Cloning of the human cDNA for the U1 RNA‐associated 70K protein.
Cloning of the human cDNA for the U1 RNA‐associated 70K protein.
复制标题
U1 RNA 相关 70K 蛋白的人类 cDNA 克隆。
DOI：
10.1002/j.1460-2075.1986.tb04631.x
复制
发表时间：
1986
期刊：
影响因子：
--
通讯作者：
L. Philipson
中科院分区：
文献类型：
--
作者：
H. Theissen;M. Etzerodt;R. Reuter;C. Schneider;F. Lottspeich;P. Argos;R. Lührmann;L. Philipson
用抗RNP血清分离出U1SnRNP的最大多肽--摩尔蛋白的cDNA克隆。WT 70kd，命名为70K，从构建在表达载体pEX1中的人肝脏cDNA文库中获得。CRO-β-半乳糖苷酶-70K融合蛋白可与多种抗RNP患者血清、兔抗70K抗血清以及针对该蛋白的单抗反应。测定了4个70K多肽的序列，它们与p70.1的1.3 kb插入片段的部分氨基酸序列相匹配，表明它是一个真正的70K基因。从p70.1克隆的HeLa细胞多聚体mRNA构建的新的cDNA文库中筛选出一个重叠的克隆Fl70K，该克隆长2.7kb，除了推测起始密码子上游的680个核苷酸外，还覆盖了70K蛋白的完整编码和3‘-未翻译序列。编码蛋白的WT约为70kd。对纯化的HeLa 70K蛋白进行氨基酸分析，得到的值与全长cDNA的核苷酸序列相近或相同。70K蛋白富含精氨酸(20%)和酸性氨基酸(18%)。在蛋白质的羧基末端发现了含有混合电荷氨基酸簇的极亲水区域，这可能是RNA结合的功能。与最近克隆的两个RNA结合蛋白的序列比较表明，该蛋白与U1RNP 70K蛋白中的一个区域同源。这个结构域也可能负责RNA结合。
Anti‐RNP sera were used to isolate a cDNA clone for the largest polypeptide of the U1 snRNP, a protein of mol. wt 70 kd designated 70K, from a human liver cDNA library constructed in the expression vector pEX1. The cro‐beta‐galactosidase‐70K fusion protein reacted with various anti‐RNP patient sera, a rabbit anti‐70K antiserum, as well as with a monoclonal antibody specific for this protein. The sequences of four 70K peptides were determined and they match parts of the deduced amino acid sequence of the 1.3 kb insert of p70.1 indicating that it is a genuine 70K cDNA. Screening of a new cDNA library constructed from polysomal mRNA of HeLa cells with the p70.1 clone yielded an overlapping clone, FL70K, which was 2.7 kb long and covered the complete coding and 3′‐untranslated sequence of the 70K protein in addition to 680 nucleotides upstream of the putative initiation codon, The predicted mol. wt of the encoded protein is approximately 70 kd. Amino acid analysis of the purified HeLa 70K protein yielded values close or identical to those deduced from the nucleotide sequence of the full‐length cDNA. The 70K protein is rich in arginine (20%) and acidic amino acids (18%). Extremely hydrophilic regions containing mixed‐charge amino acid clusters have been identified at the carboxyl‐terminal half of the protein, which may function in RNA binding. A sequence comparison with two recently cloned RNA binding proteins revealed homology with one region in the U1 RNP 70K protein. This domain may also be responsible for RNA binding.