Long term effects of chernobyl contamination on dna repair function and plant resistance to biotic and abiotic stress factors.

Thirty years after the Chernobyl explosion we still lack information regarding the genetic effects of radionuclide contamination on the plant population. For example, are plants adapting to the low dose of chronic ionising irradiation and showing improved resistance to radiation damage? Are they coping with changing/increased pathogenicity of fungi and viruses in the Chernobyl exclusion zone? Are plant populations rapidly accumulating mutational load and should we expect rapid microevolutionary changes in plants in the Chernobyl area? This review will try to summarise the current knowledge on these aspects of plant genetics and ecology and draw conclusions on the importance of further studies in the area around Chernobyl.

[A new thermostable proteinase from Thermoactinomyces SP.27a. Cloning and gene expression]. / Novaia termostabil'naia proteinaza iz Thermoactinomyces SP.27a. Klonirovanie i ékspressiia gena.

A library of Thermoactinomyces sp. 27a, producer of thermostable proteases of different groups, has been created. Gene coding for thermostable neutral proteinase was cloned and expressed in Bac. subtilis cells. Restriction map for cloned DNA fragment was created and physicochemical parameters of recombinant proteinase were characterized. The thermostability and optimum of proteolytic activity of the enzyme was lower than in the natural Thermoactinomyces sp. strain, which can be due to heterologous expression of the gene coding for thermostable protein in the mesophilic host.

[Effect of increased pressure and changed gaseous media on biological properties of microorganisms]. / Issledovanie vliianiia povyshennogo davleniia i izmenennoi gazovoi sredy na biologicheskie svoistva mikroorganizmov.

Bacterial contamination of diving outfit and inner surfaces of pressure complex increases at the expense of expansion of gram-negative microflora during long exploitation of undersea complexes. Gram-negative bacteria were hypothesized to effectively adapt to hyperbaric conditions. We investigated the effect of hyperbaric conditions and changed gaseous environment on the cultural and morphological characteristics of colonies, growth rate and time of generation of test cultures (P. aeruginosa ATCC 10145, E. coli Tg1, and Bac. subtilis GB2036). Phenotypically modified clones were selected for subsequent analysis of changes at a genetic level. Experiments revealed no essential changes in the studied properties under the effect of extreme conditions.

[Medium for the production of Bacillus intermedius glutamyl endopeptidase by the recombinant Bacillus subtilis]. / Sreda dlia biosintheza glutamiléndopeptidazy Bacillus intermedius rekombinantnym shtammom Bacillus subtilis.

A nutrient medium was elaborated for the efficient production of glutamyl endopeptidase by the recombinant Bacillus subtilis strain AJ73 bearing the Bacillus intermedius 3-19 glutamyl endopeptidase gene within a multicopy plasmid. Optimal concentrations of the main nutrients, peptone and inorganic phosphate, were found using a multifactor approach. To provide for active growth and efficient glutamyl endopeptidase production, the cultivation medium of the recombinant strain should be enriched in phosphorus, organic and inorganic nitrogen sources, and yeast extract. Complex protein substrates, such as casein and gelatin, enhanced the biosynthesis of glutamyl endopeptidase. At the same time, easily metabolizable carbon sources suppressed it. The production of glutamyl endopeptidase was stimulated by the bivalent cations Ca2+, Mg2+, and Co2+.

A highly conserved plasmid from the extreme thermophile Thermotoga maritima MC24 is a member of a family of plasmids distributed worldwide.

We have screened Thermotoga strains, isolated from hydrothermal vents near the Kuril Islands, for the presence of plasmid DNA. The miniplasmid pMC24 was isolated from the extreme thermophilic eubacteria Thermotoga maritima and sequenced, showing it to be a plasmid of 846 bp. It was found, from a search of the databases, to be closely related to the previously described Thermotoga miniplasmid pRQ7, isolated from a strain found on the Azore Islands, and was distinguished by only two point mutations. These changes resulted in two consecutive frameshifts altering a region encoding 9 amino acids in the Rep-coding region. We have also shown that pMC24, as with pRQ7, is negatively supercoiled. It seems that negatively supercoiled miniplasmids related to pRQ7 are spread worldwide and strongly maintained among Thermotoga strains.

[Expression of secreted guanyl-specific ribonuclease genes from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis cells]. / Ekspressiia genov sekretiruemykh guanilspetsifichnykh ribonukleaz iz Bacillus intermedius i Bacillus pumilus v kletkakh Bacillus subtilis.

Plasmids with whole genes for ribonucleases from B. intermedius (binase) and B. pumilis (RNase Bp) assembled with the whole gene of barstar, a specific intracellular inhibitor, are constructed. The resultant plasmids pMZ55 and pMZ56 effectively express binase and RNase Bp genes in B. subtilis cells. A medium for maximum expression of RNase genes by recombinant strains is developed. The expression of binase and RNase Bp genes in B. subtilis cells is negatively regulated by exogenic inorganic phosphate.

[Cloning and expression of a serine proteinase gene from Thermoactinomyces sp. in Bacillus subtilis cells]. / Klonirovanie i ékspressiia gena serinovoi proteinazy Thermoacinomyces sp. v kletkakh Bacillus subtilis.

A gene coding for thermostable serine protease from Thermoactinomyces sp. K50 is cloned and expressed in Bacillus subtilis cells. Restriction map of cloned DNA fragment is determined. Thermostability and temperature optimum of proteolytic activity of the cloned gene product are lower than those of the natural proteinase of Thermoactinomyces sp. K50. Serine protease, a product of cloned gene, is highly sensitive to proteolysis and its degradation can be prevented by Ca2+ ions.

Bacillus intermedius glutamyl endopeptidase. Molecular cloning and nucleotide sequence of the structural gene.

The glutamyl endopeptidase gene of Bacillus intermedius was cloned from a genomic library expressed in Bacillus subtilis and sequenced (EMBL accession number Y15136). The encoded preproenzyme contains 303 amino acid residues; the mature 23-kDa enzyme consists of 215 residues. The mature enzyme reveals 38% of identical residues when aligned with the glutamyl endopeptidase from Bacillus licheniformis, whereas only five invariant residues were found among all known glutamyl endopeptidases. The amino acid residues that form the catalytic triad (H47, D98, and S171) as well as H186 participating in the binding of the substrate carboxyl group were identified. It seems that the structural elements responsible for the function of glutamyl endopeptidases from various sources are highly variable.

[Cloning and expression of thermostable secretory metalloproteinase genes from two Bacillus brevis strains in Bacillus subtilis cells]. / Klonirovanie i ékspressia genov termostabil'nykh sekretornykh metalloproteinaz dvukh shtammov Bacillus brevis v kletkakh Bacillus Subtilis.

Two closely related genes of thermostable Bac. brevis metalloproteases were cloned and expressed in Bac. subtilis cells. Their restriction maps and directions of transcription were determined. Thermostability and thermal optimum of proteolytic activity of cloned gene products are significantly lower than those of native enzymes. The authors believe that alteration of the enzymes' characteristics may be due to uncorrected folding of thermostable protein in case of its expression in mesophilic bacterial strains.

[Cloning and gene expression of Bacillus cereus neutral proteinase in Bacillus subtilis cells]. / Klonirovanie i ékspressiia gena neitral'noi proteinazy Bacillus cereus v kletkakh Bacillus subtilis.

The neutral proteinase gene of Bacillus cereus was cloned. Its restriction map and the direction of transcription was determined. It was shown that the neutral proteinase gene could be expressed in Bacillus cells. The thermostability of the product coded by the neutral proteinase gene and its natural analogue was explored. The obtained data indicate that the neutral proteinase of Bacillus cereus is closely related to the enzyme of Bacillus amyloliquefaciens by these parameters. It was found that the neutral proteinase of Bacillus cereus has a high sensitivity to autolysis and that leads to the decrease in the enzymes concentration in the cultural medium at the late stages of cell growth. But the possibility of stabilization the neutral proteinase by Ca2+ ions has been demonstrated.

Temperature-dependent activation of Bacillus brevis metalloprotease expressed in mesophilic Bacillus subtilis.

When the Bacillus brevis secretory metalloprotease is expressed from the npr gene on a plasmid vector in the mesophile B. subtilis, grown at 37 degrees C, the enzyme was found to be properly processed, but secreted into the culture medium in a low-active conformation. Secreted metalloprotease can by heat-treatment (70 degrees C for 30 min) be converted into fully active enzyme.

[Analysis of the structure of Bacillus brevis neutral proteinase and its biosynthesis in Bacillus subtilis cells]. / Analiz struktury neitral'noi proteinazy Bacillus brevis i ee biosintez v kletkakh Bacillus subtilis.

Amino acid sequence of neutral metalloprotease from Bac. brevis has been compared with that of Bac. amyloloquefaciens, Bac. cereus, Bac. subtilis, Bac. stearothermophilis, Bac. thermoproteolyticus (thermolysine). A sequence region from N-40 to N-1 with a significant degree of homology allowed to predict the processing site of the propart of Bac. brevis enzyme. The sequence comparison allows to put Bac. brevis enzyme within the evolutionary branch of enzymes, which includes thermolysin and proteases of Bac. cereus and Bac. stearothermophilus. Using automated Edman degradation the N-terminal sequence of Bac. brevis protease has been determined. It does not differ from the sequence predicted from the nucleotide sequence of the gene. It was shown that, when Bac. brevis gene coding for thermostable protease is expressed on a plasmid vector in Bac. subtilis cells at 37 degrees C, enzyme forms possessing low activity are secreted. The enzyme may be significantly activated without an additional cleavage or processing and the activation includes numerous conformation transition states of the protein molecule.