Expression of the human apolipoprotein A-I gene in rat myogenic L6E9 cells. DNA methylation and regulation of gene activity.
An 11-kilobase EcoRI fragment containing the human apoA-I gene was cloned in the pSV2gpt vector. The recombinant plasmid was then used to transfect a rat myogenic cell line (L6E9) and HeLa cells. Selection and analysis of cell clones resistant to hypoxanthine/aminopterin/thymidine and mycophenolic acid showed that the apoA-I gene is expressed in L6E9 but not in HeLa cells. The apoA-I mRNA transcribed by either the transfected L6E9 or control human liver cell line, HepG2, has a major transcription initiation site located 26 nucleotides downstream from the CATAAA box. The apoA-I synthesized by the L6E9 cells is secreted into the culture medium and is indistinguishable from the apoA-I secreted by HepG2 cells. These results demonstrate that the L6E9 cells contain the necessary factors required for the expression of the apoA-I gene. Further analysis showed that the endogenous rat apoA-I gene is not expressed in L6E9 cells and exists in a hypermethylated configuration. In contrast, the exogenous human apoA-I gene was found to be hypomethylated in the transfected L6E9 cells and in other apoA-I-producing cells. These findings are consistent with the hypothesis that the expression of the endogenous rat apoA-I gene in L6E9 cells is prevented by hypermethylation, suggesting that compared to other mechanisms which control initiation of transcription, DNA methylation may play a higher hierarchical role in the regulation of expression of eukaryotic genes.
Ruiz-Opazo
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Department of Medicine, Boston University Medical Center, Massachusetts 02118.
Zannis
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D000818:Animals; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D004262:DNA Restriction Enzymes / Q000378:metabolism; D005786:Gene Expression Regulation; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D009132:Muscles / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D004262:DNA Restriction Enzymes