Young E T, Sloan J, Miller B, Li N, van Riper K, Dombek K M
Department of Biochemistry, University of Washington, Seattle, WA, USA.
Gene. 2000 Mar 21;245(2):299-309. doi: 10.1016/s0378-1119(00)00035-4.
To determine when a glucose-repressed alcohol dehydrogenase isozyme and its regulatory gene, ADR1, arose during evolution, we surveyed species of the genus Saccharomyces for glucose-repressed ADH isozymes and for ADR1 homologues. Glucose-repressed ADH isozymes were present in all species of Saccharomyces sensu strictu and also in Saccharomyces kluyveri, the most distant member of the Saccharomyces clade. We cloned and characterized ADH promoters from S. bayanus, S. douglasii, and S. kluyveri. The ADH promoters from S. bayanus and S. douglasii had conserved sequences, including upstream regulatory elements, and an extended polydA tract. The expression of a reporter gene driven by the S. bayanus promoter was glucose-repressed and dependent on the major activator of transcription, ADR1, when it was introduced into S. cerevisiae. One S. kluyveri promoter was also glucose-repressed and ADR1-dependent in S. cerevisiae. The other S. kluyveri ADH promoter was expressed constitutively and was ADR1-independent. Although showing little sequence conservation with the S. cerevisiae ADH2 promoter, the glucose-repressed S. kluyveri promoter contains numerous potential binding sites for Adr1. The glucose-repressed ADH from S. kluyveri is a mitochondrial isozyme most closely related to S. cerevisiae ADHIII. ADR1 homologues from S. douglasii and S. paradoxus contain a trinucleotide repeat encoding polyAsn that is lacking in S. cerevisiae and S. bayanus. No ADR1 homologue could be detected in S. kluyveri, suggesting that the potential for Adr1 regulation may have arisen first, before ADR1 evolved.
为了确定葡萄糖抑制型乙醇脱氢酶同工酶及其调控基因ADR1在进化过程中出现的时间，我们调查了酿酒酵母属的物种，以寻找葡萄糖抑制型乙醇脱氢酶同工酶和ADR1同源物。葡萄糖抑制型乙醇脱氢酶同工酶存在于所有狭义酿酒酵母物种中，也存在于酿酒酵母进化枝中最遥远的成员克鲁维酵母中。我们克隆并鉴定了巴氏酵母、道格拉斯酵母和克鲁维酵母的乙醇脱氢酶启动子。巴氏酵母和道格拉斯酵母的乙醇脱氢酶启动子具有保守序列，包括上游调控元件和一个延伸的多聚腺苷酸区域。当将由巴氏酵母启动子驱动的报告基因导入酿酒酵母时，其表达受到葡萄糖抑制且依赖于转录的主要激活因子ADR1。克鲁维酵母的一个启动子在酿酒酵母中也受到葡萄糖抑制且依赖于ADR1。克鲁维酵母的另一个乙醇脱氢酶启动子组成型表达且不依赖于ADR1。尽管与酿酒酵母ADH2启动子的序列保守性较低，但葡萄糖抑制型克鲁维酵母启动子含有许多Adr1的潜在结合位点。克鲁维酵母的葡萄糖抑制型乙醇脱氢酶是一种线粒体同工酶，与酿酒酵母ADHIII关系最为密切。道格拉斯酵母和奇异酵母的ADR1同源物含有一个编码多聚天冬酰胺的三核苷酸重复序列，而酿酒酵母和巴氏酵母中则没有。在克鲁维酵母中未检测到ADR1同源物，这表明Adr1调控的潜力可能在ADR1进化之前就已出现。
