CLONING OF THE BSSHII RESTRICTION-MODIFICATION SYSTEM IN ESCHERICHIA COLI - BSSHII METHYLTRANSFERASE CONTAINS CIRCULARLY PERMUTED CYTOSINE-5 METHYLTRANSFERASE MOTIFS

摘要

BssHII restriction endonuclease cleaves 5'-GCGCGC-3' on double-stranded DNA between the first and second bases to generate a four base 5' overhang. BssHII restriction endonuclease was purified from the native Bacillus stearothermophilus H3 cells and its N-terminal amino acid sequence was determined. Degenerate PCR primers were used to amplify the first 20 codons of the BssHII restriction endonuclease gene. The BssHII restriction endonuclease gene (bssHIIR) and the cognate BssHII methyltransferase gene (bssHIIM) were cloned in Escherichia cell by amplification of Bacillus stearothermophilus genomic DNA using PCR and inverse PCR. BssHII methyltransferase (M.BssHII) contains all 10 conserved cytosine-5 methyltransferase motifs, but motifs IX and X precede motifs I-VIII. Thus, the conserved motifs of M.BssHII are circularly permuted relative to the motif organizations of other cytosine-5 methyltransferases. M.BssHII and the noncognate multi-specific phi BssHII methyltransferase, M.phi BssHII [Schumann, J. et al, (1995) Gene, 157, 103-104] share 34% identity in amino acid sequences from motifs I-VIII, and 40% identity in motifs IX-X. A conserved arginine is located upstream of a TV dipeptide in the N-terminus of M.BssHII that may be responsible for the recognition of the guanine 5' of the target cytosine. The BssHII restriction endonuclease gene was expressed in E. coli via a T7 expression vector.