CARBOXY TERMINUS OF POLYOMA MIDDLE-SIZED TUMOR-ANTIGEN IS REQUIRED FOR ATTACHMENT TO MEMBRANES, ASSOCIATED PROTEIN-KINASE ACTIVITIES, AND CELL-TRANSFORMATION
CARBOXY TERMINUS OF POLYOMA MIDDLE-SIZED TUMOR-ANTIGEN IS REQUIRED FOR ATTACHMENT TO MEMBRANES, ASSOCIATED PROTEIN-KINASE ACTIVITIES, AND CELL-TRANSFORMATION
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DOI：
10.1073/pnas.79.11.3579
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发表时间：
1982-01-01
期刊：
影响因子：
--
通讯作者：
BENJAMIN, TL
中科院分区：
文献类型：
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作者：
CARMICHAEL, GG;SCHAFFHAUSEN, BS;BENJAMIN, TL
构建了一种转化缺陷型多瘤病毒突变体（Py 1387-T），该突变体可指导正常小肿瘤抗原、功能性大肿瘤抗原和COOH末端缺失37个氨基酸的截断（51,000道尔顿）中型肿瘤（mT）抗原的合成。缩短的mT多肽缺少疏水尾部，该尾部被认为负责将该蛋白锚定到质膜中，实际上存在于细胞质溶胶中。这种截短的mT多肽在体外蛋白激酶测定中是无活性的，并且在体内的磷酸化被改变。突变体1387- t与野生型病毒的不同之处在于在核苷酸位置1387上有一个t.c cntdot. a碱基对而不是c.c cntdot. g碱基对。这种变化是通过使用合成的十一核苷酸作为特定的诱变剂引入病毒DNA的。利用分子克隆技术将野生型多瘤DNA转化为单链。该寡核苷酸与待改变位点的错配杂交，用于启动双链闭合环状DNA的合成。通过DNA序列分析筛选重组噬菌体的后代以获得所需的碱基变化。用含有突变体的DNA分子重组噬菌体复制来重建多瘤突变体。
A transformation-defective polyoma virus mutant (Py 1387-T) that directs the synthesis of a normal small tumor antigen, a functional large tumor antigen and a truncated (51,000-dalton) middle-sized tumor (mT) antigen that lacks 37 amino acids at its COOH terminus was constructed. The shortened mT polypeptide is missing the hydrophobic tail thought to be responsible for the anchorage of this protein into the plasma membrane and is in fact in cytosol fractions. This truncated mT polypeptide is inactive in an in vitro protein kinase assay and is altered in its phosphorylation in vivo. Mutant 1387-T differs from wild-type virus in having a T.cntdot.A base pair instead of a C.cntdot.G base pair at nucleotide position 1387. This change was introduced into viral DNA by using a synthetic undecanucleotide as a specific mutagen. Wild-type polyoma DNA was rendered single stranded by molecular cloning into coliphage M13. The oligonucleotide, which hybridizes with a mismatch at the site to be altered, was used to prime the synthesis of double-stranded closed circular DNA. Progeny recombinant phages were screened by DNA sequence analysis for the desired base change. The polyoma mutant was reconstructed from recombinant phage replicative form DNA molecules containing the mutation.