[The development of yeast vector system for study of regulational functions of eukaryotic noncoding elements].

The original yeast vector system was proposed for study of regulation functions of eukaryotic noncoding sequences. This system consists of two reporter genes that arranges in opposition to one another. The promoter activity of LTR HERV-K of locus 22-19 in 7p22 human chromosome was studied. It was shown that the LTR initiates transcription of reporter gene in yeasts in forward and reverse orientation with respect to the reporter. It was displayed that the LTR 22-19 HERV-K possesses bidirectional promoter activity in the yeast vector system. Comparison of the promoter activity LTR 22-19 and strong GAL1 and TDH promoters in yeasts Saccharomyces cerevisiae shown that promoter activity of the LTR amount approximately 0.34% for promoter activity of the inducible GAL1 promoter and 0.26%--of the constitutive TDH promoter.

[Construction of the synthetic genes for protein analogs of spider silk carcass spidroin 1 and their expression in tobacco plants]. / Konstruirovanie sinteticheskikh genov, kodiruiushchikh belki-analogi belka karkasnoi niti pautiny spidroina 1, i ikh ékspressiia v rasteniiakh tabaka.

To obtain transgenic tobacco plants expressing recombinant analogs of spider dragline silk spidroin 1, artificial 1f5 and 1f9 coding for spidroin 1 analogs were 3'-fused in-frame with the reporter lichenase gene. The Tr2' weak constitutive promoter of Agrobacterium tumefaciens T-DNA and the strong constitutive promoter of the cauliflower mosaic virus 35S RNA gene were used as regulatory elements. The expression cassettes were used to transform agrobacteria and then introduced in tobacco leaf disks. On evidence of Southern hybridization, transgenic plants each carried a single copy of a hybrid gene, which corresponded in size to the constructed one. Zymography and Western blotting revealed full-length hybrid proteins in leaf extracts of transgenic plants. The results testified that plants can maintain and express synthetic genes for spider silks and, consequently, may be used as a convenient producer of recombinant silk analogs.

[Expression of a thermostable bacterial cellulase in transgenic tobacco plants]. / Ekspressiia termostabil'noi bakterial'noi tselliulazy v transgennykh rasteniiakh tabaka.

The bacterial gene of the thermostable endo-beta-1,4-glucanase (cellulase) was shown to retain its activity and substrate specificity when expressed in transgenic tobacco plants. The leader peptide of the carrot extensin was efficient in transferring the bacterial enzyme into the apoplast. The expression of the bacterial cellulase gene leads to changes in the plant tissue morphology. In the transgenic plant lines, regeneration of primary shoots from callus occurred at the three to five times higher cytokinin (6-BAP) concentration than in control plants. The transgenic plants that expressed the bacterial gene exhibited increased business and altered leaf shape. The transgenic plants developed can be used as models for studying the cellulases role and function in plants.

[Bifunctional reporter genes: construction and expression in prokaryotic and eukaryotic cells]. / Bifunktsional'nye reporternye geny: konstruirovanie i ékspressiia v kletkakh pro- i éukariot.

Bifunctional reporter proteins were constructed to combine Clostridium thermocellum lichenase (LicBM2) with Aequorea victoria green fluorescent protein (GFP) or with Escherichia coli beta-glucuronidase (GUS). The major properties of the initial proteins were preserved in the hybrid ones: LicBM2 was active at 65 degrees C, GFP fluoresced, and GUS hydrolyzed its substrates. LicBM2 remained active after extension of its C of N end. Bifunctional reporter systems were shown to provide a convenient tool for studying the gene expression regulation in prokaryotic (E. coli) and eukaryotic (Saccharomyces cerevisiae, mammalian) cells, advantages of one reporter compensating for drawbacks of the other.

[A thermostable Clostridium thermocellum lichenase-based reporter system for studying the gene expression regulation in prokaryotic and eukaryotic cells]. / Reporternaia sistema, osnovannaia na termostabil'nosti likhenazy Clostridium thermocellum, dlia izucheniia reguliatsii ékspressii genov v kletkakh pro- i éukarioticheskikh organizmov.

A new reporter system was developed to study the gene expression regulation in prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and mammalian) cells. The system was based on the modified bacterial lichenase gene (licBM2), which was shown to meet the requirements for a reporter. The gene product was active and did not undergo modification in heterologous hosts. Simple and sensitive methods were used to detect and to quantitate the lichenase activity. Inducible licBM2 expression was demonstrated with E. coli and yeast cells, allowing the system to be employed in dynamic studies.

[Transgenic Tobacco plants expressing bacterial genes encoded the thermostable glucanases]. / Transgennye rasteniia tabaka, ékspressiruiushchie bakterial'nye geny termostabil'nykh gliukanaz.

It is shown that bacterial genes for thermostable beta-glucanases are expressed retaining their activity and substrate specificity. The leader peptide of the carrot extensin exerts effective secretion of the bacterial enzymes into the intercellular space of the plant tissue. Expression of the bacterial gene for beta-1,3-glucanase in plant tissues alters their morphogenetic potential. Regeneration of shoots from the calli of these plant lines requires a six- to eightfold increase in cytokinin (6-BAP) concentration in comparison with the control lines and the transgenic lines expressing beta-1,3-1,4-glucanase. Rooting of transgenic plants expressing the bacterial gene for beta-1,3-glucanase occurs much faster. The transgenic plants obtained in the study are proposed as model objects for investigating the role of glucanases in plants.