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首页> 外文期刊>Molecular and Cellular Biology >Definition of the human raf amino-terminal regulatory region by deletion mutagenesis.
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Definition of the human raf amino-terminal regulatory region by deletion mutagenesis.

机译：通过缺失诱变定义人类raf氨基末端调节区。

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Activation of transforming potential of the cellular raf gene has uniformly been associated with the deletion of amino-terminal coding sequences. In order to determine whether 5' truncation alone could activate cellular raf, we constructed 21 human c-raf-1 cDNAs with variable BAL 31-generated deletions distal to a Moloney murine sarcoma virus long terminal repeat and a consensus translation initiation sequence. The deletions ranged from 136 to 1,399 nucleotides of coding sequence and shortened the 648-amino-acid raf protein by 44 to 465 amino acids. The full-length c-raf-1 cDNA was nontransforming upon transfection of NIH 3T3 cells, as were four mutants with deletions of 142 or fewer amino acids. Seven of nine mutants with deletions of 154 to 273 amino acids induced transformation with efficiencies ranging from 0.25 to 70 foci per micrograms of DNA. Mutants with deletions of 303 to 324 amino acids displayed high transforming activities (comparable with that of v-raf), with a peak activity of 2,400 foci per microgram of DNA when 305 amino acids were deleted. Deletions of greater than 383 amino acids, extending into the raf kinase domain, lacked transforming activity. Northern (RNA) blotting and immunoprecipitation assays indicated that transfected NIH cells expressed raf RNAs and proteins of the expected sizes. Thus, 5' truncation alone can activate raf transforming potential, with a sharp peak of activation around amino acid 300. Analysis of three raf genes previously detected by transfection of tumor DNAs indicated that these genes were activated by recombination in raf intron 7 and encoded fusion proteins containing amino-terminal non-raf sequences. The extend of deletion of raf sequences in these recombinant genes corresponded to BAL 31 mutants which did not display high transforming activity, suggesting that the fused non-raf coding sequences may also contribute to biological activity.
机译：细胞raf基因的转化潜能的激活一直与氨基末端编码序列的缺失有关。为了确定单独的5'截短是否可以激活细胞raf，我们构建了21个人c-raf-1 cDNA，其在Moloney鼠肉瘤病毒长末端重复序列的末端具有可变的BAL 31产生的缺失和共有翻译起始序列。缺失范围为编码序列的136至1,399个核苷酸，并且将648个氨基酸的raf蛋白缩短了44至465个氨基酸。全长c-raf-1 cDNA在转染NIH 3T3细胞后不会发生转化，这是四个缺失142个或更少氨基酸的突变体。 9个突变体中有7个突变体缺失154至273个氨基酸，诱导转化的效率为每微克DNA 0.25至70个焦点。缺失303至324个氨基酸的突变体显示出高转化活性（与v-raf相当），当缺失305个氨基酸时，每微克DNA的峰值活性为2,400个焦点。延伸到raf激酶结构域的大于383个氨基酸的缺失缺乏转化活性。 Northern（RNA）印迹和免疫沉淀分析表明，转染的NIH细胞表达了预期大小的raf RNA和蛋白质。因此，单独的5'截短可以激活raf转化潜能，在300位氨基酸附近具有一个急剧的激活峰值。对先前通过转染肿瘤DNA所检测到的三个raf基因的分析表明，这些基因在raf内含子7中重组并编码了融合蛋白含有氨基末端非raf序列的蛋白质。在这些重组基因中raf序列的缺失延伸对应于不显示高转化活性的BAL 31突变体，表明融合的非raf编码序列也可能有助于生物学活性。

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