Mutation Analysis of Saccharomyces cerevisiae CDC6 Promoter: Defining Its UAS Domain and Cell Cycle Regulating Element
AbstractUsing Î²-galactosidase as the reporter gene, we carried out mutagenesis experiments to investigate the 5â² promoter region of the CDC6 gene. Our results showed that the DNA element, between â262 and â170, is important for the upstream activating sequence (UAS) activities. On the basis of the DNA sequence, there is a Mlu I (â204) and a Mlu I-like (â216) element located within the middle of the UAS region. Insertion and deletion mutagenesis analysis of the Mlu I sequence has indicated that the internal CGCG sequence of the Mlu I site (ACGCGT) is important for gene expression. Furthermore, when DNA elements containing the Mlu I sites were subcloned into the tester plasmid, periodic expression of a reporter gene throughout the cell cycle was observed, as evidenced by the Î²-galactosidase activities and lacZ mRNA. Because the possible transcriptional initiation sites of the CDC6 transcript have been previously defined (Zhou and Jong, 1990, J. Biol. Chem. 264, 9022â9029), we propose a model regarding the construct of the CDC6 promoter region. This 5â² promoter construct contains a UAS region and a Mlu I element (MCB box) typical of a family of cell cycleregulated genes involved in DNA metabolism. Previous genetic studies have not completely defined the CDC6 execution point in the functional yeast cell cycle map. Our results favor the possibility that the CDC6 gene is required, and directly involved, in the initiation of DNA replication.