CHARACTERIZATION OF EVOLUTIONARY INDUCED GENES IN YEAST DEBARYOMYCES HANSENII

汉逊酵母中进化诱导基因的表征

• 批准号: 5211653
• 负责人: GOVIND S NADATHUR
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5211653
• 关键词: biochemical evolution; biological signal transduction; complementary DNA; computer assisted sequence analysis; electrolyte balance; fungal genetics; gene expression; genetic library; genetic regulatory element; glycerol 3 phosphate dehydrogenase; molecular genetics; northern blottings; nucleic acid probes; nucleic acid sequence; subtraction hybridization; yeasts

The long term goals of this investigation ar to understand the molecular mechanisms involved in the transduction of environmental signals by the yeast Debaryomyces hansenii. For this research we have chosen salt as an external signal. One of the major reasons for this is the fact that Debaryomyces is capable of survival both in the absence of salt as well hypersaline environments. Three approaches will be employed for the isolation of salt-induced genes. The first involves the construction of a subtracted cDNA library for the enrichment of salt-induced cDNA. The second approach will isolate the gene for the glycerol-3-phosphate dehydrogenase utilizing a synthetic oligodeoxynucleotide complementary to a conserved region of the gene. Finally, since we have identified a 150 kD. protein present only in salt-induced cultures, we will purify this protein and raise polyclonal antibodies against it. We will use these antibodies to screen cDNA expression libraries and isolate the corresponding cDNA. This isolated cDNA will be used as a probe to screen genomic libraries for the isolation of the gene to this protein. Once the genes are isolated, they will be sequenced with special emphasis on the 5' non-transcribed sequences. We will analyze these sequences and identify cis-acting regions that could be involved in the regulation. With the help of these regulatory sequences we will also construct vectors that will allow the expression of any gene in Debaryomyces hansenii.

这项研究的长期目标是了解分子水平， 参与环境信号转导的机制， 汉逊德巴利酵母 在这项研究中，我们选择了盐作为 外部信号。 其中一个主要原因是， 德巴利酵母菌也能在无盐条件下存活 高盐环境 三种方法将用于分离盐诱导的 基因. 第一个是构建消减cDNA文库 用于盐诱导cDNA的富集。 第二种方法将 利用一种分离甘油-3-磷酸脱氢酶的基因的方法， 合成的寡脱氧核苷酸，其互补于所述多肽的保守区。 基因 最后，因为我们已经确定了一个150 kD。仅蛋白质存在 在盐诱导的培养中，我们将纯化这种蛋白质， 我们将用这些抗体来筛选 cDNA表达文库，并分离相应的cDNA。这 分离的cDNA将被用作探针来筛选基因组文库， 分离这种蛋白质的基因。 一旦这些基因被分离出来， 5'非转录序列。 我们将分析这些序列， 确定可能参与调控的顺式作用区域。 在这些调控序列的帮助下，我们还将构建 允许德巴利酵母中任何基因表达的载体 汉森尼。