摘要
应用PCR技术，对P16抑癌基因（CDKN2）进行体外定点突变．在P16cDNA中引入第48位密码子CCG（Pro）→CTG（Leu）和第74位密码子GAC（Asp）→AAC（Asn）突变，构建了p16－P48L和p16－D74N突变体，并把它们导入纯合缺失P16基因的人肺癌细胞株H460．经RNA点杂交、Northern印迹、Western印迹和细胞免疫化学染色，检测到P16表达．通过比较表达野生型和突变型P16的H460细胞在3H－TdR掺入及细胞所处周期的差异，证实P16表达抑制细胞进入S期，而P48L和D74N突变体对细胞进入S期没有影响．为了确证P48L和D74N突变体丧失抑制细胞增殖的功能是因为与CDK4结合功能下降，将野生型和突变型P16cDNA克隆于酵母表达载体plexA，用酵母双杂交筛选实验研究野生型和突变型p16蛋白与CDK4的结合．结果野生型P16和CDK4在酵母中表达并相互作用．而突变型P16cDNA和CDK4在酵母中相互作用受到影响．说明P48L和D74N突变影响了p16蛋白与CDK4的结合，从而影响了其调控细胞周期的功能．
The tumor suppressor P16 is a cyclin-dependent kinase inhibitor that inhibits cell proliferation. The P16 cDNA was cloned from HeLa cell by RT-PCR. Two P16 mutants,P48L and D74N, were obtained by site-directed mutagenesis using two step PCR method,and the sequences were confirmed. Then they were cloned into the mammalin expression vector pcDNA3 to construct P16 expression vectors pcDNA3-P16, pcDNA3-P16P48L and pcDNA3-P16D74N, respectively. After introduction of these expression construction into human lung cancer cell lines H460 in which P16 gene was homozygously deleted, exogenous P16 pression were detected in G418-resistant cell clones by Northern blotting, Western blotting and immunohistochemistry staining. Overexpression of the wild type P16 inhibited cell proliferation by causing G1 arrest. In contrast, both of two mutants showed a defect in their ability to arrest cell cycle. In order to confirm that these two mutants with affected p16 function in cell cycle arrest were caused by affected interaction with CDK4, the wild type and mutant P16 cDNA were cloned into yeast expression vector pLexA,and cotransformed yeast EGY48 [p8op-lacZ] with pB42-CDK4 construction respectively. the interaction mating experiments showed that, as previously reported the wild-type p16 interacted with CDK4, but the two P16 mutants, P16P48L and P16D74N, showed a decrease in affinities for CDK4.This result suggested that these two P16 missense mutants were functionally of deficiency.
出处
《复旦学报（自然科学版）》
CAS
CSCD
北大核心
1998年第4期387-394,共8页
Journal of Fudan University：Natural Science
关键词
P16基因
P48L
D74N
肿瘤抑制基因
P16 gene
function studying
site-directed mutagenesis
gene expression
cell cycle
yeast two-hybrid protein interaction screen
