The 5′-cytosine in CCGG¹is methylated in two eukaryotic DNAS and Msp I is sensitive to methylatlon at this site

Abstract

Novikoff rat hepatoma and bovine liver DNAs were digested with Msp I or Hpa II. Restriction fragments were end-labeled using [alpha-32P]-dCTP and the Klenow fragment of E. coli DNA polymerase I and then digested to 2'-deoxyribonucleoside-3'-monophosphates using micrococcal nuclease and spleen phosphodiesterase. Mononucleotides were separated by two-dimensional thin layer chromatography, localized by radioautography, and the [32P]-label quantitated by scintillation spectrometry. This method, based on known specificities of Msp I and Hpa II, shows that CCGG, CMGG, and MCGG (M refers to 5-methylcytosine) occur at frequencies of 89.6%, 1.4%, and 9.0%, respectively, in the rat DNA and at 41.6%, 48.3%, and 10.0%, respectively, in the bovine DNA. [32P] recovery in 3'-5-MedCMP from end-labeled Msp I digests was negligible compared to recovery from Hpa II digests. Hence, Msp I is sensitive to methylation at the 5' cytosine in the sequence CCGG.

Clonal inheritance of the pattern of DNA methylation in mouse cells.

“…This cytosine is partially methylated in higher plants. The results of this experiment are especially puzzling because it has been reported that this cytosine is methylated in a tissue-specific manner at Msp I sites (23), including those at the 5' ends of the human -A and yv genes (21 (26). Furthermore, it has been shown that on the average, active genes are about 50% undermethylated at all C-G residues (27).…”

Active beta-globin gene transcription occurs in methylated, DNase I-resistant chromatin of nonerythroid chicken cells.

“…The bands at 1.22 and 3.7 kb are probably explained by polymorphism at a second MspI site at the other end of the intron (Fig. 7), although the presence of mCpC in some of the sites may also be the explanation (68).…”

DNA Methylation--A Regulatory Signal in Eukaryotic Gene Expression

“…In eukaryotes the modified base 5-mC (C*) occurs predominantly in the sequence 5'-C*pG-3' (Dosko~il & ~orm, 1962;Grippo et al, 1968), but 5-mC has also been found in 5'-C*pC-3' (Salomon & Kaye, 1970;Sneider, 1980;Gruenbaum et al, 1981;Romanov & Vanyushin, 1981) and 5'-C*pT-3' sequences (Salomon & Kaye, 1970;Sneider, 1972). The degree of methylation of C in these sequences varies greatly depending on many parameters, such as species, type of gene and stage of development.…”

“…Whenever the internal C-residue of the sequence is modified to 5'-CC*GG-3' by methylation, the endonuclease HpaII cannot cut, whereas MspI does cut at such sites. Conversely, methylation of the external C, 5'-C*CGG-3', renders MspI refractory, while the endonuclease HpaII remains fully active (Sneider, 1980). The availability of this isoschizomeric pair of restriction enzymes with a complementary refractoriness for methylation facilitates the elucidation of methylation patterns in specific genes.…”