HineI is an isoschizomer of HinfIII restriction endonuclease

HineI is a restriction enzyme isolated from Haemophilus influenzae strain Re. Like other type III restriction endonucleases it requires ATP for cleavage and S-adenosyl-methionine for methylation of DNA. This enzyme recognises the same sequence as HinfIII (Piekarowicz et al., 1981) and cleaves and methylates DNA in a manner similar to all type III restriction enzymes.     

Isolation of a new restriction enzyme,ApaCI, an isoschizomer ofBamHI produced byAcetobacter pasteurianus

A new Type II restriction endonucleaseApaCI purified fromAcetobacter pasteurianus is an isoschizomer ofBamHI that cleaves at the nucleotide sequence 5′-G/GATCC-3′ of double-stranded DNA. The single restriction activity present in this strain permits rapidly purified 30 000 units of cleavage activity from 10 g of freshly harvested cells. The resultingApaCI preparation is free of contaminant nuclease activities that might interfere within vitro manipulation of DNA.     

Purification and characterization of the restriction endonuclease RsrI, an isoschizomer of EcoRI

Rhodobacter sphaeroides strain 630 produces restriction enzyme RsrI which is an isoschizomer of EcoRI. We have purified this enzyme and initiated a comparison with the EcoRI endonuclease. The properties of RsrI are consistent with a reaction mechanism similar to that of EcoRI: the position of cleavage within the -GAATTC-site is identical, the MgCl2 optimum for the cleavage is identical, and the pH profile is similar. Methylation of the substrate sequence by the EcoRI methylase protects the site from cleavage by the RsrI endonuclease. RsrI cross-reacts strongly with anti-EcoRI serum indicating three-dimensional structural similarities. We have determined the sequence of 34 N terminal amino acids for RsrI and this sequence possesses significant similarity to the EcoRI N terminus.     

Bst71I: an isoschizomer of the type-IIS restriction enzyme, BbvI, recognizing the GCAGC(8/12) site.

A new type-IIS restriction enzyme, Bst71I, with the specificity 5'-GCAGC(N)8/3'-CGTCG(N)12 was isolated from Bacillus stearothermophilus (Promega No. 71). This enzyme is an isoschizomer of BbvI with somewhat improved characteristics for use by molecular biologists.     

Isolation of restriction enzyme EcoVIII, an isoschizomer of HindIII, produced by Escherichia coli E1585-68

A restriction endonuclease designated EcoVIII, an isoschizomer of HindIII, was isolated from Escherichia coli E1585-68 and purified by dextran-polyethylene glycol (DPG) phase partition, ammonium sulfate precipitation, phospho- and DEAE-cellulose chromatography, and hydroxylapatite chromatography. The purified EcoVIII was stable during the purification procedure and its high specific activity required 10 mM Mg²⁺. Unlike HindIII, Eco VIII exhibited a high specific activity even at low pH (pH 6.3) and showed the highest activity at 48° C. Transformation of purified plasmid DNA from E. coli E1585-68 into K-12 indicated that the EcoVIII gene was carried on a multicopy 4.4-kb miniplasmid. EcoVIII seems to be preferable to HindIII for its production and use because of easier handling of producer cells and a wider range of activity.     

A new isoschizomer, BnaI, of the BamHI restriction endonuclease

By assaying the yield of phage SPO1 we have identified a new restriction-modification activity in the Bacillus natto B3364 strain. A class II restriction endonuclease, BnaI, isolated from the crude extract of B3364 cells was shown to be a true isoschizomer of the BamHI endonuclease. The Mr, stability and optimal conditions required for DNA digestion were determined for BnaI. Although both enzymes show the same specificity, BnaI and BamHI differ from each other in all the properties specified above.     

Site-specific endonuclease NspLKI is an isoschizomer of endonuclease HaeIII

Site-specific endonuclease NspLKI has been isolated and purified to functionally pure state from soil bacterium Nocardia species LK by successive chromatography on columns with phosphocellulose, HTP hydroxyapatite, and heparin-Sepharose. The isolated enzyme recognizes the 5'-GG downward arrowCC-3' sequence on DNA and cleaves it as indicated by the arrow, i.e., it is an isoschizomer of HaeIII. The final enzyme yield is 1.105 units per gram of wet biomass. The enzyme is active in the temperature range of 25-60 degrees C with an optimum at 48-55 degrees C; it does not lose activity on storage for three days at room temperature. An optimal buffer is HRB containing 10 mM Tris-HCl, pH 7.4, 200 microgram/ml albumin, 10 mM MgCl2, and 100 mM NaCl.     

Widespread occurrence of the restriction endonuclease YenI, an isoschizomer of PstI, in Yersinia enterocolitica serotype O8.

The cold-active restriction endonuclease YenI, an isoschizomer of PstI, was found in 12 of 14 Yersinia enterocolitica serotype O8 strains of different origins, but not in other serotypes of Y. enterocolitica, Yersinia pseudotuberculosis, or Yersinia pestis. In spite of the limited number of strains tested, the result suggests that the detection of YenI endonuclease or the gene might result in more rapid determination of the prominently pathogenic serotype of Y. enterocolitica.     

Widespread occurrence of the restriction endonuclease YenI, an isoschizomer of PstI, in Yersinia enterocolitica serotype O8

The cold-active restriction endonuclease YenI, an isoschizomer of PstI, was found in 12 of 14 Yersinia enterocolitica serotype O8 strains of different origins, but not in other serotypes of Y. enterocolitica, Yersinia pseudotuberculosis, or Yersinia pestis. In spite of the limited number of strains tested, the result suggests that the detection of YenI endonuclease or the gene might result in more rapid determination of the prominently pathogenic serotype of Y. enterocolitica.     

High-performance liquid chromatography for the purification of restriction endonucleases, application to BanII, SacI, and SphI

Conventional fractionation methods are time consuming, thus they prolong the time required to process low-stability restriction enzymes. We now report a rapid and effective two-step chromatographic method that affords high purity endonucleases in a short time. Accordingly, an inexpensive chromatographic adsorbent such as phosphocellulose or dyed agarose in the first step is coupled to a high-performance ion exchanger, namely, MonoQ, in the second step. The purification schemes reported here are now in routine use to prepare high-purity BanII, SacI, and SphI as judged by the "overdigestion" and "cut-ligate-recut" stringent quality tests.