ApaCl, an isoschizomer of BamHl isolated fromAcetobacter pasteurianus

ApaCI, type n restriction endonuclease has been isolated from Acetobacter pasteurianus species. Strain was grown at 28°C in YPG cultivation medium. The cells were harvested and disrupted by sonication. The enzyme was separated and purified by chromatography on Phosphocellulose P-23, Blue-Sepharose, and DEAE cellulose DE-52. The enzyme was free of contaminating nuclease activity. Optimal conditions for enzyme activity are 100 mM NaCl, 10 mM Tr i s -HCl (pH 8.0), 10 mM 2-mercaptoethanol at 37°C. The enzyme cleaved lambda, M13mpl8, pBR322, and pUC18 DNAs at 5, 1, 1 and 1 site respectively (Figure 1). A comparison a cleavage patterns of lambda, pBR322, pUC18 and 0X174 by ApaCI to BamHI restriction endonucleases confirmed the recognition sequence 5'-GGATCC-3'. The cleavage site within the recognition sequence a pTZ19 derivative with an insert containing a ApaCI cleavage site, was digested by the enzyme and 5'-terminal nucleotides were labeled with [T 3 2 P]ATP using T4 polynucleotide kinase. The dideoxy sequencing reactions (1) were performed at this region using universal primer and run in parallel with the labeled fragment. Result in Figure 2 show that the labeled fragment 0ane I) comigrates with the band corresponding to the G in the 5'...GGATCC... 3' sequence. From the results obtained above, the restriction endonuclease ApaCI from Acetobacter pasteurianus strain C is an isoschizomer of BamHI (2) and recognizes and cleaves the following palindromatic hexanucleotide DNA sequences: Figure 1. ApaCI digest, lane 3, lambda DNA; lane 4, pBR322; lane 5, pUC18; lane 6, M13mpl8. lane 1, lambda DNA-HindDH molecular size standard; lane 2, lambda DNA-BamHI.