Cloning and expression of the ApaLI, NspI, NspHI, SacI, ScaI, and SapI restriction-modification systems in Escherichia coli

The genes encoding the ApaLI (5'-G TGCA-C-3'), NspI (5'-RCATG Y-3'), NspHI (5'-RCATG Y-3'), SacI (5'-GAGT C-3'), SapI (5'-GCTCTTCN1 -3', 5'- N4GAAGAGC -3') and ScaI (5'-AGT ACT-3') restriction-modification systems have been cl oned in E. coli. Amino acid sequence comparison of M .ApaLI, M. NspI, M. Ns pHI, and M. SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI r estriction-modification systems are highly homologous in amino acid sequenc e. The C-termini of the NspI and NlaIII (5'-CATG-3') restriction endonuclea ses share significant similarity. 5mC modification of the internal C5 in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the s econd base in the sequence 5'-GCTCTTC-3' blocks SapI digestion. N4mC modifi cation of the other cytosines in the SapI site does not affect SapI digesti on. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase ho molog was found adjacent to the ApaLI restriction-modification system. A DN A transposase subunit homolog was found upstream of the SapI restriction en donuclease gene.