CELL CYCLE-REGULATED TRANSCRIPTION IN S CEREVISIAE

酿酒酵母HO基因的转录紧随其后 取决于细胞周期的开始。启动子元件CACGAAAA (或SCB)和所需的两个反式作用调节器(SWI4和SWI6 对于HO转录的细胞周期调控已经被确定 (Breeden和Nasmyth，1987A)。这些SCB元素是最近发现的 启动子中有三个Gl细胞周期蛋白基因。此外，我们已经表明， SWI4和一大群SWI4的周期性表达需要SWI6 含有不同细胞周期调控启动子的DNA合成基因 元素ACGCGTNA(或MCB)。这导致我们将SWI6视为一个关键环节 在G1后期发生的所有已知周期性转录事件之间， 提示SWI6活性的调节可能定义了Gl/S 过渡。 我们建议继续努力了解START的机制-- 依赖的跨脚本，特别关注合作伙伴和 SWI4和SWI6蛋白的活性。我们将寻找依赖于Start的 SWI4和SWI6的磷酸化变化。我们还将描述 我们已经分离出的sw6突变的抑制因子，并鉴定了其他 与SWI6相互作用的基因产物。 我们发现Gl/S的一个重要原因--特异性转录 HO和CLN基因是SWI4转录的细胞周期调控基因。 SWI4启动子的必需上游激活序列(UAS) 位于140个碱基对的DNA片段内。它对细胞周期是足够的 法规，并包含三个MCB元素。令人惊讶的是，删除所有 三个MCB元件不能消除细胞周期调控的转录。 我们建议对SWI4启动子进行进一步研究以鉴定新的细胞 周期调节要素。140 BP SWI4无人机的不同寻常之处还在于它 不会以相反方向激活转录。我们建议 筛选与此抑制有关的序列中的突变体 然后确定此序列是否在其 正常的染色体定位，使SWI4启动子与上游隔离 如果是这样，我们将获得导致这种情况的因素的突变 边界活动。 最后，我们生成了一个稳定的、独立于SWI6的SWI4形式 在整个过程中导致CLN1和CLN2高水平转录的蛋白质 细胞周期。这对细胞生长有明显的负面影响。我们建议 来描述这种生长缺陷并分离出代偿性突变 和这一表型的高拷贝抑制因子。通过这样做，我们将确定 参与Gl细胞周期蛋白代谢和激活的其他细胞因子。

Transcription of the S.cerevisiae HO gene occurs immediately after and is dependent upon the Start of the cell cycle. The promoter element CACGAAAA (or SCB) and two trans-acting regulators (SWI4 and SWI6) that are required for the cell cycle regulation of HO transcription have been identified (Breeden and Nasmyth, 1987a). These SCB elements have recently been found in the promoters three Gl cyclin genes. In addition, we have shown that SWI6 is required for the periodic expression of SWI4 and a large group of DNA synthesis genes that contain a different cell cycle regulated promoter element ACGCGTNA (or MCB). This leads us to view SWI6 as a critical link between all the known periodic transcription events that occur in late Gl, and suggests that the regulation of SWI6 activity may define the Gl/S transition. We propose to continue our efforts to understand the mechanisms of Start- dependent tran-scription, with a particular focus on the partners and activities of the SWI4 and SWI6 proteins. We will look for Start-dependent changes in phosphorylation of SWI4 and SWI6. We will also characterize suppressors of swi6 mutations that we have isolated, and identify other gene products that interact with SWI6. We have found that one important cause of Gl/S-specific transcription of HO and the CLN genes is the cell cycle regulation of SWI4 transcription. The essential upstream activation sequence (UAS) in the SWI4 promoter resides within a 140 bp DNA fragment. It is sufficient for cell cycle regulation and contains three MCB elements. Surprisingly, deletion of all three MCB elements does not eliminate cell cycle regulated transcription. We propose further studies of the SWI4 promoter to identify the novel cell cycle regulatory element. The 140 bp SWI4 UAS is also unusual in that it does not activate transcription in the reverse orientation. We propose to screen for mutants in the sequence responsible for this repression and then to determine if this sequence performs a boundary function in its normal chromosomal location, insulating the SWI4 promoter from upstream UAS's. If so, we will obtain mutations in the factors responsible for this boundary activity. Finally, we have generated a stable, SWI6-independent form of the SWI4 protein which causes high level CLN1 and CLN2 transcription throughout the cell cycle. This has a dominant negative effect on cell growth. We propose to characterize this growth defect and to isolate compensatory mutations and high copy suppressors of this phenotype. In so doing, we will identify other cellular factors involved in Gl cyclin metabolism and activation.