Hepworth S R, Ebisuzaki L K, Segall J
Department of Biochemistry, University of Toronto, Ontario, Canada.
Mol Cell Biol. 1995 Jul;15(7):3934-44. doi: 10.1128/MCB.15.7.3934.
Sporulation of the yeast Saccharomyces cerevisiae represents a simple developmental process in which the events of meiosis and spore wall formation are accompanied by the sequential activation of temporally distinct classes of genes. In this study, we have examined expression of the SPS4 gene, which belongs to a group of genes that is activated midway through sporulation. We mapped the upstream boundary of the regulatory region of SPS4 by monitoring the effect of sequential deletions of 5'-flanking sequence on expression of plasmid-borne versions of SPS4 introduced into a MATa/MAT alpha delta sps4/delta sps4 strain. This analysis indicated that the 5' boundary of the regulatory region was within 50 bp of the putative TATA box of the gene. By testing various oligonucleotides that spanned this boundary and the downstream sequence for their ability to activate expression of a heterologous promoter, we found that a 15-bp sequence sufficed to act as a sporulation-specific upstream activation sequence. This 15-bp fragment, designated UASSPS4, activated expression of a CYC1-lacZ reporter gene midway through sporulation and was equally active in both orientations. Extending the UAS fragment to include the adjacent 14-bp enhanced its activity 10-fold. We show that expression of SPS4 is regulated in a manner distinct from that of early meiotic genes: mutation of UME6 did not lead to vegetative expression of SPS4, and sporulation-specific expression was delayed by mutation of IME2. In vivo and in vitro assays suggested that a factor present in vegetative cells bind to the UASSPS4 element. We speculate that during sporulation this factor is modified to serve as an activator of the SPS4 gene or, alternatively, that it recruits an activator to the promoter.
酿酒酵母的孢子形成代表了一个简单的发育过程，在此过程中，减数分裂和孢子壁形成事件伴随着时间上不同类别的基因的顺序激活。在本研究中，我们检测了SPS4基因的表达，该基因属于一组在孢子形成过程中被激活的基因。我们通过监测5'侧翼序列的连续缺失对导入MATa/MATαδsps4/δsps4菌株的质粒携带的SPS4版本的表达的影响，绘制了SPS4调控区域的上游边界。该分析表明，调控区域的5'边界在该基因假定的TATA框的50 bp范围内。通过测试跨越该边界和下游序列的各种寡核苷酸激活异源启动子表达的能力，我们发现一个15 bp的序列足以作为孢子形成特异性上游激活序列。这个15 bp的片段，命名为UASSPS4，在孢子形成过程中激活了CYC1-lacZ报告基因的表达，并且在两个方向上都具有同等活性。将UAS片段延伸至包括相邻的14 bp可使其活性提高10倍。我们表明，SPS4的表达调控方式与早期减数分裂基因不同：UME6的突变不会导致SPS4的营养型表达，而IME2的突变会延迟孢子形成特异性表达。体内和体外实验表明，营养细胞中存在的一种因子与UASSPS4元件结合。我们推测，在孢子形成过程中，这种因子被修饰以作为SPS4基因的激活剂，或者，它将一种激活剂招募到启动子上。
