Amplification of the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa protein, in UT-1 cells.

K. L. Luskey, J. R. Faust, D. J. Chin, M. S. Brown, J. L. Goldstein

Research output: Contribution to journalArticle

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Abstract

32P-labeled cDNA probes were used to study levels of genomic DNA and regulation of mRNA for 3-hydroxy-3-methylglutaryl coenzyme A reductase in UT-1 cells, a clone of compactin-resistant Chinese hamster ovary cells that have a 100-1000-fold increase in the amount of reductase protein. Similar measurements were made for the 53-kDa protein, a cytosolic protein of unknown function that is also expressed at high levels in UT-1 cells. The number of copies of the gene for reductase was increased by 15-fold in UT-1 cells as compared to the parental Chinese hamster ovary cells, as judged from Southern gel analysis of restriction endonuclease-digested genomic DNA. In contrast, there was no detectable increase in the number of gene copies for the 53-kDa protein. The amount of cytoplasmic mRNA for both proteins was markedly elevated in UT-1 cells, as determined by filter hybridization studies using 32P-labeled cDNA probes. The amount of mRNA for both reductase and the 53-kDa protein declined in parallel after addition of low density lipoprotein, 25-hydroxycholesterol, or mevalonate to the culture medium. The decline in reductase mRNA was associated with a marked decrease in the rate of [3H]uridine incorporation into hybridizable cytoplasmic mRNA. When UT-1 cells were grown for 3-4 months in the absence of compactin, the level of reductase mRNA and enzymatic activity decreased markedly, but the number of copies of the reductase gene did not decline. When the compactin-withdrawn cells were rechallenged with compactin, high levels of reductase mRNA and enzymatic activity promptly returned. We conclude that the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa protein, has been stably amplified in UT-1 cells. Despite this differential gene amplification, the levels of cytoplasmic mRNA for both gene products are markedly elevated, and both are reduced in parallel by either sterols (low density lipoprotein-cholesterol or 25-hydroxycholesterol) or mevalonate, the product of the reductase-catalyzed reaction.

Original languageEnglish (US)
Pages (from-to)8462-8469
Number of pages8
JournalJournal of Biological Chemistry
Volume258
Issue number13
StatePublished - Jul 10 1983

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Gene Amplification
Amplification
Oxidoreductases
Genes
Messenger RNA
Proteins
Gene Dosage
Mevalonic Acid
Cricetulus
Ovary
Complementary DNA
Cells
3-hydroxy-3-methylglutaryl-coenzyme A
Uridine
DNA
DNA Restriction Enzymes
Sterols
LDL Lipoproteins
LDL Cholesterol
Culture Media

ASJC Scopus subject areas

  • Biochemistry

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Amplification of the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa protein, in UT-1 cells. / Luskey, K. L.; Faust, J. R.; Chin, D. J.; Brown, M. S.; Goldstein, J. L.

In: Journal of Biological Chemistry, Vol. 258, No. 13, 10.07.1983, p. 8462-8469.

Research output: Contribution to journalArticle

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abstract = "32P-labeled cDNA probes were used to study levels of genomic DNA and regulation of mRNA for 3-hydroxy-3-methylglutaryl coenzyme A reductase in UT-1 cells, a clone of compactin-resistant Chinese hamster ovary cells that have a 100-1000-fold increase in the amount of reductase protein. Similar measurements were made for the 53-kDa protein, a cytosolic protein of unknown function that is also expressed at high levels in UT-1 cells. The number of copies of the gene for reductase was increased by 15-fold in UT-1 cells as compared to the parental Chinese hamster ovary cells, as judged from Southern gel analysis of restriction endonuclease-digested genomic DNA. In contrast, there was no detectable increase in the number of gene copies for the 53-kDa protein. The amount of cytoplasmic mRNA for both proteins was markedly elevated in UT-1 cells, as determined by filter hybridization studies using 32P-labeled cDNA probes. The amount of mRNA for both reductase and the 53-kDa protein declined in parallel after addition of low density lipoprotein, 25-hydroxycholesterol, or mevalonate to the culture medium. The decline in reductase mRNA was associated with a marked decrease in the rate of [3H]uridine incorporation into hybridizable cytoplasmic mRNA. When UT-1 cells were grown for 3-4 months in the absence of compactin, the level of reductase mRNA and enzymatic activity decreased markedly, but the number of copies of the reductase gene did not decline. When the compactin-withdrawn cells were rechallenged with compactin, high levels of reductase mRNA and enzymatic activity promptly returned. We conclude that the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa protein, has been stably amplified in UT-1 cells. Despite this differential gene amplification, the levels of cytoplasmic mRNA for both gene products are markedly elevated, and both are reduced in parallel by either sterols (low density lipoprotein-cholesterol or 25-hydroxycholesterol) or mevalonate, the product of the reductase-catalyzed reaction.",
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AU - Faust, J. R.

AU - Chin, D. J.

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AU - Goldstein, J. L.

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