Spiegelman B M, Frank M, Green H
J Biol Chem. 1983 Aug 25;258(16):10083-9.
We have constructed a recombinant bacterial library containing cDNA prepared from mRNA of adipose 3T3 cells. We have screened this library by several methods and isolated colonies containing sequences complementary to mRNAs for glycerophosphate dehydrogenase, two other major differentiation-dependent proteins of Mr = 28,000 and 13,000, and actin. These recombinants were identified by hybrid selection of total adipocyte mRNA, translation in vitro and subsequent immunoprecipitation, and two-dimensional electrophoresis of translated proteins. Three of the four cloned cDNAs hybridized to single adipocyte mRNA species of size close to that expected from the size of the polypeptide it specifies, but the mRNA for glycerophosphate dehydrogenase contained 3,550 bases, far larger than necessary to code for its polypeptide (Mr = 34,000). The increase in amount of mRNA during differentiation was 150-fold for the protein of Mr = 13,000 and considerably greater for glycerophosphate dehydrogenase and the protein of Mr = 28,000. The time course of mRNA accumulation was different for each of these mRNAs, indicating they do not respond synchronously during differentiation.
我们构建了一个重组细菌文库，其中包含从脂肪3T3细胞的mRNA制备的cDNA。我们通过多种方法筛选了该文库，并分离出了含有与甘油磷酸脱氢酶、另外两种分子量分别为28,000和13,000的主要分化依赖性蛋白以及肌动蛋白的mRNA互补序列的菌落。这些重组体通过对脂肪细胞总mRNA进行杂交选择、体外翻译及随后的免疫沉淀以及对翻译产物进行二维电泳来鉴定。四个克隆的cDNA中有三个与大小接近其指定多肽大小预期的单个脂肪细胞mRNA种类杂交，但甘油磷酸脱氢酶的mRNA含有3550个碱基，远远大于编码其多肽（分子量34,000）所需的碱基。在分化过程中，分子量为13,000的蛋白质的mRNA量增加了150倍，甘油磷酸脱氢酶和分子量为28,000的蛋白质的mRNA量增加得更多。这些mRNA中每一种的mRNA积累时间进程都不同，表明它们在分化过程中并非同步反应。
