In vitro expression of human p53 cDNA clones and characterization of the cloned human p53 gene
In vitro expression of human p53 cDNA clones and characterization of the cloned human p53 gene
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人 p53 cDNA 克隆的体外表达和克隆人 p53 基因的表征
DOI：
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发表时间：
1985
影响因子：
5.3
通讯作者：
V. Rotter
中科院分区：
文献类型：
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作者：
D. Wolf;Z. Laver;V. Rotter
克隆人p53基因，并通过使用一系列p53 DNA克隆进行表征。用我们以前克隆的人p53-H14（2.35-脱氢酶）cDNA探针分离了一系列不同大小和相对于mRNA分子定位的人cDNA克隆。一种这样的分离物，克隆p53-H7（2.65个内切酶），跨越整个人成熟p53 mRNA分子。在pSP 65 RNA转录载体中构建人cDNA克隆有利于SP 6噬菌体RNA聚合酶产生p53转录物。在无细胞系统中将未经进一步处理获得的p53特异性RNA转录物翻译成p53蛋白。通过使用这种快速的体外转录-翻译测定，我们发现克隆p53-H7（2.65个酶）编码成熟大小的p53蛋白，而较短的cDNA克隆p53-H13（1.8个酶）决定合成较小大小的p53蛋白（45千道尔顿）。体外合成的p53蛋白与人特异性抗p53抗体有效地免疫沉淀。人类DNA的基因组分析显示，存在一个单一的p53基因驻留在两个EcoRI片段。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7个主要外显子。
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.