(sh) chromosomes was revealed. In addition, we found a decrease in NOR signal intensity on both 6S(sh) chromosomes in a portion of the S(3) and S(4) allopolyploid plants. As Southern hybridization showed, the allopolyploid plants demonstrated additive inheritance of parental rDNA units together with contraction of copy number of some rDNA families inherited from Ae. sharonensis. Also, we identified a new variant of amplified rDNA unit with MspAI1 restriction sites characteristic of Ae. umbellulata. These genetic alterations in the allopolyploid were associated with comparative hypomethylation of the promoter region within the Ae. umbellulata-derived rDNA units. The fast uniparental elimination of rDNA observed in the synthetic allopolyploid agrees well with patterns observed previously in natural wheat allotetraploids.