DNA methylation patterns associated with asparagine synthetase expression in asparagine-overproducing and -auxotrophic cells.
In Chinese hamster ovary cells, the gene for asparagine synthetase, which spans 20 kilobase pairs, was found to contain a cluster of potential sites for CpG methylation in a 1-kilobase-pair region surrounding the first exon. Fourteen of the sites that could be assayed for methylation by MspI-HpaII digestions were found in this region, with an additional nine MspI sites spread throughout the remainder of the gene. The methylation status of the gene was analyzed in a series of cell lines that differed in the amount of asparagine synthetase activity. The level of expression showed a direct correlation with the extent of methylation of a subset of the MspI sites found in the 5' region of the gene. The rest of the gene was completely methylated in most cell lines. Wild-type cells, which expressed a basal level of asparagine synthetase activity, were partially demethylated in the 5' region. In contrast, asparagine-requiring N3 cells, which lacked detectable mRNA for asparagine synthetase, were methylated throughout the entire gene. Spontaneous revertants of strain N3, selected for growth in asparagine-free medium, exhibited extensive hypomethylation of the asparagine synthetase gene. The methylation pattern of the gene in cell lines that overproduced the enzyme was also examined. Albizziin-resistant cell lines, which had amplified copies of the gene, were extensively demethylated in the 5' region. Overexpression of asparagine synthetase in beta-aspartyl hydroxamate-resistant lines without amplified copies of the gene was also correlated with DNA hypomethylation.
Andrulis
I L
IL
Research Institute, Mt. Sinai Hospital, Ontario, Canada.
Barrett
M T
MT
D000818:Animals; D001216:Asparagine / Q000378:metabolism; D001217:Aspartate-Ammonia Ligase / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D015342:DNA Probes; D005091:Exons; D005784:Gene Amplification; D005786:Gene Expression Regulation; D015151:Immunoblotting; D008025:Ligases / Q000096:biosynthesis*; D008745:Methylation; D009154:Mutation; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics
D015342:DNA Probes; D012333:RNA, Messenger; D001216:Asparagine; D004247:DNA; D008025:Ligases; D001217:Aspartate-Ammonia Ligase