Mutant clone of Chinese hamster ovary cells lacking 3-hydroxy-3-methylglutaryl coenzyme A reductase

S. T. Mosley, M. S. Brown, R. G W Anderson, J. L. Goldstein

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Abstract

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) converts HMG-CoA to mevalonate, a key intermediate in the formation of cholesterol and several nonsterol isoprenoid compounds. Using the bromodeoxyuridine/bisbenzimide photosensitization technique, we isolated a mutant clone of Chinese hamster ovary cells that requires mevalonate for growth. This mutant, designated UT-2 cells, expresses 2-5% of the HMG-CoA reductase activity of parental Chinese hamster ovary cells, even after growth for 9 months in the absence of selective pressure. By immunoblotting, no cross-reactive HMG-CoA reductase protein was detected in UT-2 cells. Incorporation of [14C]acetate and [14C]pyruvate into cholesterol was less than 5% of that in parental Chinese hamster ovary cells. In contrast, [3H]mevalonate incorporation into cholesterol was normal. The activities of acetoacetyl-CoA thiolase and HMG-CoA synthase, the two enzymes that precede HMG-CoA reductase in the cholesterol biosynthetic pathway, were normal or slightly elevated in UT-2 cells. No gross deletions or rearrangements in the gene for HMG-CoA reductase were apparent when DNA from UT-2 cells was digested with restriction endonucleases, subjected to Southern blotting, and probed with a 32P-labeled cDNA for HMG-CoA reductase. We conclude that UT-2 cells have a mutation that specifically prevents the production of normal amounts of HMG-CoA reductase.

Original languageEnglish (US)
Pages (from-to)13875-13881
Number of pages7
JournalJournal of Biological Chemistry
Volume258
Issue number22
StatePublished - 1983

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Cricetulus
Ovary
Oxidoreductases
Clone Cells
Cells
Mevalonic Acid
Cholesterol
Hydroxymethylglutaryl-CoA Synthase
Acetyl-CoA C-Acetyltransferase
Bisbenzimidazole
Hydroxymethylglutaryl CoA Reductases
Photosensitivity Disorders
DNA Restriction Enzymes
Terpenes
Bromodeoxyuridine
3-hydroxy-3-methylglutaryl-coenzyme A
Gene Rearrangement
Pyruvic Acid
Biosynthetic Pathways
Growth

ASJC Scopus subject areas

  • Biochemistry

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Mutant clone of Chinese hamster ovary cells lacking 3-hydroxy-3-methylglutaryl coenzyme A reductase. / Mosley, S. T.; Brown, M. S.; Anderson, R. G W; Goldstein, J. L.

In: Journal of Biological Chemistry, Vol. 258, No. 22, 1983, p. 13875-13881.

Research output: Contribution to journalArticle

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abstract = "3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) converts HMG-CoA to mevalonate, a key intermediate in the formation of cholesterol and several nonsterol isoprenoid compounds. Using the bromodeoxyuridine/bisbenzimide photosensitization technique, we isolated a mutant clone of Chinese hamster ovary cells that requires mevalonate for growth. This mutant, designated UT-2 cells, expresses 2-5{\%} of the HMG-CoA reductase activity of parental Chinese hamster ovary cells, even after growth for 9 months in the absence of selective pressure. By immunoblotting, no cross-reactive HMG-CoA reductase protein was detected in UT-2 cells. Incorporation of [14C]acetate and [14C]pyruvate into cholesterol was less than 5{\%} of that in parental Chinese hamster ovary cells. In contrast, [3H]mevalonate incorporation into cholesterol was normal. The activities of acetoacetyl-CoA thiolase and HMG-CoA synthase, the two enzymes that precede HMG-CoA reductase in the cholesterol biosynthetic pathway, were normal or slightly elevated in UT-2 cells. No gross deletions or rearrangements in the gene for HMG-CoA reductase were apparent when DNA from UT-2 cells was digested with restriction endonucleases, subjected to Southern blotting, and probed with a 32P-labeled cDNA for HMG-CoA reductase. We conclude that UT-2 cells have a mutation that specifically prevents the production of normal amounts of HMG-CoA reductase.",
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T1 - Mutant clone of Chinese hamster ovary cells lacking 3-hydroxy-3-methylglutaryl coenzyme A reductase

AU - Mosley, S. T.

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AU - Anderson, R. G W

AU - Goldstein, J. L.

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AB - 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) converts HMG-CoA to mevalonate, a key intermediate in the formation of cholesterol and several nonsterol isoprenoid compounds. Using the bromodeoxyuridine/bisbenzimide photosensitization technique, we isolated a mutant clone of Chinese hamster ovary cells that requires mevalonate for growth. This mutant, designated UT-2 cells, expresses 2-5% of the HMG-CoA reductase activity of parental Chinese hamster ovary cells, even after growth for 9 months in the absence of selective pressure. By immunoblotting, no cross-reactive HMG-CoA reductase protein was detected in UT-2 cells. Incorporation of [14C]acetate and [14C]pyruvate into cholesterol was less than 5% of that in parental Chinese hamster ovary cells. In contrast, [3H]mevalonate incorporation into cholesterol was normal. The activities of acetoacetyl-CoA thiolase and HMG-CoA synthase, the two enzymes that precede HMG-CoA reductase in the cholesterol biosynthetic pathway, were normal or slightly elevated in UT-2 cells. No gross deletions or rearrangements in the gene for HMG-CoA reductase were apparent when DNA from UT-2 cells was digested with restriction endonucleases, subjected to Southern blotting, and probed with a 32P-labeled cDNA for HMG-CoA reductase. We conclude that UT-2 cells have a mutation that specifically prevents the production of normal amounts of HMG-CoA reductase.

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