Mammalian alpha-polymerase: cloning of partial complementary DNA and immunobinding of catalytic subunit in crude homogenate protein blots.
Mammalian alpha-polymerase: cloning of partial complementary DNA and immunobinding of catalytic subunit in crude homogenate protein blots.
复制标题
哺乳动物 α 聚合酶：部分互补 DNA 的克隆和粗匀浆蛋白印迹中催化亚基的免疫结合。
DOI：
10.1021/bi00377a041
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Wilson,SH
中科院分区：
文献类型：
--
作者：
SenGupta,DN;Kumar,P;Zmudzka,BZ;Coughlin,S;Vishwanatha,JK;Robey,FA;Parrott,C;Wilson,SH
摘要：用均相HeLa细胞α-聚合酶制备了一种新的抗α-聚合酶催化多肽的多克隆抗体。该抗体中和α-聚合酶活性，并且在HeLa细胞粗提取物的Western印迹分析中对α-聚合酶催化多肽（MT 183000）是强的和特异性的。用该抗体筛选新生大鼠脑poly（A+）RNA的cDNA文库。对阳性噬菌体进行鉴定和噬斑纯化。这种噬菌体，命名为阿波罗。2，也被发现与抗果蝇α-聚合酶的抗体阳性。阿波罗的插入物。2（1183个碱基对）在3 '端含有一个poly（A）序列，在5'端含有一个短的同相开放阅读框。用与开放阅读框架相对应的人工合成寡肽（8个氨基酸）在兔中制备抗血清。从该血清中纯化的抗体亲和力被发现是对纯化的α-聚合酶的酶联免疫吸附试验的免疫反应，并能够免疫沉淀α-聚合酶。这表明阿波罗。2插入片段编码α-聚合酶表位，提示该cDNA对应于α-聚合酶mRNA。这在使用含有cDNA插入物和来自新生大鼠脑的多聚（A+）RNA的pUC 9的杂交选择实验中得到证实;插入物与能够编码α-聚合酶催化多肽的mRNA杂交。大鼠脑poly（A+）RNA的北方印迹分析显示该mRNA约为5.4个碱基。
Revised Manuscript Received October 8, 1986 abstract: A new polyclonal antibody against the a-polymerase catalytic polypeptide was prepared by using homogeneous HeLa cell a-polymerase. The antibody neutralized a-polymerase activity and was strong and specific for the a-polymerase catalytic polypeptide (MT 183000) in Western blot analysis of crude extracts of HeLa cells. The antibody was used to screen a cDNA library of newborn rat brain poly (A+) RNA in Agtll. A positivephage was identified and plaque purified. This phage, designated Apolal. 2, also was found to be positive with an antibody against Drosophila a-polymerase. The insert in Apolal. 2 (1183 base pairs) contained a poly (A) sequence at the 3'terminus and a short in-phase open reading frame at the 5'terminus. A synthetic oligopeptide (eight amino acids) corresponding to the open reading frame was used to raise antiserum in rabbits. Antibody affinity purified from this serum was found to be immunoreactive against purified a-polymerase by enzyme-linked immunosorbent assay and was capable of immunoprecipitating a-polymerase. This indicatedthe Apolal. 2 insert encoded an a-polymerase epitope andsuggested that the cDNA corresponded to an a-polymerase mRNA. This was confirmed in hybrid selection experiments using pUC9 containing the cDNA insert and poly (A+) RNA from newborn rat brain; the insert hybridized to mRNA capable of encoding a-polymerase catalytic polypeptides. Northern blot analysis of rat brain poly (A+) RNA revealed that this mRNA is~ 5.4 kilobases.