ABSTRACT
The distribution and amount of 5-methylcytosine (5-MeCyt) in DNA was measured for early embryos of mouse strain CF1 (2 to 4 cell stage to blastocyst) and mouse teratocarcinoma cells. In each case, the pattern of methylation was examined by use of the restriction enzymes Hha I and HPA II HPA II, which cut DNA at the sites 5'GCGC and 5'CCGG respectively, when the cytosines at these sites are not methylated. Mouse embryo DNA was found to have the same level of methylation as adult mouse tissues, and no changes in methylation were seen during differentiation of the teratocarcinoma cells. The ratio of 5-MeCyt/Cyt in DNA was measured by high performance liquid chromatography for the differentiating teratocarcinoma cells and for several adult mouse and rabbit tissues. The variation between tissues or between teratocarcinoma cells at different stages of differentiation was less than 10 percent. These results are discussed in view of proposals that 5-MeCyt plays a role in differentiation.
Subject(s)
Cytosine/analogs & derivatives , DNA, Neoplasm , DNA , Teratoma/analysis , Animals , Base Sequence , Blastocyst , Cytosine/analysis , Embryo, Mammalian , Female , Male , Methylation , Mice , Neoplasms, Experimental/analysis , Pregnancy , Rabbits , Sex Factors , Species Specificity , Tissue DistributionABSTRACT
The restriction enzymes Hpa II and Msp I both recognize the sequence 5'-CCGG (C, cytosine; G, guanine). However, Hpa II cuts mouse liver DNA to fragments four times larger than does Msp I. The size of DNA cut by Msp I is close to that predicted from base composition and nearest neighbor analysis. The most probable explanation of these results is that in mouse the site 5'-CCGG is highly methylated.
Subject(s)
DNA/metabolism , Animals , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Restriction Enzymes/metabolism , Liver/metabolism , Methylation , Mice , Molecular Weight , Substrate SpecificityABSTRACT
The aim of these experiments was to test whether incorporation of bromodeoxyuridine into DNA affects DNA methylation. Rat hepatoma (HTC) cells in culture were labeled for two generations with [14C]bromodeoxyuridine and [3H]thymidine to yield DNA which was 2.1, 20.6, 52.6, and 95.0% bromodeoxyuridine-substituted in the newly made strands. The DNA then was fractionated into highly repetitive, moderately repetitive, and single copy sequences. As determined by a comparison of 14C and 3H counts per min, the percentage of substitution with bromodeoxyuridine was found to be the same in each repetition class. The 5-methylcytosine content of each fraction was determined using high pressure liquid chromatography. It was found that bromodeoxyuridine, even at a level of substitution into newly mad DNA of 95%, has no effect on the 5-methylcytosine content of DNA. At all levels of bromodeoxyuridine substitution, highly repetitive DNA has slightly more 5-methylcytosine (3.0% of total cytosine) than does single copy DNA or moderately repetitive DNA (2.3%). The 5-methylcytosine content of whole HTC DNA is the same as that of rat liver DNA (2.4%).
