REGULATION OF HISTIDINE BIOSYNTHESIS IN YEAST

酵母中组氨酸生物合成的调控

• 批准号: 3484814
• 负责人: GERALD R FINK
• 金额: $ 38.16万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3484814
• 关键词: Saccharomyces; aminoacid biosynthesis; cell growth regulation; chemical binding; chromosome aberrations; chromosome disorders; enzyme mechanism; enzyme substrate complex; eukaryote; fungal genetics; gene expression; gene induction /repression; genetic manipulation; genetic mapping; genetic promoter element; genetic recombination; genetic transcription; genetic translation; histidine; laboratory rabbit; molecular cloning; molecular genetics; mutagen testing; nucleic acid hybridization; nucleic acid sequence; proteins; regulatory gene; site directed mutagenesis; structural genes; synthetic nucleic acid; temperature sensitive mutant; transfer RNA; transposon /insertion element

Our goal is to understand the regulation of gene activity in the yeast Saccharomyces cerevisiae. The studies will focus on histidine biosynthesis as well as the transposable genetic element, Ty1. Both cis and trans-acting elements affecting expression of the histidine genes will be studied using fusions of regulatory sequences to E. coli Beta-galactosidase. The cis-acting sequences within the promoter will be dissected by a combination of site-directed mutagenesis and oligonucleotide synthesis. Various synthetic oligonucleotides will be substituted both at HIS4 and CYC1 to separate the promoter elements required for regulation, initiation, and maintenance of the basal level of transcription. Each of these promoter segments will be used as probes (both genetic and biochemical) for the trans-acting elements that interact with each of the cis-acting elements. In addition, genetic and biochemical analysis of the components required for permeation and cellular localization of histidine will be carried out. The mechanisms by which Ty elements transpose and activate genes can now be dissected. Again, both cis and trans-acting elements will be studied. The genes required in trans for transposition will be cloned and their role in the transposition process determined. The intermediates in transposition as well as the enzymes responsible for transposition will be isolated. Since the Ty element transposes through an RNA intermediate, we will try to isolate and characterize both the reverse transcriptase and RNase's involved in reverse transcription. Our assay system permits the analysis of the structural features of the Ty element that are required for transposition. By making mutations of various modified elements important cis-acting sequences will be identified: The ends of the element, the polypurine stretch, the polymerase binding site. Furthermore, mutations in the element should permit the isolation of intermediates in transposition and ultimately the reconstruction of the pathway of transposition.

我们的目标是了解酵母中基因活性的调节。 酿酒酵母。这些研究将集中在组氨酸的生物合成上。 以及可转座的遗传元件Ty1。配置项和 影响组氨酸基因表达的反式作用元件将是 利用调节序列与大肠杆菌融合的研究 β-半乳糖苷酶。启动子内的顺式作用序列将是 通过定点突变和寡核苷酸的组合进行解剖 综合。各种人工合成的寡核苷酸将在 HIS4和Cyc1来分离调控所需的启动子元件， 启动和维持转录的基础水平。每一个 这些启动子片段将被用作探针(包括基因和 生化)与每一个相互作用的反式作用元素 顺式作用元素。此外，还进行了遗传和生化分析。 组氨酸渗透和细胞定位所需的成分 将会被执行。 Ty元件转座和激活基因的机制现在可以 被解剖了。同样，顺式作用元件和反式作用元件都将被研究。这个 转座所需的反式基因将被克隆，它们在 换位过程决定了。转位过程中的中间体 以及负责转座的酶将被分离出来。 由于Ty元素通过RNA中间体转置，我们将尝试 逆转录酶和核糖核酸酶的分离和鉴定 参与逆转录的。我们的化验系统允许分析 Ty元素的结构特征是什么 换位。通过使各种修饰元素的突变变得重要 顺式作用序列将被识别：元件的末端、 聚合酶结合部位多聚嘌呤的伸展。此外，基因突变 该元素应允许在转位过程中分离中间体 最终重建转位途径。