Isolation of constitutive mutations affecting the proline utilization pathway in Saccharomyces cerevisiae and molecular analysis of the PUT3 transcriptional activator.
Isolation of constitutive mutations affecting the proline utilization pathway in Saccharomyces cerevisiae and molecular analysis of the PUT3 transcriptional activator.
复制标题
影响酿酒酵母脯氨酸利用途径的组成型突变的分离和 PUT3 转录激活因子的分子分析。
DOI：
10.1128/mcb.9.11.4696-4705.1989
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发表时间：
1989
影响因子：
5.3
通讯作者：
Brandriss,MC
中科院分区：
文献类型：
--
作者：
Marczak,JE;Brandriss,MC
酿酒酵母利用脯氨酸途径的酶(PUT1和PUT2基因产物)受PUT1和PUT3转录激活子的协同调节。为了更多地了解这一途径的控制，人们寻找了PUT3以及其他调控因子中的结构性突变。建立了一种利用PUT1(酿酒酵母脯氨酸氧化酶)和GALK(大肠杆菌)之间的基因融合来直接筛选影响PUT1启动子的结构性突变的方案。利用Δ1-吡咯啉-5-羧酸脱氢酶(PUT2)基因与大肠杆菌β-半乳糖苷酶(LacZ)基因融合，对这些突变在PUT2基因启动子上的作用进行二次筛选。分离出了三种不同类别的突变。主要由半显性组成的PUT3突变组成，这些突变导致PUT2-lacZ的表达水平从未诱导水平的2到22倍不等。一个名为PUT5的新基因座上的单个显性突变导致了PUT2-LacZ的低水平结构性表达；该突变对隐性的、非诱导的put3-75等位基因是上位的。分离出具有多效性生长缺陷的隐性构成突变；这些突变可能不是特定于脯氨酸利用途径的，而是可能存在于控制几个途径的基因中。由于PUT3基因似乎在这一途径的调节中起着重要作用，因此进行了分子分析。该基因是通过对put3-75突变进行功能互补而克隆的。携带该基因完全缺失的菌株是活的，不利用脯氨酸，在表型上与原始的put3-75等位基因没有区别。PUT3基因编码一个2.8kb的转录本，在RNA积累水平上不受脯氨酸的调控。该基因在高拷贝数质粒上的存在并没有改变其靶基因之一PUT2-lacZ的调节，这表明PUT3基因产物不是限制性的，并且可滴定的阻遏子不参与这一途径的调节。
The enzymes of the proline utilization pathway (the products of thePUT1andPUT2genes) inSaccharomyces cerevisiaeare coordinately regulated by proline and thePUT3transcriptional activator. To learn more about the control of this pathway, constitutive mutations inPUT3as well as in other regulators were sought. A scheme using a gene fusion betweenPUT1(S.cerevisiaeproline oxidase) andgalK(Escherichia coligalactokinase) was developed to select directly for constitutive mutations affecting thePUT1promoter. These mutations were secondarily screened for their effects intranson the promoter of thePUT2(Δ1-pyrroline-5-carboxylate dehydrogenase) gene by using aPUT2-lacZ(E. coliβ-galactosidase) gene fusion. Three different classes of mutations were isolated. The major class consisted of semidominant constitutivePUT3mutations that causedPUT2-lacZexpression to vary from 2 to 22 times the uninduced level. A single dominant mutation in a new locus calledPUT5resulted in low-level constitutive expression ofPUT2-lacZ; this mutation was epistatic to the recessive, noninducibleput3-75allele. Recessive constitutive mutations were isolated that had pleiotropic growth defects; it is possible that these mutations are not specific to the proline utilization pathway but may be in genes that control several pathways. Since thePUT3gene appears to have a major role in the regulation of this pathway, a molecular analysis was undertaken. This gene was cloned by functional complementation of theput3-75mutation. Strains carrying a complete deletion of this gene are viable, proline nonutilizing, and indistinguishable in phenotype from the originalput3-75allele. ThePUT3gene encodes a 2.8-kilobase-pair transcript that is not regulated by proline at the level of RNA accumulation. The presence of the gene on a high-copy-number plasmid did not alter the regulation of one of its target genes,PUT2-lacZ, suggesting that thePUT3gene product is not limiting and that a titratable repressor is not involved in the regulation of this pathway.