Self-catalyzed site-specific depurination of G residues mediated by cruciform extrusion in closed circular DNA plasmids

A major variety of "spontaneous" genomic damage is endogenous generation of apurinic sites. Depurination rates vary widely across genomes, occurring with higher frequency at "depurination hot spots." Recently, we discovered a site-specific self-catalyzed depurinating activity in short (14-18 nucleotides) DNA stem-loop-forming sequences with a 5'-G(T/A)GG-3' loop and T·A or G·C as the first base pair at the base of the loop; the 5'-G residue of the loop self-depurinates at least 10(5)-fold faster than random "spontaneous" depurination at pH 5. Formation of the catalytic intermediate for self-depurination in double-stranded DNA requires a stem-loop to extrude as part of a cruciform. In this study, evidence is presented for self-catalyzed depurination mediated by cruciform formation in plasmid DNA in vitro. Cruciform extrusion was confirmed, and its extent was quantitated by digestion of the plasmid with single strand-specific mung bean endonuclease, followed by restriction digestion and sequencing of resulting mung bean-generated fragments. Appearance of the apurinic site in the self-depurinating stem-loop was confirmed by digestion of plasmid DNA with apurinic endonuclease IV, followed by primer extension and/or PCR amplification to detect the endonuclease-generated strand break and identify its location. Self-catalyzed depurination was contingent on the plasmid being supercoiled and was not observed in linearized plasmids, consistent with the presence of the extruded cruciform in the supercoiled plasmid and not in the linear one. These results indicate that self-catalyzed depurination is not unique to single-stranded DNA; rather, it can occur in stem-loop structures extruding from double-stranded DNA and therefore could, in principle, occur in vivo.     

An electron microscopic study of Borrelia in the body of the female ixodid tick Ixodes persulcatus

Borrelia burgdorferi s. lato in naturally infected females of tick Ixodes persulcatus were examined by transmission electron microscopy. The Borreliae were found in midgut and ovary. Location and ultrastructure of bacteria indicate extracellular migration through the midgut epithelium as a preferential way. In gonad, the borreliae intracellular situate in ovarian epithelium and oocytes before and at the beginning of vitellogenesis. The demonstration of numerous spirochetes in the oocytes provides the support for transovarial transmission of the agent. Two morphological types of borreliae were observed.     

The Diversity of Karyotypes and Genomes within Section Syllinum of the Genus Linum (Linaceae) Revealed by Molecular Cytogenetic Markers and RAPD Analysis

The wide variation in chromosome number found in species of the genus Linum (2n = 16, 18, 20, 26, 28, 30, 32, 36, 42, 72, 84) indicates that chromosomal mutations have played an important role in the speciation of this taxon. To contribute to a better understanding of the genetic diversity and species relationships in this genus, comparative studies of karyotypes and genomes of species within section Syllinum Griseb. (2n = 26, 28) were carried out. Elongated with 9-aminoacridine chromosomes of 10 species of section Syllinum were investigated by C- and DAPI/С-banding, CMA and Ag-NOR-staining, FISH with probes of rDNA and of telomere repeats. RAPD analysis was also performed. All the chromosome pairs in karyotypes of the studied species were identified. Chromosome DAPI/C-banding patterns of 28-chromosomal species were highly similar. Two of the species differed from the others in chromosomal location of rDNA sites. B chromosomes were revealed in all the 28-chromosomal species. Chromosomes of Linum nodiflorum L. (2n = 26) and the 28-chromosomal species were similar in DAPI/C-banding pattern and localization of several rDNA sites, but they differed in chromosomal size and number. The karyotype of L. nodiflorum was characterized by an intercalary site of telomere repeat, one additional 26S rDNA site and also by the absence of B chromosomes. Structural similarities between different chromosome pairs in karyotypes of the studied species were found indicating their tetraploid origin. RAPD analysis did not distinguish the species except L. nodiflorum. The species of section Syllinum probably originated from a common tetraploid ancestor. The 28-chromosomal species were closely related, but L. nodiflorum diverged significantly from the rest of the species probably due to chromosomal rearrangements occurring during evolution.     

A tissue-specific decrease in the pre-mRNA level of L1- and alu-containing alleles of human genes

LINE1 and Alu retroelements occupy approximately 17 and 13% of the human genome, respectively. They include the evolutionarily youngest element groups Ta-L1, AluYa5, and AluYb8, many inserts of which are polymorphous in the Homo sapiens population. Despite the data on the ability of L1 and Alu elements to cause various modifications of the genome, the effects of these retroelements on gene expression have yet not been studied. Using the RT PCR method, we analyzed the pre-mRNA (heterogeneous nuclear RNA) content of allele pairs of four genes in five human cell lines, heterozygous with respect to intronic inserts of L1 and Alu elements. We showed for the first time a tissue-specific decrease in the pre-mRNA content of the gene allele bearing L1 or Alu inserts relative to the other allele of the same gene lacking the retroelement.     

Analysis of tyrphostin AG1478 effect on behavior of internalized EGF receptor at different stages of endocytosis

In the work, the effect of tyrphostin AG1478, a specific inhibitor of the receptor tyrosine kinase, on the behavior of an internalized EGF receptor at different stages upon the stimulation of endocytosis has been analyzed. It was found that tyrphostin added 30 min after the stimulation of endocytosis resulted in recycling of a significant portion of ¹²⁵I-EGF onto the cell surface. This portion decreased with time. EGF-receptor complexes, which are recycled under the action of AG1478, did not dissociate, possibly due to the ability of tyrphostin AG1478 to initiate receptor oligomerization in the absence of ligand and, therefore, probably affect dissociation constants. It was found that only a portion of the EGF receptor localized in early endosomes was able to recycle upon TK inhibition. The addition of inhibitor 30 and 60 min after the stimulation of endocytosis resulted in a decrease in the labeled EGF degradation. At early stages, internalized EGF-receptor complexes were mostly blocked in early endosomes, while, at late stages, their accumulation occurred in incompletely matured late endosomes. These data indicate that there is the late endocytic stage transition that depends on the receptor TK. Furthermore, the addition of tyrphostin after 90 min of endocytosis did not lead to a decrease, but rather an increase in degradation, which indicates the existence of mechanisms that create a temporal window during which receptor TK can carry out functions that are not directly connected with endocytosis.     

Most recent AluY insertions in human gene introns reduce the content of the primary transcripts in a cell type specific manner

Being the most effectively transposed primate-specific SINEs, Alu elements are present in more than one million copies in the human genome and include most recently transposed subsets of AluY elements that are polymorphic in humans. Although Alu elements are commonly thought to play an essential role in shaping and functioning of primate genomes, the understanding of the impact of recent Alu insertions on human gene expression is far from being comprehensive. Here we compared hnRNA contents for allele pairs of genes heterozygous for AluY insertions in their introns in human cell lines of various origins. We demonstrated that some AluY insertions correlated with decreased content of the corresponding hnRNAs. The effect observed does not depend on sequences of Alu elements and their orientation but is likely to be cell type specific.     

Genome differentiation in Aegilops. 3. Evolution of the D-genome cluster

 Six polyploid Aegilops species containing the D genome were studied by C-banding and fluorescence in situ hybridization (FISH) using clones pTa71 (18S-5.8S-26S rDNA), pTa794 (5S rDNA), and pAs1 (non-coding repetitive DNA sequence) as probes. The C-banding and pAs1-FISH patterns of Ae. cylindrica chromosomes were identical to those of the parental species. However, inactivation of the NOR on chromosome 5D with a simultaneous decrease in the size of the pTa71-FISH site was observed. The N^v and D^v genomes of Ae. ventricosa were somewhat modified as compared with the N genome of Ae. uniaristata and the D genome of Ae. tauschii. Modifications included minor changes in the C-banding and pAs1-FISH patterns, complete deletion of the NOR on chromosome 5D^v, and the loss of several minor 18S-5.8S-26S rDNA loci on N^v genome chromosomes. According to C-banding and FISH analyses, the D^cr1 genome of Ae. crassa is more similar to the D^v genome of Ae. ventricosa than to the D genome of Ae. tauschii. Mapping of the 18S-5.8S-26S rDNA and 5S rDNA loci by multicolor FISH suggests that the second (X^cr) genome of tetraploid Ae. crassa is a derivative of the S genome (section Emarginata of the Sitopsis group). Both genomes of Ae. crassa were significantly modified as the result of chromosomal rearrangements and redistribution of highly repetitive DNA sequences. Hexaploid Ae. crassa and Ae. vavilovii arose from the hybridization of chromosomal type N of tetraploid Ae. crassa with Ae. tauschii and Ae. searsii, respectively. Chromosomal type T1 of tetraploid Ae. crassa and Ae. umbellulata were the ancestral forms of Ae. juvenalis. The high level of genome modification in Ae. juvenalis indicates that it is the oldest hexaploid species in this group. The occurrence of hexaploid Ae. crassa was accompanied by a species-specific translocation between chromosomes 4D^cr1 and 7X^cr. No chromosome changes relative to the parental species were detected in Ae. vavilovii, however, its intraspecific diversity was accompanied by a translocation between chromosomes 3X^cr and 3D^cr1. Received July 24, 2001 Accepted October 1, 2001     

Comparative Genome Analysis in Two Flax Species by C-Banding Patterns

C-banding patterns of the karyotypes of two closely related wild flax species, Linum austriacumL. (2n= 18) and Linum grandiflorumDesf. (2n= 16), were studied. The karyotypes of both species were similar in the chromosome morphology and size. In each species, metacentric and acrocentric chromosomes (1.7–4.3 m) and one satellite chromosome were observed. In the karyotypes of the species studied, all homologous chromosome pairs were identified, and quantitative idiograms were constructed. Eight chromosome pairs in the two species had similar C-banding patterns. A low level of intraspecific polymorphism in the intercalary and telomeric C-bands was shown in both species. The results indicate that the genomes of two flax species originated from one ancestral genome with the basic chromosome number of 8 or 9. Apparently, the duplication or loss of one chromosome with subsequent redistribution of the chromosome material in the ancestral form resulted in the divergence into two species,L. austriacumL. and L. grandiflorumDesf. A considerable similarity of chromosomes in these species provides evidence for their close phylogenetic relatedness, which makes it possible to place them in one section within the Linumgenus.     

Ultrastructure of salivary glands of the chicken red mite <Emphasis Type="Italic">Dermanyssus gallinae</Emphasis> (Acarina,Gamasina, Dermanyssidae)

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