Mosley S T, Brown M S, Anderson R G, Goldstein J L
J Biol Chem. 1983 Nov 25;258(22):13875-81.
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.
3-羟基-3-甲基戊二酰辅酶A还原酶（HMG-CoA还原酶）将HMG-CoA转化为甲羟戊酸，甲羟戊酸是胆固醇和几种非甾醇类异戊二烯化合物形成过程中的关键中间体。利用溴脱氧尿苷/双苯甲酰亚胺光敏技术，我们分离出了一株中国仓鼠卵巢细胞的突变克隆株，该克隆株的生长需要甲羟戊酸。这个突变体被命名为UT-2细胞，即使在无选择压力的情况下生长9个月后，其HMG-CoA还原酶活性也仅为亲代中国仓鼠卵巢细胞的2%-5%。通过免疫印迹法，在UT-2细胞中未检测到交叉反应性的HMG-CoA还原酶蛋白。[14C]乙酸盐和[14C]丙酮酸掺入胆固醇的量不到亲代中国仓鼠卵巢细胞的5%。相比之下，[3H]甲羟戊酸掺入胆固醇的量是正常的。在胆固醇生物合成途径中位于HMG-CoA还原酶之前的两种酶，即乙酰乙酰辅酶A硫解酶和HMG-CoA合酶的活性，在UT-2细胞中正常或略有升高。当用限制性内切酶消化UT-2细胞的DNA、进行Southern印迹分析并用32P标记的HMG-CoA还原酶cDNA进行探针杂交时，未发现HMG-CoA还原酶基因有明显的大片段缺失或重排。我们得出结论，UT-2细胞发生了一种突变，该突变特异性地阻止了正常量HMG-CoA还原酶的产生。
