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: 3734293
• 负责人: GOVIND S NADATHUR
• 金额: --
• 依托单位: UNIVERSITY OF PUERTO RICO MAYAGUEZ
• 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/3734293
• 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 环境信号转导机制的研究进展 汉森德巴利酵母。在这项研究中，我们选择了盐作为 外部信号。造成这种情况的主要原因之一是 变形霉菌在无盐条件下也能存活 高盐度环境。 将采用三种方法来分离盐诱导的 基因。第一个涉及构建消减的cdna文库。 用于富集盐诱导的cDNA.第二种方法将 利用基因工程技术分离3-磷酸甘油脱氢酶基因 合成的寡聚脱氧核苷酸，互补于保守的区域 吉恩。最后，由于我们已经确定了一个150kD的。仅存在蛋白质 在盐诱导的培养中，我们将提纯这种蛋白质并提高 抗它的多克隆抗体。我们将使用这些抗体进行筛查 构建基因表达文库，并分离相应的基因。这 分离的cdna将作为探针用于筛选基因组文库 分离出该蛋白质的基因。 一旦基因被分离出来，它们将被特别强调地进行测序 在5‘端非转录序列上。我们将分析这些序列并 确定可能参与监管的顺式作用区域。 在这些调控序列的帮助下，我们还将构建 允许在Debarymyces中表达任何基因的载体 汉森。