Somatic cell genetic and biochemical characterization of cell lines resulting from human genomic DNA transfections of Chinese hamster ovary cell mutants defective in sterol-dependent activation of sterol synthesis and LDL receptor expression.
We have isolated several non-leaky mutant Chinese hamster ovary (CHO) cell clones (M4, M19, and M21) requiring cholesterol and unsaturated fatty acid for growth. These mutants belong to the same complementation group as the mutant M1 cells previously reported from this laboratory. M19 cells reverted to lipid prototrophy at very low frequency and were chosen as recipients to perform DNA-mediated gene-transfer experiments using total human genomic DNAs. Biochemical characterization of these transfectant clones indicated that, unlike their parental M19 cells, they were able to exhibit activation of cholesterol biosynthesis and LDL receptor expression in response to sterol removal from the growth medium. RNA blotting analysis indicated that these transfectants were able to increase HMG-CoA synthase gene transcripts in response to sterol removal. From the genomic DNAs of a representative secondary transfectant cells, we cloned a unique human DNA fragment (designated as h lambda 2) and showed that h lambda 2 closely linked with the presumptive human M1 gene.
Hasan
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Department of Biochemistry, Dartmouth Medical School, Hanover, New Hamsphire 03755-3844.
Chang
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Chang
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D000666:Amphotericin B / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media; D004247:DNA / Q000235:genetics; D005720:Gamma Rays; D005786:Gene Expression Regulation; D005816:Genetic Complementation Test; D008040:Genetic Linkage; D006801:Humans; D006822:Hybrid Cells; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000235:genetics; D016296:Mutagenesis; D009154:Mutation; D011973:Receptors, LDL / Q000096:biosynthesis* / Q000235:genetics; D013261:Sterols / Q000096:biosynthesis / Q000378:metabolism*; D014162:Transfection
D003470:Culture Media; D011973:Receptors, LDL; D013261:Sterols; D000666:Amphotericin B; D004247:DNA; D006903:Hydroxymethylglutaryl CoA Reductases; D006904:Hydroxymethylglutaryl-CoA Synthase