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首页> 外文期刊>Molecular and Cellular Biology >Expression of a molecularly cloned human c-src oncogene by using a replication-competent retroviral vector.
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Expression of a molecularly cloned human c-src oncogene by using a replication-competent retroviral vector.

机译：通过使用具有复制能力的逆转录病毒载体表达分子克隆的人c-src癌基因。

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We studied the expression of a molecularly cloned human c-src gene, c-src-1, localized on chromosome 20, whose coding region consists of 11 exons and spans a 19.5-kilobase (kb) distance. Using a replication-competent retroviral vector derived from molecularly cloned Rous sarcoma virus DNA (pSRA-2), we obtained two constructs: one (pSR-CS) carrying the unmodified human c-src coding sequence and another (pSR-CVS) with a chimeric gene formed between the human c-src gene and the carboxy-terminal 12-amino acid v-src-specific coding sequence. From chicken embryo fibroblasts transfected with these DNA constructs, infectious viruses designated as WO CS and WO CVS, respectively, were recovered. WO CS virus did not cause cell transformation, whereas WO CVS induced cell transformation. Analyses of the proviral DNAs indicated that all introns were spliced out such that the 19-kb inserts were converted to 1.7-kb cDNA forms. Analyses of src proteins in infected cells, using monoclonal antibody MAb327 against v-src protein, showed the following results. The CVS and CS src proteins were about 60 and 61 kilodaltons in size, respectively; the specific protein kinase activity assayed in vitro of the CVS src protein was about 20-fold higher than that of the CS src protein and comparable to that of the v-src protein; the transforming CVS src protein reacted to an antibody against a v-src-specific peptide, whereas the CS src protein did not. These results indicate that the human c-src gene has a potential transforming ability and suggest that the v-src-specific sequence played an important role in the generation of Rous sarcoma virus.
机译：我们研究了分子克隆的人类c-src基因c-src-1的表达，该基因位于20号染色体上，其编码区由11个外显子组成，跨度为19.5千碱基（kb）。使用衍生自分子克隆的劳斯肉瘤病毒DNA（pSRA-2）的具有复制能力的逆转录病毒载体，我们获得了两个构建体：一个（pSR-CS）携带未修饰的人c-src编码序列，另一个（pSR-CVS）带有人c-src基因和羧基末端12个氨基酸的v-src特异性编码序列之间形成的嵌合基因。从用这些DNA构建体转染的鸡胚成纤维细胞中，回收分别称为WO CS和WO CVS的感染性病毒。 WO CS病毒不引起细胞转化，而WO CVS诱导细胞转化。对原病毒DNA的分析表明，剪接了所有内含子，使得19kb的插入片段被转化为1.7kb的cDNA形式。使用针对v-src蛋白的单克隆抗体MAb327分析受感染细胞中的src蛋白，得出以下结果。 CVS和CS src蛋白的大小分别约为60和61千道尔顿。 CVS src蛋白的体外比蛋白激酶活性比CS src蛋白高约20倍，与v-src蛋白相当。转化的CVS src蛋白与针对v-src特异性肽的抗体反应，而CS src蛋白没有。这些结果表明人c-src基因具有潜在的转化能力，并且表明v-src特异性序列在劳斯肉瘤病毒的产生中起重要作用。

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