Andrews B J, Moore L
Department of Molecular and Medical Genetics, University of Toronto, Ont., Canada.
Biochem Cell Biol. 1992 Oct-Nov;70(10-11):1073-80. doi: 10.1139/o92-152.
Entry of budding yeast cells into the mitotic cell cycle requires the activity of a conserved regulatory kinase encoded by the CDC28 gene. The kinase is thought to trigger entry into the cell cycle or START, through association with a number of regulatory subunits known as G1 cyclins. A number of genes whose transcription is dependent on CDC28 and thus linked to START are controlled by two transcription factors, SWI4 and SWI6. The genes controlled by SWI4 and SWI6 include two known G1 cyclins (CLN1 and CLN2), a putative new G1 cyclin (HCS26), and the HO gene whose product initiates cell type switching. SWI4 and SWI6 act through a repeated sequence element, SCB (SWI4,6-dependent cell cycle box), found 2-10 times in the upstream regulatory sequences of target genes. We have constructed a library of mutants in the SCB using doped oligonucleotide mutagenesis. All single base pair changes examined compromised the ability of the SCB to activate transcription in vivo. Analysis of the behaviour of the mutant SCBs in an in vitro DNA binding assay shows that the inability to activate transcription can be explained by reduced binding of SWI4 and SWI6 to the mutant SCBs. This analysis, together with a consideration of the SCBs found upstream of known SWI4,6-dependent genes, leads to the proposal of a revised consensus sequence for this important regulatory element.
出芽酵母细胞进入有丝分裂细胞周期需要由CDC28基因编码的一种保守的调节激酶的活性。这种激酶被认为通过与一些被称为G1细胞周期蛋白的调节亚基结合来触发进入细胞周期或起始点（START）。许多转录依赖于CDC28从而与起始点相关的基因受两个转录因子SWI4和SWI6的控制。受SWI4和SWI6控制的基因包括两个已知的G1细胞周期蛋白（CLN1和CLN2）、一个假定的新G1细胞周期蛋白（HCS26）以及其产物启动细胞类型转换的HO基因。SWI4和SWI6通过一个重复序列元件SCB（SWI4,6依赖的细胞周期框）起作用，该元件在靶基因的上游调控序列中出现2至10次。我们利用掺杂寡核苷酸诱变构建了一个SCB突变体文库。所有检测的单碱基对变化都损害了SCB在体内激活转录的能力。对突变SCB在体外DNA结合试验中的行为分析表明，转录激活能力的丧失可以通过SWI4和SWI6与突变SCB的结合减少来解释。这一分析，连同对已知的SWI4,6依赖基因上游发现的SCB的考虑，导致了对这一重要调控元件的修订共有序列的提议。
