Genomic organization and promoter characterization of the rat cyclin B1 gene

Cyclin B1 is a key regulatory protein involved in cellular mitosis. We have cloned 1.8 kb of DNA sequence upstream of the rat cyclin B1 gene translati on start site from Rattus norvegicus liver genomic DNA and a commercial rat testis genomic library. The mRNA transcription start point (tsp) was deter mined by primer extension and mRNA end ligation followed by RT-PCR across t he ligated 3' and 5' ends. An authentic tsp was confirmed similar to 100 bp upstream of the translation start site. A second potential tsp was also de tected similar to 32 bp downstream from the first. RT-PCR analysis of rat l iver poly(A)(+) RNA using 5'-derived oligonucleotide primers indicated that the 5' end sequence was present in both the 1.6 and 2.4 kb rat liver cycli n 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 transcripti on factor binding sites, some of which are also reported in other cyclin pr omoters. 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 promote r 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 importan t for transcription, and that there may be negative regulatory elements pre sent between 1800 and 1100 bp upstream of the translation start site. (C) 2 000 Elsevier Science B.V. All rights reserved.