Abstract
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%).
Highlights
Frsm the *Division ofBiology, City ofHope National Medical Center, Duarte, California 91010 and the mepartment of Biochemistry, School of Medicine, University of Southern California, Los Angeles, California 90033
In Escherichia coli, both the lac repressor and restriction enzymes are known to be sensitive to subtle changes in the major groove of the DNA helix [25,26,27,28]
We have argued that the introduction of a &methyl group in the major groove by formation of 5methylcytosine does affect the binding of regulatory proteins and that DNA methylation may have a regulatory function [1]
Summary
Frsm the *Division ofBiology, City ofHope National Medical Center, Duarte, California 91010 and the mepartment of Biochemistry, School of Medicine, University of Southern California, Los Angeles, California 90033 The aim of these experiments was to test whether incorporation of bromodeoxyuridine into DNA affects DNA methylation. The DNA was fractionated into highly repetitive, moderately repetitive, and single copy sequences. 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%). We have measured the extent of incorporation of BrdUrd into repetitive and single copy DNA, since it has been reported previously that in rat and chick embryo cells [13, 14] low concentrations of BrdUrd are selectively incorporated into repetitive and “intermediate”
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