Identification and regulation of rat squalene synthetase mRNA.
Identification and regulation of rat squalene synthetase mRNA.
复制标题
大鼠角鲨烯合成酶mRNA的鉴定和调控。
DOI：
10.1006/abbi.1993.1215
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发表时间：
1993
影响因子：
3.9
通讯作者：
Ness,GC
中科院分区：
文献类型：
--
作者：
Keller,RK;Cannons,A;Vilsaint,F;Zhao,Z;Ness,GC
角鲨烯合成酶在类异戊二烯途径中占据关键分支点，催化甾醇生物合成中的关键步骤（1）。有人提出，角鲨烯合成酶与3-羟基-3-甲基戊二酰辅酶A还原酶的协调调节控制了甾醇和非甾醇的融合（2）。最近，从大鼠肝脏中纯化了角鲨烯合成酶（3），并获得了cDNA序列（4）。这些进展使得研究角鲨烯合成酶基因表达的调控成为可能。在这份报告中，我们描述的cDNA探针的制备，mRNA的组织分布，mRNA的大小，和肝角鲨烯合成酶mRNA水平的调节喂养胆固醇，考来替泊（胆汁酸结合树脂），和3-羟基-3-甲基戊二酰辅酶A还原酶的各种抑制剂。先前的工作表明，这些饮食条件显着影响角鲨烯合成酶活性（3，5）。我们发现该mRNA以2.0和3.5kb两种形式存在。令人惊讶的是，睾丸角鲨烯合成酶mRNA表达水平高于其他组织检查，包括肝脏。胆固醇喂养抑制肝脏mRNA水平至对照的60%，而喂食考来替泊使这些水平增加了四倍。喂养O. 04%的洛伐他汀或氟伐他汀使肝角鲨烯合成酶mRNA水平增加3 ~ 4倍，而普伐他汀基本上没有影响。这些变化类似于对3-羟基-3-甲基戊二酰辅酶A还原酶mRNA观察到的那些变化（6，7），表明一定程度的协同调节。使用公开的序列（4）通过PCR制备角鲨烯合成酶cDNA探针。图1显示了观察到的预期大小的独特产物。通过双脱氧核苷酸测序验证该产物的身份。该cDNA代表核苷酸519-1101，用于探针北方印迹。
Squalene synthetase occupies a key branchpoint in the isoprenoid pathway, catalyzing the committed step in the biosynthesis of sterols (1). It has been proposed that coordinate regulation of squalene synthetase with 3-hydroxy-3-methylglutaryl coenzyme A reductase controls fux into sterols and nonsterols (2). Recently, squalene synthetase was purified from rat liver (3) and a cDNA sequence was obtained (4). These advances now make it possible to study the regulation of squalene synthetase gene expression. In this report, we describe the preparation of a cDNA probe, the tissue distribution of the mRNA, the size of the mRNA, and the regulation of hepatic squalene synthetase mRNA levels by feeding cholesterol, colestipol (a bile acid binding resin), and various inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Previous work has shown that these dietary conditions markedly affect squalene synthetase activity (3, 5). We found that the mRNA exists in two forms of 2.0 and 3.5 kb. Surprisingly, testis expressed higher levels of squalene synthetase mRNA than the other tissues examined, including liver. Cholesterol feeding suppressed hepatic mRNA levels to 60% of control, vvhile feeding colestipol increased these levels fourfold. Feeding O. 04% lovastatin or fluvastatin increased hepatic squalene synthetase mRNA leveis three-to fourfold while feeding pravastatin has essentially no effect. These changes are similar to those observed for 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA (6, 7), suggesting a degree of co-ordinate regulation.A squalene synthetase cDNA probe was prepared by PCR" using the published sequence (4). Figure 1 shows that a distinct product of the expected size was observed. The identity of this product was verified by dideoxynucleotide sequencing. This cDNA, representing nucleotides 519–1101, was used to probe Northern blots.