【摘要】 以前的工作曾用人胃癌基因Ha-ras转化了大鼠全胚细胞系Ratl细胞,得到转化细胞Rat3-3。克隆了Ha-ras癌基因6.6kb及其上游区2.5kb DNA片段,并发现2.5kb有Alu重复顺序,说明这个片段是来源于人胃癌细胞,虽族观察到p21蛋白编码12位点突变,我们又发现转化的Rat3-3细胞的Ha-ras mRNA水平比未转化的Rat1高大约五倍;通过DNase I超敏感实验证明只有转化细胞核中的Ha-ras基因对DNaseI敏感,1μg/mL的DNaseI就有明显的降解,而未转化细胞Rat1细胞核的Ha-ras基因在15μg/mL的DNaseI中也未发现有任何降解;另外还发现转化细胞核有一种能为Ha-ras基因上游区2.5kb特异结合的核蛋白,分子量大约35kD,此核蛋白不能与6.6kb Ha-ras基因本身结合,在未转化细胞中未发现此蛋白。从这些结果推测,癌基因Ha-ras的活化,除了点突变外,还可能存在另一条活化途径,即它的上游区可能有类似增强子的调控区。更多还原

【Abstract】 In the previous study (Cancer Res. 47, 3195, 1987) we have found three Bam H 1 digested fragments, namely 8.8 kb, 6.6 kb and 2.5 kb, from the clone A 120, which contains the c-Ha ras oncogene of human gastric carcinoma cell line BGC-823. The 6.6 kb fragment has been cloned and a part of its sequence was analyzed. The 2.5 kb fragment, which contains the Alu Sequence, was found at the upstream of the 6.6 kb fragment, which does not contain the Alu sequence, In the present study both the 2.5 kb and the 6.6 kb fragments were labeled with 32P-dCTP and were used as probes to detect the DNA-binding nuclear proteins of the Rat 3-3 transformed cells and the Rat 1. untransformed cells.Both the Western and Southern blottings and also the gel shift techniques showed that only in the Rat 3-3 transformed cells there was a nuclear protein fraction strongly bound to the 2.5 kb DNA, but not to the 6.6 kb DNA. The Mr. of this nuclear protein is about 35 KD. The c-Ha-ras in the Rat 3-3 cell nuclei was hypersensitive to DNase I at concentration of 1 μg/mL, while that in the Rat 1 cell nuclei was not degraded by DNase I even at concentration of 15 μg/ml. In the Northern blotting for mRNAs extracted from Rat 3-3 and Ret 1 cells, by using 6.6 kb DNA as a probe, it was found that the expression of Ha-ras in the former was five times higher than the latter From the results shown above it seems that the 2.5 kb stretch has a function to regulate the expression of c-Ha-ras by binding to the 35 KD protein, thus enhancing the transcription of the activated c-Ha-ras oncogene.更多还原