Clostridium thermocellum thermostable lichenase with circular permutations and modifications in the N-terminal region retains its activity and thermostability.

The Clostridium thermocellum lichenase (endo-ß-1,3;1,4-glucan-D-glycosyl hydrolase) displays a high thermostability and specific activity and has a compact protein molecule, which makes it attractive, in particular, for protein engineering. We have utilized in silico analysis to construct circularly permuted (CP) variants and estimated the retained activity and thermostability. New open termini in the region of residues 53 or 99 in two lichenase CP variants (CN-53 and CN-99) had no effect on their activity and thermal tolerance versus another variant CP variant, CN-140 (cut in the region of residue 140), which displayed a dramatic decrease in the activity and thermostability. Construction and further activity and thermostability testing of the modified lichenase variants (M variants) and CP variants with peptides integrated via insertion fusion have demonstrated that the N-terminal regions in the lichenase catalytic domain (53 and 99 amino acid residues) that permit circular permutations with retention of activity and thermostability of the enzyme as well as the region between the C and N termini of the native lichenase in thermostable and active lichenase variants (CN-53 and CN-99) may be used for integrating small peptides without the loss of activity and thermostability. These findings not only suggest that CP predictions can be used in search for internal integration sites within protein molecule, but also form the background for further enzymatic engineering of the C. thermocellum thermostable lichenase aiming to create new fusion proteins.

[Expression of plant antimicrobial peptide pro-SmAMP2 gene increases resistance of transgenic potato plants to Alternaria and Fusarium pathogens].

The chickweed (Stellaria media L.) pro-SmAMP2 gene encodes the hevein-like peptides that have in vitro antimicrobial activity against certain harmful microorganisms. These peptides play an important role in protecting the chickweed plants from infection, and the pro-SmAMP2 gene was previously used to protect transgenic tobacco and Arabidopsis plants from phytopathogens. In this study, the pro-SmAMP2 gene under control of viral CaMV35S promoter or under control of its own pro-SmAMP2 promoter was transformed into cultivated potato plants of two cultivars, differing in the resistance to Alternaria: Yubiley Zhukova (resistant) and Skoroplodny (susceptible). With the help of quantitative real-time PCR, it was demonstrated that transgenic potato plants expressed the pro-SmAMP2 gene under control of both promoters at the level comparable to or exceeding the level of the potato actin gene. Assessment of the immune status of the transformants demonstrated that expression of antimicrobial peptide pro-SmAMP2 gene was able to increase the resistance to a complex of Alternaria sp. and Fusarium sp. phytopathogens only in potato plants of the Yubiley Zhukova cultivar. The possible role of the pro-SmAMP2 products in protecting potatoes from Alternaria sp. and Fusarium sp. is discussed.

Control of meiotic crossing over in plant breeding.

Meiotic crossing over is the main mechanism for constructing a new allelic composition of individual chromosomes and is necessary for the proper distribution of homologous chromosomes between gametes. The parameters of meiotic crossing over that have developed in the course of evolution are determined by natural selection and do not fully suit the tasks of selective breeding research. This review summarizes the results of experimental studies aimed at increasing the frequency of crossovers and redistributing their positions along chromosomes using genetic manipulations at different stages of meiotic recombination. The consequences of inactivation and/or overexpression of the SPO11 genes, the products of which generate meiotic double-strand breaks in DNA, for the redistribution of crossover positions in the genome of various organisms are discussed. The results of studies concerning the effect of inactivation or overexpression of genes encoding RecA-like recombinases on meiotic crossing over, including those in cultivated tomato (Solanum lycopersicum L.) and its interspecific hybrids, are summarized. The consequences of inactivation of key genes of the mismatch repair system are discussed. Their suppression made it possible to significantly increase the frequency of meiotic recombination between homeologues in the interspecific hybrid yeast Saccharomyces cerevisiae × S. paradoxus and between homologues in arabidopsis plants (Arabidopsis thaliana L.). Also discussed are attempts to extrapolate these results to other plant species, in which a decrease in reproductive properties and microsatellite instability in the genome have been noted. The most significant results on the meiotic recombination frequency increase upon inactivation of the FANCM, TOP3α, RECQ4, FIGL1 crossover repressor genes and upon overexpression of the HEI10 crossover enhancer gene are separately described. In some experiments, the increase of meiotic recombination frequency by almost an order of magnitude and partial redistribution of the crossover positions along chromosomes were achieved in arabidopsis while fully preserving fecundity. Similar results have been obtained for some crops.

[Analysis of the meiotic recombination frequency in transgenic tomato hybrids expressing recA and NLS-recA-licBM3 genes].

To study and induce meiotic recombination in plants, we generated and analyzed transgenic tomato hybrids F1-RecA and F1-NLS-recA-LicBM3 expressing, respectively, the recA gene of Escherichia coli and the NLS-recA-licBM3 gene. It was found that the recA and NLS-recA-licBM3 genes are inherited through the maternal and paternal lineages, they have no selective influence on the pollen and are contained in tomato F1-RecA and F1-NLS-RecA-LicBM3 hybrids outside the second chromosome in the hemizygous state. The comparative analysis of the meiotic recombination frequency (rf) in the progenies of the transgenic and nontransgenic hybrids showed that only the expression of the recA gene of E. coli in cells of the F1-RecA plants produced a 1.2-1.5-fold increase in the frequency of recombination between some linked marker genes of the second chromosome of tomato.

[Transgenic tomato plants expressing recA and NLS-recA-licBM3 genes as a model for studying meiotic recombination].

Homologous DNA recombination in eukaryotes is necessary to maintain genome stability and integrity and for correct chromosome segregation and formation of new haplotypes in meiosis. At the same time, genetic determination and nonrandomness of meiotic recombination restrict the introgression of genes and generation of unique genotypes. As one of the approaches to study and induce meiotic recombination in plants, it is recommended to use the recA gene of Escherichia coli. It is shown that the recA and NLS-recA-licBM3 genes have maternal inheritance and are expressed in the progeny of transgenic tomato plants. Plants expressing recA or NLS-recA-licBM3 and containing one T-DNA insertion do not differ in pollen fertility from original nontransgenic forms and can therefore be used for comparative studies of the effect of bacterial recombinases on meiotic recombination between linked genes.

[Compartmentalization of Spo11p in vegetative cells of yeast Saccharomyces cerevisiae].

Double-stranded DNA breaks are currently thought to initiate homologous DNA recombination during meiosis. These breaks are mediated by several proteins, the key protein is Spol1p. Spo11 proteins being encoded by the highly conserved orthologs of SPO11 are present in most eukaryotes ranging from plants to man and are structurally similar to the subunit A of the archaea topoisomerase VI. The SPO11 of S. cerevisiae is currently known to be expressed during prophase I. It encodes a topoisomerase II that is apparently active as a dimer. Neither its localization in the native cells nor its nuclear localisation signals have been described in the literature. We report the expression of the coding region of SPO11 and its truncated variants C-terminally tagged by the egfp reporter in yeast. As judged by the EGFP fluorescence, the Spo11 p-EGFP fusion was localized in vegetative yeast nuclei whereas Spo11pdelta-EGFP lacking 25 N-terminal amino acids of Spollp was localized in cytoplasm. Nineteen N-terminal amino acids of Spo11p fused to EGFP made some reporter to be localized in the nucleus. Thus, we conclude that N-terminal part of Spo11p is a nuclear localization signal that is not specific for prophase I and is used to import proteins in vegetative yeast cells.

[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.

[Comparative study of the expression of the native and modified cry3a genes of Bacillus thuringiensis in prokaryotic and eukaryotic cells].

The cry3a gene of Bacillus thuriengiensis was cloned. Based on sequence analysis of this gene, a modified gene, cry3aM, was constructed, which has the optimal codon composition for effective expression in eukaryotic cells. Hybrid genes cry3a-licBM2 and cry3aM-licBM2 were constructed, in which the sequences of the native and modified genes are fusedfused with the reorter gene for thermostable lichenase in the reading frame. We have shown that the expression levels of hybrid genes cry3a-licBM2 and cry3aM-licBM2 in Escherichia coli are comparable, being 5% of those for reporter gene licBM2. In cells of a lower eukaryote Saccharomyces cerevisiae, the expression of hybrid gene cry3aM-licBM2? Which contains the modified gene, considerably exceeded the level of expression of cry3a-licBM2 containing the native gene. The presence of lichenase in the composition of hybrid proteins was shown to facilitate selection and analysis of the expression level of hybrid proteins in transgenic organisms.

[Thermostable lichenase as a translational reporter].

Hybrid genes containing the reporter gene for thermostable lichenase and model genes recA, recA1, cry3a, cry3aM, and ssp1 were constructed. The expression of these genes was studied in prokaryotic and eukaryotic cells. The presence of lichenase in the hybrid proteins was shown to facilitate analysis of the hybrid protein expression in transgenic organisms. Owing to high relative activity and thermostability of lichenase, the activity of this enzyme can be measured by simple, rapid and sensitive qualitative and quantitative methods that do not require costly equipment and reagents. Using the zymograms method, molecular masses of the lichenase-containing hybrid proteins can be precisely estimated. This method is proposed instead of Western blotting using lichenase as a translational reporter. Our results showed that the use of thermostable lichenase as a translational reporter yields the data that are problematic to obtain using traditional methods of gene expression analysis, which is of importance for fundamental and applied research.