[Analysis of point mutations in BRCA1 gene using hybridization on hydrogel microchips].

Germ-line mutations in BRCA1 gene account for a substantial proportion of inherited breast and ovarian cancers. Identification of these mutations allows molecular diagnosis for breast cancer susceptibility. We have developed method for identification of 185delAG, 300T>G, 4153delA, 4158A>G and 5382insC mutations in BRCA1 gene using hybridization with microarray of geL-immobilized oligonucleotides (microchip). The microchip was tested with 36 control samples, carrying the above-mentioned mutations and 65 clinical cases with breast cancer. Our data demonstrated that developed microchip can be very effective and realible tool, easily introduced in ordinary medical and genetic laboratories.

Transformation of methylotrophic yeast Hansenula polymorpha: cloning and expression of genes.

We developed a host-vector system for transformation and gene cloning experiments using the methylotrophic yeast Hansenula polymorpha. Regeneration of protoplasts in a medium containing polyethylene glycol before plating made transformation more efficient and reproducible (2 to 3 x 10(4) micrograms DNA). The frequency of transformation was significantly lower when dominant resistance marker Cup1r was used for transformant selection. The transformation system developed was used to clone the DNA fragment which complements functionally the defect in the dihydroxyacetone kinase (DHAK*) activity of a H. polymorpha mutant strain. The DNA insert isolated was shown to increase by up to ten times the activity of DHAK in transformants carrying recombinant plasmids. When recombinant plasmids were introduced into S. cerevisiae, the transformants obtained acquired the ability to grow on the medium with dihydroxyacetone as a sole carbon source and the activity of DHAK was observed.

Methods for improving the reliability and information content of chromatographic analyses of biologically active compounds.

A series of methods was developed for improving the reliability of chromatographic analyses and to augment their information content and reproducibility. In high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) the possibility of utilizing azeotropic and isobaric solvent systems was substantiated. To control variations of the composition of the eluent systems during storage and analysis in TLC and HPLC, gas chromatography (GC) was found to be applicable. To improve the reliability of GC analyses, a membraneless assembly for introduction of samples, which allows false peaks in a chromatogram to be removed, is recommended. In order to augment the reliability and information content of chromatographic analyses of several bioactive compounds, complex chromatographic methods based on GC, TLC and HPLC were found to be desirable.

Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha.

For the transformation of the yeast Hansenula polymorpha we have constructed a set of hybrid plasmids carrying the LEU2 gene of Saccharomyces cerevisiae as a selective marker and fragments of mitochondrial DNA of Candida utilis and H. polymorpha or chromosomal DNA fragments of H. polymorpha as replicator sequences. The replication properties of chimeric plasmids in the yeast H. polymorpha were investigated. We showed that for plasmids propagated autonomously in this yeast the plasmid monomers could be detected in the transformants only during the immediate time after the transformation event. Further growth under selective conditions led to the selection of polymeric forms of plasmid DNA as it was clearly shown for transformants carrying cosmid pL2 with mtDNA fragment of C. utilis. Such transformants carrying polymerized plasmids showed a remarkably increased stability of the transformed phenotype. Cosmid pL2 was able to shuttle between Escherichia coli, S. cerevisiae and H. polymorpha, whereas plasmids with DNA fragments from H. polymorpha did not transform S. cerevisiae effectively.

[Radiobiological method of studying the structure of the partially diploid area in T4 bacteriophage]. / Radiobiologicheskii metod izucheniie struktury chastichno diploidnoi oblasti u bacteriofaga T4.

The mechanism of cross-reactivation after UV-irradiation was examined using different deletion mutants as recipients. A model of the rescue mechanism of partly diploid area was suggested. The method of cross-reactivation in demonstrated to permit an easy determination of the orientation of duplicated genes.

Construction of a hybrid col E1 plasmid carrying the gene for bacteriophage lambda repressor.

A biologically active hybrid DNA molecule was constructed from plasmid Col E1 and the Eco R1 fragment of lambda DNA containing the gene for lambda repressor. The presence of this gene in the hybrid molecule was demonstrated genetically. The hybrid plasmid contains two closely located targets for restriction endonuclease Hind 111 in the integrated fragment. Thus, the plasmid may be used as a vector not only for Eco R1 fragments but also for Hind 111 fragments.

[Identification of partial hydrolysis products of lambda bacteriophage DNA by restriction endonuclease EcoRI]. / Identifikatsiia produktov nepolnogo gidroliza DNK bakteriofaga lambda endonukleazoi EcoR1.

The relationship between the electrophoretic mobility of double stranded DNA fragments electrophoresed in agarose gel and their molecular weights within the range from 1.10(6) to 8.10(7) daltons and agarose concentration 0.3--2.0% has been studied. Partial hydrolysis products of lambda phage DNA obtained by restriction endonuclease EcoRI have been separated. Partial hydrolysis products have been identified by determining the fragments of full cleavage as well as by genetic methods using a system of transformation of E. coli cells treated with CaCl2, which have been infected with different helper-phages containing definite gene mutations.

Sepcific fragmentation of DNA heteroduplex molecules of two bacteriophage lambda mutants with endonuclease Si from Aspergillus oryzae.

Heteroduplex DNA molecules of two bacteriophage mutants (lambda b2 and lambda i434ct68) were obtained by the method of molecular hybridization. These heteroduplexes possessed two types of loops formed as a result of: a) deletion in one of the DNA strands; and b) substitution of a DNA fragment for nonhomological one. The digestion of heteroduplexes with single-stranded specific nuclease SI from Aspergillus oryzae produced two fragments at 37 degrees C and three ones at 55 degrees C. The separation of fragments and determination of their molecular weight were carried out by means of electrophoresis in agarose. The molecular weights both measured and preliminarily calculated proved to be close. One of the fragments was identificated by its biological activity in CaCl2-dependent infectious system with helperphage.

[Lethal and mutagenic photodynamic effect of acridine orange on bacteriophage cd]. / Letal'noe i mutagennoe fotodinamicheskoe deistvie akridinovogo oranzhevogo na bakteriofag sd

Both acridine-sensitized inactivation and mutagenesis in phage sd have been studied and compared with the effect of other mutagens. Inactivation curve was not stictly exponential, with a small shoulder at short light doses and deviation of survival lower than 10(-5). The lethal effect was not reactivated by multiplicity reactivation. Photodynamic damage in phage sd was accompanied by the increase of the rise (but not of the latent) period in the one-step curve of phage multiplication and increase of the burst size. Experiments were carried out at dye concentration of 1-10(-5) M or lower; a strong dark effect of the dye being observed under 2-fold increase of the dye concentration. Plaque-type mutants were formed up to the maximum approximately 1% at the survival approximately 2--10(-5); in comparison with other mutagens photo-sensitized mutagenesis in phage sd was lower. The significant increase in the number of plaque mutants was observed after the illumination of phage fraction surviving the pre-treatment with higher acridine orange concentration (greater than or equal to 2-10(-5)M).

[Biological activity of different forms of bacteriophage lambda DNA]. / Biologicheskaia aktivnost' razlichiykh form DNK bakteriofaga lambda

Hershey circles and linear tandem aggregated forms of DNA have been obtained in vitro and treated with polynucleotide ligase to form phosphodiester bond. Using zone centrifugation in glycerol gradient covalently closed circles and linear dimers have been purified and their biological activity investigated. It was found that closed circular molecules lost most, if not all, of their activity in CaCl2-dependent system. In order to investigate the biological activity of tandem dimer molecules, hybrid dimers consisting of DNA's from lambda C1857 and lambda 1434 have been obtained. In plaque assay with the appropriate non-permissive strains of E. coli the efficiency of infectivity of hybrid dimers was measured. Biological activity of dimer molecules sealed with ligase was about 5% of the activity of linear monomers. Ig has been suggested that tandem dimers of lambda DNA joined by phosphodiester bond are able to penetrate into the CaCl2-treated host cells and both components of dimers are active during subsequent multiplication.