CLONING AND DISRUPTION OF A GENE REQUIRED FOR GROWTH ON ACETATE BUT NOT ON ETHANOL - THE ACETYL-COENZYME-A SYNTHETASE GENE OF SACCHAROMYCES-CEREVISIAE
CLONING AND DISRUPTION OF A GENE REQUIRED FOR GROWTH ON ACETATE BUT NOT ON ETHANOL - THE ACETYL-COENZYME-A SYNTHETASE GENE OF SACCHAROMYCES-CEREVISIAE
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DOI：
10.1002/yea.320081207
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发表时间：
1992-12-01
期刊：
影响因子：
2.6
通讯作者：
WIEMKEN, A
中科院分区：
文献类型：
--
作者：
DEVIRGILIO, C;BURCKERT, N;WIEMKEN, A
克隆了酿酒酵母的乙酰辅酶A(乙酰辅酶A)合成酶基因的DNA片段。测序并定位于第一染色体，它包含一个2139个核苷酸的开放阅读框，编码79-2 kDa的预测基因产物。与其子囊菌同源物相反，编码序列中没有内含子。开放阅读框的第一个ATG密码子处于翻译起始点的不同寻常的上下文中，而下一个ATG密码子，下游24个密码子，处于更常规的上下文中。讨论了两个可供选择的翻译起始点对酶的细胞定位的可能影响。通过基因阻断技术获得的该基因的一个稳定突变体，在葡萄糖条件下具有与野生型细胞相同的低基础乙酰辅酶A合成酶活性，但完全缺乏进入稳定期时的强烈活性增加，这为该基因编码酵母可诱导的乙酰辅酶A合成酶(ACS1)提供了直接证据。不出所料，该突变株不能以醋酸盐为唯一碳源生长。然而，在醋酸盐培养基上，异柠檬酸裂解酶的诱导是正常的，这表明乙酰-辅酶A合成酶的活性对于酿酒酵母乙醛酸循环的诱导是必不可少的。令人惊讶的是，A CSI基因的中断并没有影响在以乙醇为唯一碳源的培养基上的生长，这表明在这些条件下存在导致乙酰-辅酶A的替代途径。
A DNA fragment of Saccharomyces cerevisiae with high homology to the acetyl-coenzyme A (acetyl-COA) synthetase genes of Aspergillus nidulans and Neurospora crassa has been cloned. sequenced and mapped to chromosome I. It contains an open reading frame of 2139 nucleotides, encoding a predicted gene product of 79-2 kDa. In contrast to its ascomycete homologs, there are no introns in the coding sequence. The first ATG codon of the open reading frame is in an unusual context for a translational start site, while the next ATG, 24 codons downstream, is in a more conventional context. Possible implications of two alternative translational start sites for the cellular localization of the enzyme are discussed. A stable mutant of this gene, obtained by the gene disruption technique, had the same low basal activity of acetyl-CoA synthetase as wild-type cells when grown on glucose but completely lacked the strong increase in activity upon entering the stationary phase, providing direct proof that the gene encodes an inducible acetyl-CoA synthetase (ACS1) of yeast. As expected, the mutant was unable to grow on acetate as sole carbon source. Nevertheless, it showed normal induction of isocitrate lyase on acetate media, indicating that activity of acetyl-CoA synthetase is dispensable for induction of the glyoxylate cycle in S. cerevisiae. Surprisingly, disruption of the A CSI gene did not affect growth on media containing ethanol as the sole carbon source, demonstrating that there are alternative pathways leading to acetyl-CoA under these conditions.