CHARACTERIZATION OF ADDITIONAL HOST RESTRICTION-MODIFICATION SYSTEMS IN THE UNICELLULAR CYANOBACTERIUM CYANOTHECE SP

In order to develop a gene transfer system for the unicellular diazotr ophic cyanobacterium Cyanothece sp. strain BH68K, this organism has be en further investigated for the presence of additional host restrictio n-modification enzymes other than Csp68KI, previously reported for Cya nothece sp. Analysis of cell extracts by phosphocellulose and Mono Q f ast protein liquid chromatography (FPLC) has led to the identification of three new restriction endonucleases. These enzymes have been desig nated Csp68KII, Csp68KIII, and Csp68KVI. Csp68KII is an isoschizomer o f AsuII and restricts DNA at the recognition sequence 5'-TT/CGAA-3'. C leavage occurred between thymine and cytosine producing 2 bp 5' overha ng ends. The third restriction endonuclease, Csp68KIII, is an isoschiz omer of AvaIII and restricts DNA at the recognition sequence 5'-ATGCA/ T-3'. Cleavage occurred between the 3' adenosine and thymine nucleotid es producing 4 bp 3' overhang ends. The fourth enzyme identified, Csp6 8KVI, recognizes CGCG and cleaves this sequence between the internal g uanine and cytosine nucleotides producing blunt ends. (C) 1996 Academi c Press, Inc.