Molecular cloning of mRNA from 3T3 adipocytes. Regulation of mRNA content for glycerophosphate dehydrogenase and other differentiation-dependent proteins during adipocyte development.
Molecular cloning of mRNA from 3T3 adipocytes. Regulation of mRNA content for glycerophosphate dehydrogenase and other differentiation-dependent proteins during adipocyte development.
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DOI：
10.1016/s0021-9258(17)44608-4
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发表时间：
1983-08
期刊：
影响因子：
--
通讯作者：
B. Spiegelman;M. Frank;H. Green
中科院分区：
文献类型：
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作者：
B. Spiegelman;M. Frank;H. Green
我们构建了一个含有从脂肪3T3细胞的mRNA制备的cDNA的重组细菌文库。我们已经通过几种方法筛选了该文库，并分离出含有与甘油磷酸脱氢酶（Mr = 28，000和13，000的另外两种主要分化依赖性蛋白质）和肌动蛋白的mRNA互补序列的菌落。这些重组体通过总脂肪细胞mRNA的杂交选择、体外翻译和随后的免疫沉淀以及翻译蛋白的双向电泳来鉴定。四个克隆的cDNA中的三个与单个脂肪细胞mRNA种类杂交，其大小接近其指定的多肽大小的预期大小，但甘油磷酸脱氢酶的mRNA含有3，550个碱基，远远大于编码其多肽所需的碱基（Mr = 34，000）。在分化过程中，对于Mr = 13，000的蛋白质，mRNA的量增加了150倍，对于甘油磷酸脱氢酶和Mr = 28，000的蛋白质，mRNA的量增加了相当多。这些mRNA中的每一种的mRNA积累的时间过程是不同的，表明它们在分化期间不同步响应。
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.