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首页> 外文期刊>Molecular and Cellular Biology >In vitro expression of human p53 cDNA clones and characterization of the cloned human p53 gene.
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In vitro expression of human p53 cDNA clones and characterization of the cloned human p53 gene.

机译：人p53 cDNA克隆的体外表达和克隆人p53基因的表征。

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The human p53 gene was cloned and characterized by using a battery of p53 DNA clones. A series of human cDNA clones of various sizes and relative localizations to the mRNA molecule were isolated by using the human p53-H14 (2.35-kilobase) cDNA probe which we previously cloned. One such isolate, clone p53-H7 (2.65 kilobases), spans the entire human mature p53 mRNA molecule. Construction of the human cDNA clones in the pSP65 RNA transcription vector facilitated the generation of p53 transcripts by the SP6 bacteriophage RNA polymerase. The p53-specific RNA transcripts obtained without further processing were translated into p53 proteins in a cell-free system. By using this rapid in vitro transcription-translation assay, we found that whereas clone p53-H7 (2.65 kilobases) coded for a mature-sized p53 protein, a shorter cDNA clone, p53-H13 (1.8 kilobases), dictated the synthesis of a smaller-sized p53 protein (45 kilodaltons). The p53 proteins synthesized in vitro immunoprecipitated efficiently with human-specific anti-p53 antibodies. Genomic analysis of human DNA revealed the presence of a single p53 gene residing within two EcoRI fragments. Heteroduplex analysis between the full-length cDNA clone p53-H7 and the cloned p53 gene indicated the presence of seven major exons.
机译：使用一系列p53 DNA克隆克隆并鉴定了人p53基因。使用我们先前克隆的人p53-H14（2.35千碱基）cDNA探针，可以分离出一系列大小和相对于mRNA分子相对定位的人cDNA克隆。一种这样的分离株，克隆p53-H7（2.65千碱基），跨越了整个人类成熟的p53 mRNA分子。 pSP65 RNA转录载体中人cDNA克隆的构建通过SP6噬菌体RNA聚合酶促进了p53转录物的产生。无需进一步处理即可获得的p53特异性RNA转录本在无细胞系统中翻译为p53蛋白。通过使用这种快速的体外转录-翻译测定，我们发现尽管克隆p53-H7（2.65千碱基）编码一个成熟大小的p53蛋白，而较短的cDNA克隆p53-H13（1.8千碱基）却决定了较小的p53蛋白（45道尔顿）。用人特异性抗p53抗体有效地免疫沉淀了体外合成的p53蛋白。人类DNA的基因组分析揭示了存在于两个EcoRI片段中的单个p53基因的存在。全长cDNA克隆p53-H7和克隆的p53基因之间的异源双链分析表明存在七个主要外显子。

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