MOLECULAR-BASIS FOR HETEROGENEITY OF THE HUMAN P53-PROTEIN
MOLECULAR-BASIS FOR HETEROGENEITY OF THE HUMAN P53-PROTEIN
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DOI：
10.1128/mcb.6.12.4650
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发表时间：
1986-12-01
影响因子：
5.3
通讯作者：
ROTTER, V
中科院分区：
文献类型：
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作者：
HARRIS, N;BRILL, E;ROTTER, V
人p53肿瘤抗原包含几种物理上不同的蛋白质。两种p53蛋白，可通过聚丙烯酰胺凝胶电泳分离，由人转化细胞系SV-80表达。分离编码这些蛋白质的单个cDNA并构建到SP 6转录载体中。这些克隆编码的蛋白通过SP 6载体的体外转录和无细胞系统中的翻译鉴定。p53-H-1和p53-H-19 cDNA克隆分别编码SV-80的较快和较慢迁移的p53蛋白种类。p53-H-1和p53-H-19的体外表达蛋白具有相同的抗原决定性，并且在结构上与它们的体内对应物无法区分。通过体外表达定义的限制性cDNA片段，各个cDNA之间的异质性区域位于cDNA的5 ''末端。交换目的5“片段并在体外表达嵌合克隆证实了DNA异质性是这些蛋白质电泳迁移率差异的原因。两个cDNA的序列显示克隆的编码区中的单个碱基对差异（G对C）。这种序列差异导致在克隆p53-H-1中编码精氨酸，在克隆p53-H-19中在等同位置编码脯氨酸。这种变化占个别p53蛋白种类的电泳迁移率的变化。
The human p53 tumor antigen comprises several physically distinct proteins. Two p53 proteins, separable by polyacrylamide gel electrophoresis, are expressed by the human transformed cell line SV-80. The individual cDNAs which code for these proteins were isolated and constructed into the SP6 transcription vector. The proteins encoded by these clones were identified by in vitro transcription with SP6 vector and translation in a cell-free system. p53-H-1 and p53-H-19 cDNA clones code for the faster- and slower-migrating p53 protein species, respectively, of SV-80. The in vitro-expressed proteins of p53-H-1 and p53-H-19 had the same antigenic detemrinants and were structurally indistinguishable from their in vivo counterparts. By expressing defined restricted cDNA fragments in vitro, the region of heterogeneity between the respective cDNAs was located at the 5'' end of the cDNAs. Exchanging the 5'' fragments of interest and expressing the chimeric clones in vitro confirmed that the DNA heterogeneity was responsible for the difference in the electrophoretic mobility of these proteins. The sequences of the two cDNAs revealed a single base pair difference (G versus C) in the coding region of the clones. This sequence difference resulted in an arginine being coded for in clone p53-H-1 and a proline being coded for at the equivalent position in clone p53-H-19. This variation accounted for the change in the electrophoretic mobility of the individual p53 protein species.