Cloning of the MspI modification enzyme. The site of modification and its effects on cleavage by MspI and HpaII.
The gene for the MspI modification enzyme from Moraxella was cloned in Escherichia coli using the plasmid vector pBR322. Selection of transformants carrying the gene was based on the resistance of the modified plasmid encoding the enzyme to cleavage by MspI. Both chromosomal and plasmid DNA were modified in the selected clones. None of the clones obtained produced the cognate restriction enzyme which suggests that in this system the genes for the restriction enzyme and methylase are not closely linked. Crude cell extracts prepared from the recombinant strains, but not the host (E. coli HB101), contain an S-adenosylmethionine-dependent methyltransferase specific for the MspI recognition site, CCGG. Production of the enzyme is 3-4-fold greater in the transformants than in the original Moraxella strain. 5-Methylcytosine was identified as the product of the reaction chromatographically. The outer cytosine of the recognition sequence, *CCGG, was shown to be the site of methylation by DNA-sequencing methods. This modification blocks cleavage by both MspI and its isoschizomer HpaII. HpaII, but not MspI, is able to cleave the unmethylated strand of a hemimethylated substrate. The relevance of these results to the use of MspI and HpaII to analyze patterns of methylation in genomic DNA is discussed.
D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000235:genetics* / Q000378:metabolism; D019003:Deoxyribonuclease HpaII; D004926:Escherichia coli; D008780:Methyltransferases / Q000378:metabolism; D009016:Moraxella / Q000201:enzymology*; D010957:Plasmids; D012436:S-Adenosylmethionine / Q000378:metabolism
D012436:S-Adenosylmethionine; D008780:Methyltransferases; D004262:DNA Restriction Enzymes; D019003:Deoxyribonuclease HpaII