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In vitro Methylation of the Hamster Adenine Phosphoribosyltransferase Gene Inhibits Its Expression in Mouse L Cells
R. Stein, A. Razin and H. Cedar
Proceedings of the National Academy of Sciences of the United States of America
Vol. 79, No. 11, [Part 1: Biological Sciences] (Jun. 1, 1982), pp. 3418-3422
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/12293
Page Count: 5
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The effect of DNA methylation on the expression of the hamster adenine phosphoribosyltransferase (aprt) gene in mouse cells has been examined. This gene was methylated in vitro at all of its C-C-G-G sites by using Hpa II methylase and was inserted into mouse Ltk- aprt- L cells by cotransformation, with the herpes virus thymidine kinase gene as a selectable vector. Whereas clones carrying unmethylated aprt sequences were found to have an aprt+ phenotype as shown by their ability to grow in azaserine-containing medium, almost all clones carrying methylated aprt sequences were shown to be phenotypically aprt-. Blot hybridization analysis demonstrated that both the methylated and unmethylated aprt sequences were integrated into the cellular genome to the same extent and that the in vitro modification was stably maintained in these cells for many generations. When clones containing methylated aprt genes were exposed to conditions that select for the expression of the aprt gene, a low frequency of reversion to the aprt+ phenotype was observed. In all of these clones, this reversion was accompanied by reorganization and undermethylation of the aprt sequences. These results show that the expression of certain genes may be inhibited by site-specific methylation of these sequences and suggest that methylation may play a direct role in the regulation of gene expression.
Proceedings of the National Academy of Sciences of the United States of America © 1982 National Academy of Sciences