Abstract
Thai (CGCG) sites which overlap Hhal (GCGC) sites in OC174 and pBR322 DNA were methylated in vitro with Hhal methylase and S-adenosylmethionine to yield CG^G, "CGCG or "CG^CC (5-methylcytosine, m C) . Methylation of either cytosine in the Thai recognition sequence rendered the DNA resistant to Thai cleavage. Rat pituitary cell genomic DNA was digested with Thai or 2 other known methylation-sensitive enzymes, Aval or Xhol. After electrophoresis and ethldium bromide staining of the DNA, all 3 enzymes showed the infrequent DNA cleavage characteristic of methylation-sensitive enzymes. Comparison of pituitary growth hormone (GH) genes bearing strainspecific degrees of methylation showed the less methylated gene to be more frequently cut by either Aval or Thai. Thai resistant sites in GH genes were cleaved by Thai after exposing cells to 5-azacytidine, an inhibitor of DNA methylation. We conclude that Thai is a useful restriction enzyme for the analysis of "C at CGCG sequences in eukaryotlc DNA.
INTRODUCTION
Thai is a type II restriction enzyme isolated from the thermophilic mycoplasma Thermoplasm acidophilum (1) .
INTRODUCTION
This enzyme cleaves DNA in the middle of the sequence CGCG. As most of the 5-methylcytosine residues in mammalian DNA occur in CpG dinucleotide sequences (2) , Thai may be a useful probe for DNA containing methylated cytosines. A large number of restriction enzymes fail to cut DNA at recognition sequences that have been modified by the conversion of adenine to 6-N-methyladenine or cytosine to 5-methylcytosine (3) . The failure of a methylation-sensitive restriction enzyme to cut genomic DNA at a known recognition sequence can be detected after electrophoresis using Southern blotting and DNA hybridization assays (4) , and is evidence that a methylated base occurs within that sequence.
INTRODUCTION
Initial characterization of Thai showed it did not digest T. acidophilum DNA (1) . This is frequently indicative of an existing host restriction-modification system (5) . We have therefore investigated the ability of Thai to cut 5-methylcytosine modified DNA. 
MATERIALS AND METHODS

Cell

I Denotea the flret bp tn the recognition lequence for Hhal or ThaT! Poeition "0" i» the «ite of EcoRl cltavage. Sequence data ie froa (K). Tb« aequanct of the overlapping Hhal and


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