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Characterisation of BciBII, an isoschizomer of BstNI from a strain of Bacillus circulans B.

BciBU, an isoschizomer of BstM (figure 1) has been isolated from Bacillus circulans B. The enzyme was purified by precipitation with polyethylenimine, ammonium sulfate and subsequent column chromatography on DEAE-sepharose, Blue sepharose and phosphocellulose. Restriction endonuclease BciBI showed maximal activity at 37°C, pH between 7.4-7.8, MgCl2 concentration in the range of 8-12 mM and at high ionic strength. The BciBU cleavage site was sequenced according to Brown and Smith (1) using the M13mpl8 ss DNA template, the 'hybridisation primer' and T7 sequenase (2) instead of Klenow enzyme. The product of the primed synthesis reaction was digested with BciBU after heat inactivation of T7 DNA polymerase, one half of it was incubated with Klenow fragment and 4 dNTPs and both mixtures were run in parallel with four standard sequencing reactions on the 5% sequencing gel. The obtained pattern (figure 2) clearly shows that BciBI cleaves the sequence 5'-CC/ATGG-3' as indicated by the arrow and thus is a true isoschizomer of BstM.