[Site-specific BcuAI endonuclease from Bacillus cereus A]. / Sait-spetsificheskaia éndonuleaza BcuAI iz Bacillus cereus A.

A new restriction endonuclease was isolated from the Bacillus cereus BKM B-814 by means of the cell disruption with ultrasonication, ammonium sulfate fractionation of the cell-free extract, and chromatography on DEAE-Sepharose to give about 1400 U of the enzyme per gram of cells. The enzyme revealed the maximum activity at 30-37 degrees C, pH 7.6-8.2, and 5-10 mM MgCl2 under a high ionic strength (50 mM Tris-HCl, 100 mM NaCl). The site-specific endonuclease BcuAI was found to recognize the 5' G decreases G(A/T)CC sequence in double-stranded DNA and cleave it as shown with the arrow, thus being a true isoschisomer of the AvaII restriction endonuclease.

[Bsp153AI and BspM39I--new isoschizomers of PvuII restriction enzyme]. / Bsp153AI i BspM39I--novye izoshizomery restriktazy PvuII.

New restriction endonucleases, Bsp153AI and BspM39I, were isolated from Bacillus species strains 153A and M39, respectively. The enzymes recognize and cleave the nucleotide sequence [sequence: see text] and are true isoschizomers of restriction endonuclease PvuII.

[Isolation, purification, and characteristics of the new restriction enzymes BciBI and BciBII, produced by Bacillus circulans]. / Vydelenie, ochistka i kharakteristika novykh restriktaz BciBI i BciBII, produtsiruemykh Bacillus circulans.

New site-specific endonucleases BciBI and BciBII have been detected in Bacillus circulans. The enzymes were purified by fractionation of cell-free extract with polyethylene imine and ammonium sulphate (40-80% of saturation) followed by chromatography on DEAE-sepharose, blue-sepharose and phosphocellulose. The endonucleases BciBI and BciBII were separated only at the final step of the purification--by chromatography on the phosphocellulose column. The yields of BciBI and BciBII were 600 and 10,000 U/g of cells. It was found that restriction endonucleases BciBI and BciBII are isoschizomers of ClaI and BstNI, respectively.

[New site-specific endonucleases from strains of Bacillus]. / Novye sait-spetsificheskie éndonukleazy iz shtammov Bacillus.

In a search for new restriction endonucleases type II, among forty bacterial strains of the Bacillus genus two strains producing site-specific endonucleases have been found. Endonucleases BbvAIII and BspFI, isolated from B. brevis BLM B-677 and B. species F, are shown to be true isoschisomers of BspMII (Kpn2I) and Sau3AI, respectively.

[Specific endonuclease BbvBI from Bacillus brevis]. / Spetsificheskaia éndonukleza BbvBI iz Bacillus brevis.

A new restriction endonuclease BbvBI free from contaminating nonspecific nucleases and phosphatases was isolated from the Bacillus brevis cells. The enzyme was purified by fractionating the sonicated cell-free extract in a two-phase PEG/dextran system and subsequent chromatographies on DEAE-sepharose, blue sepharose and heparin sepharose. The endonuclease BbvBI displayed the maximal activity at 45 degrees C, pH between 8.0 and 8.5, MgCl2 concentration in the range of 5-10 mM and at the low ionic strength. It is shown that the enzyme cleaves the sequence G'GYPC'C, with the preferential cleavage of GGTACC and GGCACC sites as compared with GGTGCC and GGCGCC. Thus, the restriction endonuclease BbvBI is a true isoschizomer of nuclease BanI.

[BsoAI--a new site specific endonuclease from Bacillus coagulans]. / BcoAI--novaia sait-spetsificheskaia éndonukleaza iz Bacillus coagulans.

A new site-specific endonuclease was detected in toluene lysates of Bacillus coagulans AUCM B-732 and designated as BcoAI. The enzyme was purified by fractionation of the cell-free extract in the two-phase PEG/dextran system followed by chromatography on DEAE-sepharose and phosphocellulose and shown to be free of nonspecific nucleases and phosphatases. BcoAI has three cleavage sites on lambda DNA, but does not cleave SV40, pBR322 and pUC19 DNA. BcoAI recognizes the sequence 5' CAC decreases GTG 3' on double-stranded DNA and cleaves it as indicated by the arrow to yield blunt-ended DNA fragments. Thus, BcoAI is a true isoschizomer of PmaCI from Pseudomonas maltophila C.

[Site-specific endonucleases LplI and AagI]. / Sait-spetsificheskie éndonukleazy LplI i AagI.

New site-specific endonucleases LplI and AagI have been isolated from the Lactobacillus plantarum and Achromobacter agile cells, respectively. The enzymes' purification stages included treatment of cell-free extracts with polyethylenimine, fractionation in two-phase system by Albertsson's method, chromatography on blue Sepharose and DEAE-cellulose. The results of cleavage of a 5'-32P-labelled oligodeoxynucleotide duplex by restriction endonucleases LplI and AagI indicate that these enzymes recognize and cut the sequence AT decreases CGAT, being therefore true isoschizomers of the ClaI restriction endonuclease from Caryophanon latum. The L. plantarum strain has 400 fold endonuclease productivity as compared with the ClaI producent and is perspective for preparative isolation of LplI.

[The effect of monovalent cations on the activity of site-specific endonuclease PaeII]. / Vliianie monovalentnykh kationov na aktivnost' sait-spetsificheskoi éndonukleazy PaeII.

Activity of restrictase Pae II, contrary to known restriction enzymes of the II class (except of true isoshizomere Sma), depended absolutely on monovalent cations. This pattern is untypical for restrictases of the II class. At the same time, restrictase Pae II was able to hydrolyze DNA as a substrate in absence of exogenous Mg2+, in the incubation mixture contained cations K+, Rb+, Cs+ and NH4+ but not Na+ or Li+. Mg2+ was found to activate the enzyme in presence of monovalent cations. Basing on the protective effect on K+ against inactivation of restrictase Pae II by means of thiol-affecting reagents and high temperature as well as on stabilization of the enzyme by KCl during storage, monovalent cations appear to participate in formation of protein molecule structure, which is optimal for catalytic effect and resistant to inactivation.

[The search for strains producing new restriction endonucleases]. / Poisk shatmmov-produtsentov novykh éndonukleaz restriktsii.

The search for restrictases in 154 strains belonging to 104 species of 32 genera of microorganisms has been carried out by the method of rapid toluene assay. In 10 strains the activity of endonucleases specifically fragmenting the DNA of phage lambda in the presence of Mg2+ ions has been detected. Restrictases Pae I and Pae II formed by two Pseudomonas aeruginosa strains have been identified as the true isoschizomers of restriction endonucleases Sph I and Sma I respectively. The results of the screening of restrictase-producing strains indicate that the production of restrictases is widely spread among microorganisms of the genus Bacillus.

[Effect of thiol-specific reagents on the activity of PaeI and PaeII restrictases from Pseudomonas aeruginosa]. / Vliianie tiolspetsificheskikh reagnetov na aktivnost' restritaz PaeI i PaeII iz Pseudomonas aeruginosa.

5,5'-dithiobis-2-nitrobenzoic acid, N-ethylmaleimide, and parachloromercuribenzoate have been demonstrated to inhibit the activity of restrictases PaeI and PaeII from Ps. aeruginosa bacterial cells. Restrictase PaeII was more sensitive to the action of thiol-specific reagents, as compared to PaeI. The minimal concentration of reagents for SH-groups that completely inhibited the activity of restrictases PaeI and PaeII was determined. The protective effect against the inhibitory action of 5,5'-dithiobis-2-nitrobenzoic acid on the activity of PaeII was observed after preincubation of these enzymes with phage lambda DNA and Mg2+ cations. It is suggested that restrictase PaeI and PaeII molecules contain SH-groups, essential for the enzymatic activity. They are believed responsible for restrictase binding with DNA substrate.

[New specific endonucleases PaeI and PaeII from Pseudomonas aeruginosa]. / Novye spetsificheskie éndonukleazy PaeI i PaeII iz Pseudomonas aeruginosa.

Specific endonuclease activities have been found it two Pseudomonas aeruginosa strains. Isolation and purification of enzymes and determining their specific activities have permitted one to find out that PaeI is an isoshizomer of SphI and digests the sequence 5'-GCATG C-3'. Another isolated enzyme PaeII is an isoshizomer of SmaI and cleaves DNA in a fragment 5'-CCC GGG-3'. The use of PaeI and PaeII enzymes in genetical engineering and their advantages are discussed.

[Determination of restrictase activity in toluene lysates of bacterial cells]. / Opredelenie aktivnosti restriktaz v toluol'nykh lizatakh bakterial'nykh kletok.

A rapid method for determination of restrictase Bam H1 activity in bacterial cells has been devised. It is based on cell treatment with toluene followed by incubation of toluene lysates with DNA substrate. The method is unsophisticated, well reproducible and requires insignificant amount of biomass necessary for testing the restrictase activity, which makes it compare very favourably with the known methods. The treatment of microbial cells with toluene can serve the first stage in purification of restriction endonucleases.

[Polynucleotide phosphorylase from rat liver: isolation and regulation of its activity by growth hormone]. / Vydelenie polinukleotidfosforilazy iz pecheni krys i izuchenie reguliatsii ee aktivnosti gormonom rosta.

Using ion-filtration chromatography on DEAE-Sephadex A-25, a homogeneous polynucleotide phosphorylase having specific activity of 350--360 u./mg and containing no admixtures of nucleases or phosphatases was obtained. Injection of 32P phosphate to hypophysectomized animals was accompanied by the label incorporation into the enzyme molecule. The data obtained indirectly indicate that the enzyme is activated by phosphorylation and is inactivated by dephosphorylation, both processes being mediated by some factor found in liver cytosol of intact animals.

[Effect of growth hormone on polynucleotide phosphorylase activity in a primary monolayer culture of rat hepatocytes]. / Vliianie gormona rosta na aktivnost' polinukleotidfosforilazy v pervichnoi monosloinoi kul'ture gepatotsitov krys.

Growth hormone (1 microgram/ml) inoculated into the primary culture of rat hepatocytes and incubated for 3 hours at 37 degrees C decreases 2-fold the activity of polynucleotide phosphorylase (PNPase). In the culture of hepatocytes from hypophysectomized rats, the basal activity of PNPase is significantly higher as compared with that in the culture of hepatocytes from intact animals. The most pronounced inhibitory effect of growth hormone on PNPase was seen in the culture of hepatocytes from hypophysectomized animals.

[Importance of the growth hormone in regulating polynucleotide phosphorylase activity in the rat liver]. / Znachenie gormona rosta v reguliatsii aktivnosti polinukleotidfosforilazy v pecheni krys.

Hypophysectomy in rats is accompanied by a significant rise in PNPase activity in ribosomal fractions of the liver. Injection of growth hormone into operated animals produces inhibition of PNPase activity. The linear dependence "dose-response" was recorded with the use of a dosage range of 5 to 100 micrograms/animal. The action of growth hormone was most pronounced 18 hours following its single injection.

[Polyploid hybrids of industrial yeast races and the prospects for using them in production]. / Poliploidnye gibridy proizvodstvennykh ras drozhzhei i perspektivy primeneniia ikh v proizvodstve

The ploidity of the parent forms, hybrids, and two industrial yeast races was studied by determining the content of DNA per cell, the ratios during allel splitting, the dimensions and volumes of the cells. The triploid nature of the baker's race 14--2 and some hybrids was found (for example, the productive hybrid 112 obtained by crossing the distillery and baker's races). The results obtained suggest the possibility to apply the techniques of hybridization and polyploidy for selecting productive yeast strains.