The extent of methylation of the internal C in the sequence CCGG in DNA from various eukaryotic sources has been determined using the restriction enzyme MspI known to be specific for this sequence. The methylation of the CCGG sequence is reflected in the restriction pattern obtained by DNA treated with MspI and its isoschizomer HpaII and analyzed by gel electrophoresis. A direct method for detection 5-methylcytosine in the sequence CCGG has been deviced. DNA fragments obtained with MspI were radioactively labeled at their 5' ends and subsequently degraded to the corresponding 5'-deoxyribonucleoside monophosphates. 5 methylcytidylic acid has been found in most of the 5' ends of MspI fragments of calf thymus DNA (about 90%) indicating heavy methylation of the sequence CCGG in calf thymus DNA. The results also reveal a symmetric methylation of both strands at this sequence in calf thymus DNA. In contrast, the CCGG sequence in other eukaryotic DNAs from organisms like Neurospora, Drosophila and Herpes virus proved to be undermethylated at this sequence.
The restriction enzyme Msp I is inhibited by the presence of a methyl moiety at the external cytosine of the sequence CCGG, but is generally unaffected by methylation at the int...
MspI essentially fails to cut the sequence GGCmCGG at enzyme concentrations which give total digestion of CCGG, CmCGG and GGCCGG sites. This result explains why certain sites in...
Different states of eukaryotic gene expression are often correlated with different levels of methylation of DNA sequences containing structural genes and their flanking regions....
A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are gener...
A small portion of the cytosine residues in the DNA of higher eukaryotes as well as in that of many lowe eukaryotes if methylated. The resulting 5-methylcytosine residues occur ...
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