人U_1和U_2 snRNA基因5'端区域中SV_(40)T抗原结合位点的作用

ROLE OF SV40 T ANTIGEN BINDING SITES WITHIN THE 5′ FLANKING REGION OF HUMAN U_1 AND U_2 snRNA GENES

利用寡聚核苷酸指导的基因定位突变技术,从人U_1和U_2 snRNA基因5'端删去或取代能与SV_(40)T抗原结合的位点,构成基因的缺失和取代突变体。在HeLa细胞核抽提物体外转录系统中,U_1和U_2缺失及取代突变基因与野生型基因有相同的转录水平,而且RNA的合成都是从帽子位点的上游开始的。这意味着基因这一区域的突变不改变其体外转录性质。在人的HeLa和293细胞及蛙卵母细胞中,U_1和U_2缺失突变基因不被转录,而取代突变基因的转录却又恢复到野生型基因水平。这表明人的U_1和U_2基因在这三种细胞内的转录与SV_(40)T抗原结合位点的核苷酸排列顺序无关,而由于结合位点的缺失所造成的空间距离上的变化才是影响转录的主要因素。

The 5' flanking regions of the genes encoding human U_1 and U_2 snRNA each contain multiple copies of consensus pentanucleotide 5' GA/GGGC 3' which bind specifically to the SV40 T antigen. To examine the role of T antigen binding sites, the mutants deleted and substituted the pentanucleotide sequences from 5' flanking of U_1 and U_2 genes were constructed by oligonucleotide-directed DNA synthesis. A similar transcription level was observed between these mutants and wild type in cellfree transcription system. Both U_1 and U_2 snRNA synthesized in vitro initiate from upstream of the cap site identified in vivo. This imply that deletion and substitution of T antigen binding sites unaffect the transcriptional property of U_1 and U_2 genes in vitro. We have shown that deletion mutants virtually eliminated U_1 and U_2 snRNA synthcsis in HeLa, 293 and oocytes cells, while substitution mutants fully restored transcription to wild type level. Thus,while the region located between -11 and-42 of human U_1 or-58 and-90 of human U_2 DNA is required for transcription in these cells, the wild type DNA sequence in this region is not a prerequistc for transcription. This suggests that this region is required for DNA spacing rather than nucleotide sequence.