Cloning and sequence comparison of AvaI and BsoBI restriction-modification systems

Abstract

AvaI and BsoBI restriction endonucleases are isoschizomers which recognize the symmetric sequence 5'CYCGRG3' and cleave between the first C and second Y to generate a four-base 5' extension. The AvaI restriction endonuclease gene (avaIR) and methylase gene (avaIM) were cloned into Escherichia coli by the methylase selection method. The BsoBI restriction endonuclease gene (bsoBIR) and part of the BsoBI methylase gene (bsoBIM) were cloned by the "endo-blue" method (SOS induction assay), and the remainder of bsoBIM was cloned by inverse PCR. The nucleotide sequences of the two restriction-modification (RM) systems were determined. Comparisons of the predicted amino acid sequences indicated that AvaI and BsoBI endonucleases share 55% identity, whereas the two methylases share 41% identity. Although the two systems show similarity in protein sequence, their gene organization differs. The avaIM gene precedes avaIR in the AvaI RM system, while the bsoBI R gene is located upstream of bsoBI M in the BsoBI RM system. Both AvaI and BsoBI methylases contain motifs conserved among the N4 cytosine methylases.