Restriction enzyme Hincll is sensitive to methylation of cytosine that occurs 5' to the recognition sequence

摘要

In this paper we demonstrate that the Hindi restriction endo-nuclease, in addition to being sensitive to methylation of the 3' A and C residues, is also sensitive to methylation of a cytosine immediately 5' to the recognition sequence. Having encountered this property in one of the sites in the mouse c-fos gene, we confirmed the sensitivity of Hindi to the 5' cytosine methylation in in vitro methylated pUC12, pBR322 and pfos-l plasmids. Hindi is a six base-cutter which recognises the sequence GTPyPuAC, the cleavage site being between the Py/Pu. Its activity is known to be sensitive to the methylation of the A residue in the sequence (1). Recently. Bull et at. (2) used plasmid constructs having GTCGACG sequence for Hindi digestion and showed that thepresence of methylated cytosine flanked by a G at the 3' end of recognition sequence inhibits Hindi digestion. They also showed that methylation in the internal CpG did not affect the digestion. However, the Hindi sensitivity to C methylation has not been universally accepted (see ref. 3). Here. we report that in the DNA motif CGTCGACC. Hindi digestion is sensitive to methylation of the 5'C which is not a part of its recognition sequence. We also confirm that methylation of the internal C has no effect on the digestion by Hindi.