Cloning and sequence of several alpha 2u-globulin cDNAs.
Cloning and sequence of several alpha 2u-globulin cDNAs.
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几种 α2u-球蛋白 cDNA 的克隆和序列。
DOI：
10.1073/pnas.78.6.3478
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发表时间：
1981
影响因子：
11.1
通讯作者：
Feigelson,P
中科院分区：
文献类型：
--
作者：
Unterman,RD;Lynch,KR;Nakhasi,HL;Dolan,KP;Hamilton,JW;Cohn,DV;Feigelson,P
我们描述了一个简单的α 2u-球蛋白的克隆程序，既不需要富集的mRNA克隆，也不需要纯化的特定探针筛选重组菌落。以成年雄性肝脏总poly（A）+RNA为模板进行克隆，通过分别与肝脏雄性和雌性mRNA制备的放射性cDNA探针进行杂交比较，筛选随后的重组集落。几乎所有被选中的“雄性特异性”克隆后来都被证明含有α 2u-球蛋白序列。这种克隆的α 2 u-球蛋白cDNA已显示与雄性大鼠肝RNA特异性杂交，当在体外分离和翻译时，其编码与α 2 u-球蛋白免疫学上相同的21，000-道尔顿蛋白（前α 2 u-球蛋白）。当在胰腺微粒体存在下发生翻译时，这种体外合成的前α 2 u-球蛋白被加工成19，000-道尔顿成熟形式的α 2 u-球蛋白。已确定α 2u-球蛋白cDNA的核苷酸序列，从而阐明了α 2u-球蛋白的完整氨基酸序列和前α 2u-球蛋白的大部分疏水性“前导”序列。从cDNA推导的氨基酸序列与我们先前通过纯化蛋白的连续Edman降解确定的部分序列一致。α 2u-球蛋白cDNA克隆在3 ′-非翻译区内含有两个推定的多聚腺苷酸化/转录终止位点（A-A-T-A-A-A和A-A-T-T-A-A-A）之一或两者。可以使用这些中的任一种，产生两种长度的α 2u-球蛋白mRNA种类。cDNA的密码子使用分析表明，尽管所有6个亮氨酸密码子用于成熟α 2u-球蛋白中的14个亮氨酸残基，但报告的部分前导序列中的7个亮氨酸都由相同的密码子CTG编码。一级氨基酸序列含有独特的Asn-Gly-Ser序列，可能处于β-转角构象，作为该糖蛋白糖基化的可能位点。
We describe a simple cloning procedure for alpha 2u-globulin that requires neither enrichment of mRNA for cloning nor purification of a specific probe for screening recombinant colonies. Total adult male liver poly(A)+RNA was used as template for cloning, and the subsequent recombinant colonies were screened by comparing hybridization to radioactive cDNA probes prepared from hepatic male and female mRNA, respectively. Almost all of the selected "male-specific" clones were later shown to contain alpha 2u-globulin sequences. This cloned alpha 2u-globulin cDNA has been shown to specifically hybridize to male rat liver RNA, which, when isolated and translated in vitro, codes for a 21,000-dalton protein (pro-alpha 2u-globulin) immunologically identical to alpha 2u-globulin. When translation occurs in the presence of pancreatic microsomes this in vitro synthesized pro-alpha 2u-globulin is processed to the 19,000-dalton mature form of alpha 2u-globulin. The nucleotide sequence of the alpha 2u-globulin cDNA has been determined, thus elucidating the complete amino acid sequence of alpha 2u-globulin and most of the hydrophobic "leader" sequence of pro-alpha 2u-globulin. The amino acid sequence deduced from the cDNA is in agreement with the partial sequence that we previously determined by sequential Edman degradation of the purified protein. alpha 2u-Globulin cDNA clones contain within the 3'-untranslated region one or both of the two putative polyadenylylation/transcription termination sites (A-A-T-A-A-A and A-A-T-T-A-A-A). Either of these can be used, generating alpha 2u-globulin mRNA species of two lengths. A codon usage analysis of the cDNA showed that, although all six leucine codons are used for the 14 leucine residues in mature alpha 2u-globulin, the seven leucines in the partial leader sequence reported are all encoded by the same codon, CTG. The primary amino acid sequence contains a unique Asn-Gly-Ser sequence, likely to be in beta-turn conformation, as the probable site of glycosylation for this glycoprotein.