MOLECULAR CLONING AND NUCLEOTIDE SEQUENCE ANALYSIS OF A TRANSFORMING GENE FROM HUMAN GASTROCARCINOMA CELL LINE

Mouse and rat fibroblasts were transfected with total DNA from human gastrocarcinoma cell line BGC-823. It was shown by hybridization assay that the genome of one of the rat secondary foci contains transforming genes from the human gastrocarcinoma cell line, which are homologous to the protooncogene c-Ha-ras in the normal cells. The genomic library of the rat secondary foci was constructed, using λ phage EMBL3 as the vector. The transforming gene Ha-ras of the human gastrocarcinoma cell was thus cloned by screening the library with the probes of human Alu repeat sequence and c-Ha-ras. The nucleotide sequences of the first and second exons were analysed by M13-dideoxy method. The result shows that the nucleotide sequence of the transforming gene is the same as that of the normal protooncogene except one nucleotide difference in the first exon.     

INHIBITED NEOPLASTIC PHENOTYPE BY THE c-Ha-ras ANTISENSE RNA

A 2.0 kb fragment DNA plasmid which expresses antisense to the upstream first exon of c-Ha-ras oncogene was transfected into Ha-ras transformed cell lines, GCM-3T3 and REF-4.3. The transfection leads to the inhibition of malignant behaviour, shown by decreasing of growth speed, colony forming ability on soft agar, tumorigenicity in nude mice and increasing ot differentiation degree. In GCM-3T3 cells the lung metastasis frequency became much less (from 60% to 12.5%) after the transfection and the expression of ras oncogene product, and p21 protein was obviously decreased in the transfected cells. This work has first shown the inhibitory effect of antisense RNA on neoplastic behaviour in China.     

人胃癌细胞株转化基因的克隆和核苷酸序列的分析

本文用人胃癌细胞株BGC-823总DNA对小鼠及大鼠成纤维细胞进行转染。用分子杂交方法证明大鼠第二轮恶性转化细胞基因组中合有来自人胃癌的转化基因,它与存在于正常细胞中的原癌基因c-Ha-ras同源。以λ噬菌体EMBL 3为载体,构建大鼠第二轮转化细胞基因组文库,以人Alu重复序列和c-Ha-ras为探针,将人胃癌细胞转化基因Ha-ras从文库中克隆分离出。用M13——双脱氧法测定转化基因第一、第二外显子核苷酸序列。结果表明转化基因除了第一外显子中有一个碱基发生变化外,核苷酸序列与原癌基因完全相同。     

c-Ha-ras反义RNA对细胞恶性表型逆转作用的研究

本文应用表达反义c-Ha-ras癌基因上游区第一外显子2.0kb片段的RNA质粒,转染经c-Ha-ras转染的恶性细胞系GCM-3T3与REF-4.3,造成恶性行为的改变.表现在生长速度减慢,软琼脂克隆形成能力下降、裸鼠中致瘤力减小和肿瘤的病理分化程度增加,其中原来可造成转移的GCM-3T3细胞经反义RNA转染后肺转移率从60％降至12.5％,同时,两种细胞经转染后分离的克隆都有显著的ras癌基因产物p21蛋白表达的减少.本工作证明了反义RNA在癌基因表达上的调控和抑制细胞恶性行为的作用.