CHARACTERIZATION AND EXPRESSION OF THE HUMAN RHOH12 GENE-PRODUCT
CHARACTERIZATION AND EXPRESSION OF THE HUMAN RHOH12 GENE-PRODUCT
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DOI：
10.1128/mcb.9.5.2058
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发表时间：
1989-05-01
影响因子：
5.3
通讯作者：
WEINBERG, RA
中科院分区：
文献类型：
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作者：
AVRAHAM, H;WEINBERG, RA
Rho基因构成了一个进化上保守的家族，与ras癌基因家族有显著的同源性。这些基因已经在酿酒酵母、果蝇、黑腹果蝇、老鼠和人类中发现；它们的21,000道尔顿产物显示出很强的结构保守性。在人类中，已鉴定出三类Rho cDNA克隆，它们因存在可变的C-末端结构域而有所不同：RhoH12、RhoH6和RhoH9。预测的人RhoH12蛋白193个氨基酸与人RhoH6克隆的同源性为88%，与Aplysia Rho产物的同源性为96.8%，与酵母RHO1蛋白的同源性为81.8%。用含有正常的人RhoH12等位基因的克隆和编码Valine的变异体分别替代甘氨酸和亮氨酸，分别转染RAT-1和NIH3T3小鼠成纤维细胞，以取代通常位于第14位和位的谷氨酰胺。它们的替换反映了导致相关ras编码的p21蛋白致癌激活的变化。这些突变的RhoH12克隆等位基因不会在单层中形成焦点，也不会在软琼脂中生长。然而，通过与二氢叶酸还原酶基因共转染的方式扩增正常的RhoH12，导致克隆的生长对血清的依赖程度降低，生长到更高的饱和密度，并在接种到裸鼠体内时具有致瘤性。Western免疫印迹和兔抗p21rho蛋白122~135位氨基酸抗体的免疫沉淀分析表明，p21rho蛋白在转基因细胞系和正常细胞系中表达正常。
The rho genes constitute an evolutionarily conserved family having significant homology to the ras oncogene family. These genes have been found in Saccharomyces cerevisiae, Drosophila melanogaster, rat, and human; their 21,000-dalton products show strong conservation of structure. In humans, three classes of rho cDNA clones have been identified which differ by virtue of the presence of variable C-terminal domains: rhoH12, rhoH6, and rhoH9. The predicted 193 amino acids of human rhoH12 protein show 88% similarity with those of the human rhoH6 clone, 96.8% similarity with those of the Aplysia rho product, and 81.8% similarity with those of the yeast RHO1 protein. Rat-1 and NIH 3T3 mouse fibroblasts were transfected with clones containing the normal human rhoH12 allele as well as the variants encoding valine in place of the glycine and leucine in place of the glutamine normally found at residues 14 and 64, respectively. Their replacements mirror the changes responsible for oncogenic activation of the related ras-encoded p21 proteins. These mutant rhoH12 clone alleles did not cause focus formation in monolayers or growth in soft agar. However, amplification of normal rhoH12 via cotransfection with a dihydrofolate reductase gene resulted in colonies that displayed reduced dependence on serum for growth, grew to higher saturation densities, and were tumorigenic when inoculated into nude mice. Normal p21rho protein was detected in the transfected cell lines as well as in normal cell lines by Western immunoblot and immunoprecipitation analysis with rabbit antibodies raised against the peptide corresponding to amino acids 122 to 135.