[Relict microorganisms of cryolithozone as possible objects of gerontology].

Permafrost is widely distributed in the Northern Hemisphere, and its age reaches hundreds of thousands and millions of years. Permafrost contains alive microorganisms which are not frozen due to relatively high temperature of the environment (-2...-8 degrees C), but the microorganisms are immobilized and therefore aged probably similar to the age of permafrost. Longevity of the relict microbial cells is related obviously to their mechanism of protection against heat, radiation, free radicals and other damaging agents. A strain of Bacillus sp. was isolated from permafrost aged of about 3 million years, 16S rDNA sequence was identified and preliminary testing of bacterial culture on Drosophila melanogaster and mice was made. Immune stimulation and improvement of physical condition were observed, and that, together with the age of the microbial cells, presents the relict microorganisms as objects of gerontology.

[Restriction endonuclease Asi256I recognizing the nucleotide sequence 5'-GATC-3'].

A strain producing a restriction endonuclease was isolated from soil samples and identified as the Arthrobacter sp. strain Ck256. The enzyme produced by this strain was termed Asi2561. The isolation procedure for this enzyme was described, and the optimal conditions for its function were determined. It was shown that the restriction endonuclease Asi256I is a true isoschizomer of MboI, it has a temperature optimum of 6 degrees C, and can be used in molecular-biological and genetic-engineering studies performed at low temperatures.

[Consumption of hydrocarbons by psychrotolerant degrader strains].

Oil-oxidizing microorganisms have been sampled in various regions of Siberia and used in strain associations, which degrade n-alkanes of oil from various fields by 64-92% after 6 days of growth in a wide temperature range. These strains are salt-tolerant and psychrotolerant. They are compatible with aboriginal soil microflora. Promising results have been obtained in experiments on growing plants on oil-polluted soil purified with a biodegrader of this series.

New restriction endonucleases from thermophilic soil bacteria.

Using the most effective rapid method for the detection of restriction endonucleases (ENases) in microorganisms, 32 thermophilic producers have been isolated. All strains belong to the genus Bacillus. Thermostable isoschizomers of ENases, such as AvaI, BbvI, BbvII, BclI, BsaBI, BsiYI, BsrI, BstEII, BstNI, Cfr10I, ClaI, FspI, HaeIII, HpaII, Ksp632I and SfeI, were isolated.

[Determination of the substrate specificity of Bpu101 restrictase with an unusual recognition segment]. / Opredelenie substratnoi spetsifichnosti restriktazy BPu101 s neobychnym uchastkom uznavaniia.

A new enzyme Bpu10I was isolated from Bacillus pumilus. This enzyme is not an isoschizomer of any known restriction endonucleases. The search of possible recognition sequences was carried out in sequences ABCNiDEF (i = 0.6) on substrate DNA lambda CI857, T7, pBR322. The recognition sequence and cleavage sites of restriction endonuclease Bpu10I have been determined as CCTNAGC. GGANTCG

[Substrate specificity of restriction endonuclease VspI]. / Opredelenie substratnoi spetsifichnosti restriktsionnoi éndonukleazy VspI.

The recognition sequence and cleavage point of restriction endonuclease VspI have been determined as 5'-AT decreases TAAT. This enzyme is not isoschizomer of any known restriction endonucleases. DNA pBR322 contains a single VspI recognition sequence in position 3539. Therefore this enzyme may be used for cloning DNA in the VspI site in AmpR-gene of pBR322.

[New type II and IIs restriction endonucleases from thermophilic bacteria of the genus Bacillus]. / Novye éndonukleazy restriktsii tipa II i IIs iz termofil'nykh bakterii roda Bacillus.

Restriction endonucleases have been isolated from 26 strains of thermophilic strains of the Bacillus genus, their recognition sequences were determined, and for 15 of them cleavage sites identified. The enzymes proved to be isoschizomers of known endonucleases BstNI, EarI, HaeIII, HpaII, Cfr10I, BsiYI, BclI, BbvII, BbvI, BstEII, BsaBI, BsrI, FspI, ClaI, SfeI.

[Search for producers of restriction endonucleases with a given specificity]. / Poisk produtsentov éndonukleaz restriktsii zadannoi spetsifichnosti.

Six site-specific restriction endonucleases were isolated from Bacillus thuringiensis strains chosen of 58 strains ought purposefully for production of restriction enzymes. All six strains produce isoschisomers of Sau3A.

[HpyNI--a novel restriction endonuclease isolated from the pathogenic microorganism Helicobacter pylori]. / HpyNI--pervaia éndonukleaza restriktsii, vydelennaia iz patogennogo mikroorganizma Helicobacter pylori.

HpyNI is the first restriction endonuclease which has been isolated from pathogenic Helicobacter pylori. HpyNI, a new isoschizomer of ScrFI, recognizes and cleaves the palindromic sequence 5'CCNGG-3'.

[Maintenance stability of the pBR322 and pBR327 vector plasmids in Escherichia coli cells during multiple passages]. / Stabil'nost' podderzhaniia vektornykh plazmid pBR322 i pBR327 v kletkakh Escherichia coli pri mnozhestvennykh peresevakh.

Stability of pBR322 and pBR327 plasmids was studied. Plasmid-containing Escherichia coli strains were grown in liquid growth medium without selection pressure. Plasmid pBR327 was shown to be more stable in E. coli CSH54 cells than pBR322. Essential heterogenity of individual plasmid-containing clones was recognized by the maintenance stability of plasmid DNA. The indicated clones with high stability failed to be cured from pBR327 plasmid by means of acridine orange. High stability of plasmid maintenance and the failure to cure cells containing this plasmid are suggested to correlate with and to be essentially determined by the cell functions.

[Identification and classification of strains of microorganisms using genomic fingerprinting with biotinylated phage M13 DNA]. / Identifikatsiia i pasportizatsiia shtammov mikroorganizmov metodom genomnoi daktiloskopii s ispol'zovaniem biotinilirovannoi DNK faga M13.

To analyze DNA polymorphisms of various bacterial strains, a nonradioactive variant of the genomic fingerprinting method was developed. The method was based on the application of biotin-labeled single-stranded phage M13 DNA as a probe. Characteristic patterns of fingerprints obtained by MvaI, HaeIII, and HinfI restriction enzymes are presented for several species of bacilli and other bacteria. The advantages of this method in microbiology for the identification and characterization of different microbial strains are shown.

[Identification and characterization of strains of Bacillus thuringiensis by genomic fingerprinting using biotinylated phage M13 DNA]. / Identifikatsiia i pasportizatsiia shtammov Bacillus thuringiensis metodom genomnoi daktiloskopii s ispol'zovaniem biotinilirovannoi DNK faga M13]

Genomic DNA of the entomopathogenic bacterium Bacillus thuringiensis was analyzed by the genomic fingerprinting technique. The biotin-labeled single-stranded DNA of the phage M13 was used as a marker of hypervariable sequences. A procedure for analyzing the differentiation among various Bacillus thuringiensis strains was developed. Characteristic patterns of fingerprints were obtained for several strains, the main representatives of subspecies that are most frequently used in the manufacture of bacterial insecticides, such as subsp. thuringiensis, subsp. kurstaki, and subsp. galleriae. Because no essential differences were revealed in band patterns upon comparing fingerprints of crystal-producing bacterial strains with those of acrystallic mutants, it was assumed that the loss of crystal-producing ability in the insect pathogen Bacillus thuringiensis is not connected with significant rearrangement of its genome.

[Comparison of a stat method of analysis of microorganisms for the presence of site-specific restriction endonuclease activity]. / Sravnenie ékspress-metodov analiza mikroorganizmov na nalichie aktivnosti sait-spetsificheskikh éndonukleaz restriktsii.

Sensitivity of several express-methods used for detection of bacterial endonucleases was compared. The most sensitive method is that employing Triton X-100.

[The effect of various culture parameters on the yield of restriction endonuclease Sfa NI]. / Vliianie nekotorykh parametrov kul'tivirovaniia na vykhod éndonukleazy restriktsii Sfa NI.

A simple technique is proposed for testing the restriction endonuclease Sfa NI activity in lysates of Streptococcus faecalis cells. The technique was used to study the effect of inorganic phosphate and the growth phase on the enzyme yield. Conditions were chosen that provide a high yield of the Sfa NI activity and a significantly reduced level of nucleases in the cells.

[Cultivation of Mycobacterium tuberculosis in cell culture]. / Metod kul'tivirovaniia mikobakterii tuberkuleza na osnove perevivaemoi kul'tury kletok.

The method of the cultivation of M .tuberculosis in cell subcultures was tested. Five consecutive passages of 2 M. tuberculosis strains were carried out with similar inoculation and cultivation conditions. Mycobacterial cells preserved their morphological characteristics in the course of all passages. The method of M. tuberculosis cultivation in subcultured cells made it possible to study of the physiology of M. tuberculosis under conditions, most approximated to the natural tuberculosis infection in humans.

[Entomopathogenic bacteria Bacillus thuringiensis as producers of restriction endonucleases]. / Entomopatogennye bakterii Bacillus thuringiensis--produtsenty éndonukleaz restriktsii.

A total of 115 collection strains of Bacillus thuringiensis, belonging to various subspecies, have been studied for the presence of DNA restriction-modification systems. Restriction endonucleases of 13 strains have been isolated and characterized. No considerable correlations between the taxonomic positions of the bacteria and the specificities of the endonucleases isolated have been detected. It is concluded that the enzymes with identical specificities are present in both the crystalliferous and acrystalliferous strains of the same subspecies.

[Testing and isolation of high-purity restriction endonucleases]. / Testirovanie i vydelenie vysokoochishchennykh éndonukleaz restriktsii.

A new method of testing restriction nucleases is proposed. This method is based on high-temperature treatment of crude cell extracts. Disrupted cells were heated at 50-60 degrees C, centrifuged, and assayed for restrictases. This method provides the opportunity for screening new enzymes in microbial strains enriched with nonspecific restrictases. High-temperature treatment of cell extracts of certain producers reduces the number of steps of the procedure used for isolating high-purity restrictases; the resulting preparations are capable of maintaining high enzymatic activity during long-term storage. It was shown that high-temperature treatment can be applied not only to thermophilic but also to mesophilic strains of microorganisms of different taxa.