[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 role of divalent cations in DNA endonucleolysis catalyzed by restrictases]. / Rol' dvukhvalentnykh kationov v reaktsii éndonukleoliza DNK restriktazami.

Electrophoretic analysis of products obtained after hydrolysis of phage lambda DNA by means of restrictase Pae I was carried out after preincubation of DNA or the enzyme with Mg2+ as well as after preincubation of DNA simultaneously with the enzyme and the subsequent addition of the required components into the experimental samples. The analysis showed that Mg2+ were apparently not required for the enzyme-substrate complex formation and caused destabilization of the complex. Restrictase Pae I was in active form when Mg2+ was substituted by Mn2+, Ca2+, Zn2+, Co2+ but not by Cd2+, Cu2+ or Ni2+. Experiments with o-phenanthroline showed that Zn2+ cations are of importance in catalytic activity of restrictase Pae I. Possible functions of Zn2+ in protein-nucleic acids recognition are discussed.

[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.

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).

[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.

[New means of isolating restriction endonuclease preparations using organic solvents]. / Novyi sposob vydeleniia ochishchennykh preparatove restriktsionnykh éndonukleaz s ispol'zovaniem organicheskikh rastvoritelei.

A new procedure is developed for isolation of highly purified preparations of restrictional endonoucleases Bam HI and Eco RI by means of fractionation with isopropyl alcohol. Restrictional endonuclease Bam HI, practically free of unspecific nucleases, was isolated after ultrasonic destruction of cells, precipitation of the restrictases with isopropanol and chromatography on DEAE cellulose. Additional chromatography on hydroxyapatite enabled to obtain the homogenous preparation of Bam HI restrictase, as shown by polyacrylamide gel disc electrophoresis. Other organic solvents (acetone, ethanol) might be also used for purification of the restrictional endonucleases.

[Metabolism of L-albizziin in rat liver slices]. / Obmen L-al'bitstsiina v srezakh pecheni krysy.

During incubation of rat liver slices with L-albizziin (L-alpha-amino-beta-ureidopropionic acid) formation of the UV-absorbing compound was observed. This compound was isolated from the incubating medium; it possessed two peaks of maximal absorption--at 206-207 nm and 264-265 nm, but a single minimal value at 242-243 nm. The presence of ribose as a constituent of the UV-absorbing compound was demonstrated. Two possible mechanisms explaining formation of the UV-absorbing compound were proposed: 1) metabolic conversion of L-albizziin, and 2) inhibition of purine biosynthesis de novo. The latter mechanism appears to be more probable.