[Induced expression of Serratia marcescens ribonuclease III gene in transgenic Nicotiana tabacum L. cv. SR1 tobacco plants].

Transgenic Nicotiana tabacum L. cv. SR1 plants, characterized by an increase in the level of dsRNA-specific hydrolytic activity after induction by wounding, were obtained. The Solanum lycopersicum anionic peroxidase gene promoter (new for plant genetic engineering) was for the first time used for the induced expression of the target Serratia marcescens RNase III gene. Upon infection with the tobacco mosaic virus (TMV), the transgenic plants of the obtained lines did not differ significantly from the control group in the level of TMV capsid protein accumulation. In general, no delay in the development of the infection symptoms was observed in transgenic plants as compared with the control group. The obtained transgenic plants represent a new model for the study of the biological role of endoribonucleases from the RNase III family, including in molecular mechanisms of resistance to pathogens.

[Transgenic Expression of Serratia marcescens Native and Mutant Nucleases Modulates Tobacco Mosaic Virus Resistance in Nicotiana tabacum L].

Extracellular Serratia marcescens nuclease is an extremely active enzyme which non-specifically degrades RNA and DNA. Its antiviral activity was previously shown both in animals and in plants when applied exogenously. Transgenic tobacco plants (Nicotiana tabacum L cv. SR1) expressing S. marcescens chimeric, mutant, and intracellular mutant nuclease gene variants were regenerated and challenged with tobacco mosaic virus. The transgenic plants exhibited a higher level of resistance to the virus infection than the control non-transgenic plants. The resistance was evidenced by the delay of the appearance of mosaic symptoms and the retarded accumulation of viral antigen. Thus, these results reveal that modulations of both extracellular nuclease activity and intracellular RNA/DNA binding can protect plants against viral diseases.

[Evaluation of Salt Tolerance of Transgenic Tobacco Plants Bearing with P5CS1 Gene of Arabidopsis thaliana].

Arabidopsis thaliana delta1-pyrroline-5-carhoxylate synthase 1 gene (P5CS1) cDNA was cloned under the control of the potent constitutive 35S RNA promoter of the cauliflower mosaic virus and transferred into genome of tobacco cv. Petit Havana SR-1 (Nicotiana tabacum L.) plants. It is shown that the constitutive level of proline in the transgenic plants T0 exceeds that of the SR1 reference line by 1.5 to 4 times. Under conditions of salt stress (200, 300 mM NaCl) T1-generation transgenic plants in early stages of development formed a large biomass, developed more quickly, and had a higher rate of root growth compared to the control, which confirms the involvement of the P5CS1 gene in molecular mechanisms of stress resistance in plants.

[Effect of sexual maturation on DD2R gene expression in fat body of Drosophila melanogaster females].

The expression of the DD2R gene was studied by in situ hybridization in the fat body (place of the synthesis of enzymes that degrade juvenile hormone) and ovarian follicular cells (place of the synthesis of 20-hydroxyecdysone) in young and sexually mature D. melanogaster females. It was demonstrated that the DD2R gene is expressed in the fat body, and its expression is higher in young females than in sexually mature females. The DD2R gene expression was not detected in ovarian follicular cells.

[Spontaneous spectinomycin resistance mutations of the chloroplast rrn16 gene in Daucus carota callus lines].

Bioballistic transformation of carrot Daucus carota L. callus cultures with a plasmid containing the aadA (aminoglycoside 3'-adenyltransferase) gene and subsequent selection oftransformants on a selective medium containing spectinomycin (100-500 mg/l) yielded ten callus lines resistant to this antibiotic. PCR analysis did not detect exogenous DNA in the genomes of spectinomycin-resistant calluses. Resistance proved to be due to spontaneous mutations that occurred in two different regions of the chloroplast rrn16 gene, which codes for the 16S rRNA. Six lines displayed the G > T or G > C transverions in position 1012 of the rrn16 gene, and three lines had the A > G transition in position 1138 of the gene. Chloroplast mutations arising during passages of callus cultures in the presence of spectinomycin were described in D. carota for the first time. The cause of spectinomycin resistance was not identified in one line. The mutations observed in the D. carota plastid genome occurred in the region that is involved in the formation of a double-stranded region at the 3' end of the 16S rRNA and coincided in positions with the nucleotide substitutions found in spectinomycin-resistant plants of tobacco Nicotiana tabacum L. and bladderpod Lesquerella fendleri L.

[Specific features of T-DNA insertion regions in transgenic plants].

Experimental data from analysis of exogenous DNA (T-DNA) insertion sites in transgenic plants are summarized. Arguments are considered in favor and against the existence of genome DNA regions preferred for transgene integration that are determined by distinctive features characterizing the organization and nucleotide composition of the plant genome and the structure and conformational state of the chromatin. The main stages of T-DNA integration into a plant chromosome and possible molecular mechanisms of this process are discussed.

[Study of T-DNA integration loci in tobacco transgenic plants].

From the total DNA of 17 transgenic tobacco plants the DNA fragments containing T-DNA/plant DNA junctions were amplified using inverse polymerase chain reaction. Comparison of the nucleotide sequences of 34 fragments with the GENEBANK sequences revealed homology with vector sequences outside T-DNA in 10 cases and no homology with the known nucleotide sequences in most clones. The AT-content varied from 51 up to 72% that is close to the total percentage of AT pairs in tobacco genome. Alignment of the sequences truncated during embedding of the left and the right borders has shown that for the left border significant clusterization (10 bp region) of truncation sites was observed, and five sequences had identical sites of truncation (+23 T) that showed the preferable use of this nucleotide. Nine created nucleotide sequences were homologous to the repeating sequences in tobacco genome. The percentage of homology varied from 70 up to 90%. The identified repeats belong to different types.

[Species-specific features of the distribution of restriction sites in Bsp-repeats of Canidae genome]. / Vidospetsificheskie osobennosti raspredeleniia restriktsionnykh saitov v Bsp-povtorakh genomov Canidae.

Differences and similarities of the Bsp-repeats' organization in fox, dog, polar fox and raccoon dog genomes were studied. Specificity of Bsp-repeats to the Canidae family was demonstrated. The repeats are mainly organized in large clusters in all species studied. The species-specific features in restriction patterns were revealed for all five genomes, in spite of high intragenomic polymorphism exhibited in each case. This suggests that certain unique sets of structural versions of Bsp-repeats were fixed in canid genomes by amplification during the process of speciation. Fox and polar fox exhibited the highest similarity in restriction patterns of Bsp-repeats. Raccoon dog pattern is most unusual among others: its distinguishable character is the absence of large multimeric series. The EcoRI hydrolysate of raccoon dog Bsp-repeats consists mainly of one band corresponding to 1600 bp. These are in accordance with phylogenetic relations between canids.

[Heterogeneity of the Canidae Bsp-repeats family: discovery of the EcoRI subfamily]. / Geterogennost; semeistva Bsp-povtorov Canidae: obnaruzhenie EcoRI-podsemeistva.

A 1600 bp EcoRI fragment was cloned from genome of raccoon dog. The structure obtained is homologous to the Canidae Bsp-repeats family. Comparative blot hybridization of the EcoRI fragment and BamHI repeat from fox genome with restricted hydrolysates of the total of raccoon dog and fox DNAs revealed differences both in structure and genomic organization between these two Bsp-repeats versions. Evidently, the EcoRI fragment contains a sequence lacking from the BamHI fragment of the fox Bsp-repeats. Quantitative differences in contents of two Bsp versions in various canid genomes were revealed as well. The EcoRI version is most abundant in raccoon dog genome, while the BamHI fox version is most representative in polar fox genome. With other species studied, quantitative differences in version contents are not so dramatic, and the EcoRI fragment is always present in lower copy numbers. The discovery of the EcoRI subfamily of the Bsp-repeats is in accordance with the "library hypothesis" advanced by Salser in 1976. Connection of the Bsp-repeats' evolution with centric fusions and breaks characteristic of karyotype evolution of canids is being discussed. Comparative study of cloned EcoRI and BamHI fragments of Bsp-repeats in cytogenetical and molecular aspects may be useful, when investigating the role of tandem repeats in large chromosome rearrangements.