Chakraborty A K, Cichutek K, Duesberg P H
Department of Molecular & Cell Biology, University of California, Berkeley 94720.
Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2217-21. doi: 10.1073/pnas.88.6.2217.
Based on transfection into cells in culture or natural transduction into retroviruses, proto-ras genes seem to derive transforming function either from heterologous promoters or from point mutations. Here we ask how such different events could achieve the same results. To identify homologous regulatory elements, about 3 kilobases of rat DNA upstream of the first untranslated proto-Ha-ras exon was sequenced. Surprisingly, the sequence shares at -1858 a homology of 148 nucleotides with Harvey (Ha) sarcoma virus, 5' of viral ras, signaling possibly a second untranslated proto-Ha-ras exon. In addition the sequence contains a perfect repeat of 25 CA dinucleotides at -2655. A retroviral promoter, even from upstream of the poly(CA), conferred transforming function on proto-Ha-ras and increased transcription greater than 100-fold compared with that of unrearranged proto-ras. Point mutations were not necessary for transforming function of rat and human proto-Ha-ras genes with retroviral promoters but did enhance it greater than 10-fold. A unifying hypothesis proposes that proto-ras genes depend on high expression from heterologous promoters or enhancers for transforming function, which is modulated by ras point mutations. The hypothesis makes two testable predictions. (i) Unrearranged proto-ras genes with point mutations, which occur in some cancers, have no transforming function. Indeed, tumors with mutated proto-ras genes, even those that also lack hypothetical tumor-suppressor genes, are indistinguishable from counterparts with normal proto-ras genes. (ii) Proto-ras genes in transfected cells derive transforming function from heterologous promoters or enhancers acquired via illegitimate recombination from vector DNAs and particularly from viral helper genes that must be cotransfected for transformation of primary cells. Indeed, expression of exogenous proto-ras genes in cells transformed by transfection is as high as for viral ras genes and is much higher than in the cells of origin.
基于在培养细胞中的转染或逆转录病毒的自然转导，原癌基因ras似乎从异源启动子或点突变获得转化功能。在此我们探讨这些不同事件如何能产生相同结果。为鉴定同源调控元件，对原癌基因Ha-ras第一个非翻译外显子上游约3千碱基的大鼠DNA进行了测序。令人惊讶的是，该序列在-1858处与哈维（Ha）肉瘤病毒的5'端有148个核苷酸的同源性，位于病毒ras的上游，这可能表明存在第二个非翻译原癌基因Ha-ras外显子。此外，该序列在-2655处含有25个CA二核苷酸的完美重复序列。一个逆转录病毒启动子，即使来自聚（CA）序列的上游，也赋予了原癌基因Ha-ras转化功能，与未重排的原癌基因相比，转录增加了100倍以上。对于大鼠和人类原癌基因Ha-ras与逆转录病毒启动子的转化功能而言，点突变并非必需，但确实能使其增强10倍以上。一个统一的假说是，原癌基因ras的转化功能依赖于异源启动子或增强子的高表达，而ras点突变可对其进行调节。该假说有两个可检验的预测。（i）在某些癌症中出现的带有点突变的未重排原癌基因ras没有转化功能。实际上，带有突变原癌基因ras的肿瘤，即使那些也缺乏假定的肿瘤抑制基因的肿瘤，与具有正常原癌基因ras的对应肿瘤并无区别。（ii）转染细胞中的原癌基因ras通过从载体DNA的非法重组，特别是从为原代细胞转化必须共转染的病毒辅助基因获得的异源启动子或增强子获得转化功能。实际上，转染转化细胞中外源原癌基因ras的表达与病毒ras基因的表达一样高，且远高于其起源细胞中的表达。
