[Production of mycobacterial antigenes merged with cellulose binding protein domain in order to produce subunit vaccines against tuberculosis].

Protein genes Ag85A, Esat-6, and Cfp10 of Mycobacterium tuberculosis were sequenced using the database GenBank to implement selection and synthesis of primer pairs of given genes. PCR was used to obtain target amplicons of the genes. Chromosome DNA of M. tuberculosis H37Rv was used as the DNA amplification matrix. The PCR products were obtained using the plasmid pQE6, cloned, and amplified in the Escherichia coli M15 strain. Chimere products containing mycobacterial genes and cellulose binding protein domain (CBD), were obtained using the plasmid treated with restriction endonucleases. CBD fragment obtained using similar treatment of the ptt10 plasmid. The plasmids containing merged sequences of mycobacterial genes-antigenes and CBD were selected. The 3 mycobacterial genes were expressed in the E. coli M15 cells resulting in biosynthesis of corresponding recombinant proteins of expected molecular weight. Concentration of CBD, Cfp10-CBD, Ag85A-CBD, and ESAT6-CBD was 20%, 15%, and 15% total protein, respectively. The resulting chimere proteins provide high affinity for cellulose and high stability. Immobilization of CBD-containing recombinant proteins proceeds as one-stage process providing target protein purification and adsorption on cellulose. The vaccines produced using this technology are inexpensive because of low cost of cellulose sorbents as well as simultaneous use of cellulose for purification and immobilization of protein. Many cellulose preparations are not toxic, biocompatible, and widely used in medicine.

[Interaction of bacteria of the genus Bartonella with the host: inhibition of apoptosis, induction of proliferation, and formation of tumors].

The bacteria of the genus Bartonella are the causative agents for earlier not diagnosed or re-emergent diseases of the humans, danger of which increases in relation with increasing number of persons with the disturbed immune status. Bartonellae are intracellular parasites, the places of their habitation in the humans and animals are the endothelial cells of blood vessels and erythrocytes. The modern data concerning major factors of the Bartonellae virulence and host-bacteria interactions were considered and discussed in this article. The induction of the type IV secretion system, effector protein transmission, inhibition of the endothelial cells apoptosis, and induction of their proliferation lead to formation of new blood vessels and tumors.

[Molecular genetic methods of typing of the Bartonellae].

The primer systems for the PCR detection of four house-keeping genes of bartonellae in clinical material were developed and tested. The tactics of the species RFLP typing was also developed and tested. The scheme of the species RFLP typing of bartonellae was tested using as an example two strains for the first time isolated in Russia from patients with endocarditis and fever of uncertain origin. The results of the typing were supported by sequencing of the amplicons obtained. According to the sequencing the isolates were attributed to the sub species Bartonella vinsonii, subsp. arupensis. The necessity of molecular epidemiological analysis of bartonelloses in Russia was substantiated.

[Wild small mammals are the reservoir hosts of the Bartonella genus bacteria in the south of Moscow region].

A total of 103 blood samples collected from wild small mammals captured in the Prioksko-Terrasny Reserve on the south of Moscow region were studied to determine the bartonellae prevalence. The examined species were the yellow-necked mice Apodemus flavicollis (35 samples), the European wood mouse Apodemus uralensis (10 samples), the bank vole Clethrionomys glareolus (51 samples), the house mouse Mus musculus (3 samples), the common vole Microtus arvalis (2 samples), and the shrew Sorex araneus (2 samples). Initially, we obtained 76 bacterial Bartonella-like isolates after plating onto the surface of the solid nutrient media. 66 of them were PCR-positive at least for three of four targets, gltA, ftsZ, ribC and 16S RNA. Thus, the percentage of the infection in the studied community was 64%. Subsequent RFLP assay showed that obtained isolates belonged to the Bartonella grahamii and/or B. taylorii species. In 7 cases we found both bartonellae species in one animal. These data were confirmed by direct sequencing of four ftsZ, four ribC and two gltA amplicons. According to our data, there is no any marked host specificity for these bartonellae species. Now we have laid the bartonellae strain collection consisting of 31 isolates. To our knowledge, this is the first investigation of the bartonellae prevalence in wild small mammals performed in Russia. The comparison of our data with those obtained by European researchers and issues of coinfection by different bartonellae species and host specificity are discussed.

[Overexpression of a novel gene toothrin in Drosophila].

A newly found locus of the Drosophila melanogaster genome, named toothrin (tth) has been used to study the role of the conserved domain 2/3 of genes from the d4 family. In contrast to the 2/3 domain of all vertebrates studied (including humans), which is always accompanied by the d4 domain, the tth gene contains the sequence encoding the 2/3 domain but lacks that encoding the d4 domain. The tth gene overexpression has been studied using the two-component system UAS-GAL4. It has been demonstrated that the tth overexpression at the third-instar larval (prepupal) stage decreases survival rate, simultaneously causing a substantial deceleration of development in Drosophila. It is known that the change of developmental stages in Drosophila is controlled by the rates of the expression of ecdysteroid and juvenile hormones (JHs). It is supposed that the overexpression of the tth gene causes either a shift in the ecdysterone-to-JH ratio (through a decreased JH decay rate or a delayed initiation of ecdysone synthesis) or a deceleration of the release of ecdysterones synthesized.

[The origin of heterochromatin in eukaryotes]. / Proiskhozhdenie geterokhromatina u éukariot.

This study is an attempt to reconstruct the stages of the evolution of heterochromatin in eukaryotes. According to the hypothesis put forward in the work, the origin of satellite DNAs (stDNAs) was directly related to certain functional characteristics of DNA polymerases, and stDNAs themselves are products of accidental slippage at replication initiation sites. Even at the moment when the stDNAs precursors (protosatellites) appeared, they had properties of selfish DNA. Therefore, specific complex mechanisms of genetic control of their replication and recombination have developed in evolution to restrict the spread of these DNAs over the genome. The host control over protosatellites has led to the appearance of the main heterochromatic characteristics in them, such as late replication, decreased recombination, and denser chromatin packing compared to euchromatin. The next stage of heterochromatin evolution led to the union of protosatellite clusters and ordinary genes if late replication was necessary for these genes or if gene complexes already formed required protection from the destructure effect of crossing over. The known cases of location of certain genes in heterochromatic blocks in Drosophila melanogaster, the eukaryote that has been best studied genetically, confirm this hypothesis.

Xenografts of embryonic nerve tissue from Drosophila neuromutants stimulate development of neural homografts in rat brain and block glial scar formation.

The influence of xenografts of Drosophila melanogaster embryonic nerve cells on the development of embryonic neurohomografts in the adult rat brain has been investigated. Embryonic nerve cells, marked with bacterial galactosidase gene (lacZ) from D. melanogaster strain with a mutation in the Delta locus, were transplanted into adult rat brain. Drosophila cells were easily identifiable in brain histological sections by X-gal staining. Xenografts survived for at least 2-3 weeks in the recipient brain after the operation to be subsequently attacked by macrophages. Importantly, no glial scar was formed around the xenograft. The addition of Drosophila embryonic nerve cells to a homograft of rat embryonic neural tissue facilitated the survival and development of this homograft by blocking the glial scar formation, stimulating vascularization of the graft area and differentiation of the implanted embryonic nerve cells.

[Expression of genes coding NGF and BDNF human proteins in embryos of transgenic stocks of Drosophila melanogaster]. / Ekspressia genov kodiruiushchikh belki NGF i BDNF cheloveka, v émbrionakh transgennykh linii Drosophila melanogaster.

Transgenic Drosophila stocks were selected. These stocks contained human ngf and bdnf genes under Drosophila heat-shock promoter. The expression of foreign genes during Drosophila development was demonstrated with Northern and whole mount hybridization.

[Effect of the foreign gene GDNF on development of homo- and xenografts in the rat brain]. / Vliianie chuzherodnogo gena GDNF na razvitie transplantatov i ksenotransplantatov v mozge krys.

A transgenic line of Drosophila melanogaster was selected which carried the following genes: Delta, lacZ (for bacterial galactosidase), and human GDNF (for glial cell line-derived neurotrophic factor). Drosophila neuroectodermal embryonic cells were transplanted with the embryonic neurohomografts into the occipital brain region of an adult rat. Xenografts were found to block scar formation at the graft-host tissue boundary, stimulated homograft development (so that it was twice as large as the control homograft transplanted alone with no xenograft added), and noticeably improved vascularization of the homograft area.

[Transposition of the bobbed locus in Drosophila melanogaster]. / Transpozitsii lokusa bobbed u Drosophila melanogaster.

Due to the complete absence of ribosomal DNA (genetic symbol bb-), the Xbb- chromosome of Drosophila is lethal both in homozygous conditions and in compound with the Xbb- chromosome. However, in the cross between the C(1)RM/Ybb- females and the Xbb-/BSYbb+ males, characterized by the development of lethal Xbb-/Ybb- zygotes, two fertile males were detected. These males possessed all the markers of the Xbb- chromosome but lacked the Y chromosome BS marker. Genetic analysis of their progeny showed that genes responsible for restoration of viability and fertility of these exceptional males were associated with the X chromosome. The crossover tests showed that in one case these genes were tightly linked to the w locus (the bbAM1 allele), and in the second case they were located 12.6 map units to the right of the Tu locus (the bbAM7 allele). It has also been shown that the bb locus was transposed to the X chromosome within the short arm of Y chromosome. Transposition of the BSYbb+ chromosome-specific rDNA sequences to the X chromosome was confirmed by means of Southern blotting. These data indicate that replacement of the bb locus is realized by transposition rather than recombination.

[Molecular analysis of certain derivatives of mutants with the unstable white-starka allele]. / Molekuliarnyi analiz nekororykh proizvodnykh mutantov nestabil'nogo allelia white-starka.

Molecular analysis of several mutations of the white locus was performed. The mutations were obtained as a result of mutagenesis in the unstable white-starka locus described previously. A large insert (approximately 15 kb in length) downstream of the first exon of the white gene was shown to be partly excised. Simultaneous excisions of various regions from the natural regulatory region of the white locus were also observed. Restriction enzyme analysis showed that the observed mutations and reversions resulted from large deletions from the regulatory gene region. Southern and Northern blot hybridization showed the presence of unknown regulatory regions within and outside the insert.

[Molecular nature of the unstable white-starka allele]. / Molekuliarnaia priroda nestabil'nogo allelia white-starka.

In 1992, Bashkirov et al. described the case of occurrence in line C(1)RM, yw/0; Dp(1; 3) wvco/+ of a unique female with light, uniformly colored eyes. This trait was not inherited together with Dp(1; 3) wvco but was linked to the X chromosome. We denoted this new allele as white-starka (wstr). It proved to be unstable, demonstrating mutational transitions to other allelic states with frequencies of 10(-3) to 10(-5). Southern blot analysis showed that the wstr mutation was induced by an insertion of an unknown sequence with a length approximately evaluated by the authors in 1992 as 7 kb. Using a wider spectrum of restriction endonucleases and a prolonged gel running, we were able to estimate the size of this insertion more precisely. According to our data, in the case of wstr we are dealing with an insertion in the locus white of a sequence 15.1 kb long. As seen from our restriction map of the allele wstr, this insertion does not resemble retroposons with long terminal repeats, but is possibly similar to LINE mobile elements of Het-A and TART types, characteristic for heterochromatin.

A relatively small 5' regulatory region of esterase S gene of Drosophila virilis determines the specific expression as revealed in transgenic experiments.

Expression of the esterase S gene of Drosophila virilis was studied in transgenic experiments. Truncated genomic copy of this gene including 400 bp of 5' regulatory region was integrated into the genome of Drosophila melanogaster. The products of the transferred gene were detected. It was found that strict temporal and tissue specificity of the esterase S gene expression is conserved in transformed flies. The results suggest that this specificity is evidently determined by the regulatory region of the esterase S gene and controlled by cis mechanism.

The expression of esterase S gene of Drosophila virilis in Drosophila melanogaster.

Drosophila melanogaster was transformed with the esterase S gene from Drosophila virilis. This gene is strongly activated in ejaculatory bulbs of mature males of Drosophila virilis. The closely related gene from Drosophila melanogaster is activated in ejaculatory ducts. The tissue- and stage-specific expression of incomplete genomic copy of the esterase S gene integrated into the Drosophila melanogaster genome is the same as in Drosophila virilis. These data show that tissue and stage specificity is determined by relatively small 5' regulatory region of the esterase S gene. The comparison between deduced amino-acid sequences of the esterase S of Drosophila virilis and esterase 6 of Drosophila melanogaster was performed. These sequences revealed 50% homology.