Trembley J H, Chen Z, Rodrigues C M, Kren B T, Steer C J
Department of Medicine, University of Minnesota Medical School, Box 36 UMHC, 420 Delaware St. S.E., 55455, Minneapolis, MN, USA.
Gene. 2000 Sep 5;255(1):93-104. doi: 10.1016/s0378-1119(00)00319-x.
Cyclin B1 is a key regulatory protein involved in cellular mitosis. We have cloned 1.8kb of DNA sequence upstream of the rat cyclin B1 gene translation start site from Rattus norvegicus liver genomic DNA and a commercial rat testis genomic library. The mRNA transcription start point (tsp) was determined by primer extension and mRNA end ligation followed by RT-PCR across the ligated 3' and 5' ends. An authentic tsp was confirmed approximately 100bp upstream of the translation start site. A second potential tsp was also detected approximately 32bp downstream from the first. RT-PCR analysis of rat liver poly(A)(+) RNA using 5'-derived oligonucleotide primers indicated that the 5' end sequence was present in both the 1.6 and 2. 4kb rat liver cyclin B1 mRNA species. Like many other cyclin promoters, there was no apparent TATA box upstream of the transcription initiation sites. However, computer analysis of the promoter region identified a group of consensus transcription factor binding sites, some of which are also reported in other cyclin promoters. These include those for p53, p21, Ap-1, Ap-2, Ets-1, CAATT, E-Box and Yi. We also performed luciferase reporter assays using a set of promoter deletion constructs in human HuH-7 hepatoma and HeLa carcinoma cell lines. Our results suggest that an E-Box and/or CCAAT binding sites are important for transcription, and that there may be negative regulatory elements present between 1800 and 1100bp upstream of the translation start site.
细胞周期蛋白B1是一种参与细胞有丝分裂的关键调节蛋白。我们从褐家鼠肝脏基因组DNA和一个商业大鼠睾丸基因组文库中克隆了大鼠细胞周期蛋白B1基因翻译起始位点上游1.8kb的DNA序列。通过引物延伸和mRNA末端连接，随后进行跨越连接的3'和5'末端的逆转录聚合酶链反应（RT-PCR）来确定mRNA转录起始点（tsp）。在翻译起始位点上游约100bp处确认了一个真实的tsp。在第一个tsp下游约32bp处也检测到了第二个潜在的tsp。使用5'端衍生的寡核苷酸引物对大鼠肝脏多聚腺苷酸（poly(A)）(+)RNA进行RT-PCR分析表明，5'端序列存在于1.6kb和2.4kb的大鼠肝脏细胞周期蛋白B1 mRNA种类中。与许多其他细胞周期蛋白启动子一样，转录起始位点上游没有明显的TATA框。然而，对启动子区域的计算机分析确定了一组共有转录因子结合位点，其中一些在其他细胞周期蛋白启动子中也有报道。这些包括p53、p21、Ap-1、Ap-2、Ets-1、CAATT、E-Box和Yi的结合位点。我们还使用一组启动子缺失构建体在人HuH-7肝癌细胞系和HeLa癌细胞系中进行了荧光素酶报告基因测定。我们的结果表明，一个E-Box和/或CCAAT结合位点对转录很重要，并且在翻译起始位点上游1800至1100bp之间可能存在负调控元件。
