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首页> 外文期刊>Molecular and Cellular Biology >Molecular cloning and characterization of mutant and wild-type human beta-actin genes.
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Molecular cloning and characterization of mutant and wild-type human beta-actin genes.

机译：突变和野生型人β-肌动蛋白基因的分子克隆和表征。

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There are more than 20 beta-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional beta-actin genes, we used the new method of B. Seed (Nucleic Acids Res. 11:2427-2446, 1983) for selecting genomic clones by homologous recombination. A derivative of the pi VX miniplasmid, pi AN7 beta 1, was constructed by insertion of the 600-base-pair 3' untranslated region of the beta-actin mRNA expressed in human fibroblasts. Five clones containing beta-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete beta-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then used to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant beta-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived clones verified the identity of the beta-actin gene expressed in human fibroblasts.
机译：人类基因组中有20多种β-肌动蛋白特异性序列，其中许多是假基因。为了促进潜在功能性β-肌动蛋白基因的分离，我们使用了B. Seed（Nucleic Acids Res。11：2427-2446，1983）的新方法，通过同源重组来选择基因组克隆。通过插入人成纤维细胞中表达的β-肌动蛋白mRNA的600个碱基对的3'非翻译区，构建了pi VX小质粒的衍生物pi AN7 beta 1。通过文库噬菌体和小质粒之间的同源重组，从扩增的人类胎儿基因文库中选择了五个含有β-肌动蛋白序列的克隆。这些克隆之一包含完整的β-肌动蛋白基因，其编码序列与确定的人类成纤维细胞mRNA序列相同。然后，使用一个由该基因的大部分插入序列组成的DNA片段，从两个专门构建的基因库中识别出13个独立的类似基因重组拷贝，每个基因库都包含在肿瘤系中发现的两种突变型β-肌动蛋白基因之一人成纤维细胞。由未突变的基因编码的氨基酸和核苷酸序列预测，鸟嘌呤到腺嘌呤的转变是引起密码子244处甘氨酸到天冬氨酸突变的原因，并且还会导致HaeIII位点的丢失。在成纤维细胞来源的克隆中检测到这种HaeIII多态性，证实了人类成纤维细胞中表达的β-肌动蛋白基因的身份。

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