[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 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.

[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.

[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.

[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.

[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.

[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.

A site-specific endonuclease from Pseudomonas aeruginosa.

PaeI, a new restriction endonuclease from Pseudomonas aeruginosa clinical strain was isolated and characterized. It recognizes and cleaves the sequence 5'-GCATG reduced C-3' generating DNA fragments with 3'-tetranucleotide sticky ends. DNAs of pBR322, SV40 and bacteriophage lambda have one, two and six PaeI recognition sites, respectively. Seventy-two strains of Pseudomonas, Clostridium, Escherichia coli, Shigella, Proteus and Saccharomyces were screened for the presence of site-specific endonucleases. Here we describe the PaeI restriction enzyme found in Pseudomonas aeruginosa; other data will be published elsewhere. Earlier Hinkle and Miller isolated from P. aeruginosa a PaeR7 restriction endonuclease recognizing and cleaving a sequence 5'-C reduced TCGAG-3' (1). Sequence analysis of DNAs cleaved by PaeI shows that the enzyme is the isoschizomer of SphI (2).