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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Molecular cloning and characterization of cDNA encoding the alpha subunit of the rat protein synthesis initiation factor eIF-2B.
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Molecular cloning and characterization of cDNA encoding the alpha subunit of the rat protein synthesis initiation factor eIF-2B.

机译：编码大鼠蛋白质合成起始因子eIF-2B的α亚基的cDNA的分子克隆和表征。

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摘要

Eukaryotic initiation factor 2B (eIF-2B) is an essential component of the pathway of peptide-chain initiation in mammalian cells, yet little is known about its molecular structure and regulation. To investigate the structure, regulation, and interactions of the individual subunits of eIF-2B, we have begun to clone, characterize, and express the corresponding cDNAs. We report here the cloning and characterization of a 1510-bp cDNA encoding the alpha subunit of eIF-2B from a rat brain cDNA library. The cDNA contains an open reading frame of 918 bp encoding a polypeptide of 305 aa with a predicted molecular mass of 33.7 kDa. This cDNA recognizes a single RNA species approximately 1.6 kb in length on Northern blots of RNA from rat liver. The predicted amino acid sequence contains regions identical to the sequences of peptides derived from bovine liver eIF-2B alpha subunit. Expression of this cDNA in vitro yields a peptide which comigrates with natural eIF-2B alpha in SDS/polyacrylamide gels. The predicted amino acid sequence exhibits 42% identity to that deduced for the Saccharomyces cerevisiae GCN3 protein, the smallest subunit of yeast eIF-2B. In addition, expression of the rat cDNA in yeast functionally complements a gcn3 deletion for the inability to induce histidine biosynthetic genes under the control of GCN4. These results strongly support the hypothesis that mammalian eIF-2 alpha and GCN3 are homologues. Southern blots indicate that the eIF-2B alpha cDNA also recognizes genomic DNA fragments from several other species, suggesting significant homology between the rat eIF-2B alpha gene and that from other species.
机译：真核起始因子2B（eIF-2B）是哺乳动物细胞中肽链起始途径的重要组成部分，但对其分子结构和调控了解甚少。为了研究eIF-2B各个亚基的结构，调控和相互作用，我们已经开始克隆，鉴定和表达相应的cDNA。我们在这里报告从鼠脑cDNA文库克隆和表征1510 bp cDNA编码eIF-2B的α亚基。 cDNA包含918 bp的开放阅读框，编码305个氨基酸的多肽，预测分子量为33.7 kDa。该cDNA在大鼠肝脏RNA的RNA印迹上识别长度约为1.6 kb的单个RNA。预测的氨基酸序列包含与衍生自牛肝eIF-2Bα亚基的肽序列相同的区域。该cDNA的体外表达产生与SDS /聚丙烯酰胺凝胶中的天然eIF-2Bα相对应的肽。预测的氨基酸序列与酿酒酵母GCN3蛋白（酵母eIF-2B的最小亚基）推导的氨基酸序列具有42％的同一性。此外，由于无法在GCN4的控制下诱导组氨酸生物合成基因，酵母中大鼠cDNA的表达在功能上补充了gcn3缺失。这些结果有力地支持了哺乳动物eIF-2 alpha和GCN3是同源物的假说。 Southern印迹表明，eIF-2B alpha cDNA还可以识别其他几种物种的基因组DNA片段，这表明大鼠eIF-2B alpha基因与其他物种的基因具有明显的同源性。

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