MOLECULAR ANALYSIS OF PRENYLATION IN CANDIDA ALBICANS

白色念珠菌异戊二烯化的分子分析

• 批准号: 2749387
• 负责人: Theodore C. White
• 金额: $ 3.06万
• 依托单位: SEATTLE BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2749387
• 关键词: Candida albicans; Saccharomyces; fungal genetics; gene expression; genetic library; genetic promoter element; genetic terminator element; glycosylphosphatidylinositols; isoprenoid; membrane proteins; molecular cloning; northern blottings; oligonucleotides; polymerase chain reaction; protein sequence; southern blotting

DESCRIPTION: This project proposes to clone the C. albicans RAM2 gene specifying a common subunit of prenylation enzymes. Several methods are considered and the method given priority is to use PCR with degenerate oligonucleotides based on database sequences of the protein in other organisms. If this method is not successful then other methods will be used sequentially until success is achieved. In order these methods are: 1) complementation in S. cerevisiae with a C. albicans genomic library in an appropriate vector; 2) preparation of antibodies to the S. cerevisiae fusion protein, confirmation of reactivity with a candidal extract and screening of a C. albicans expression library; and 3) screening the candidal library with the S. cerevisiae gene. Aim 2 has multiple components to characterize RAM2. The gene including promoter will be sequenced. If the original cloning does not produce the whole gene, the entire sequence will be isolated from a genomic library or if a larger clone is isolated, the gene will be subcloned. The chromosomal location and number of alleles will be determined by chromosomal and Southern blotting. The expression of the gene in different strains, under different growth, morphology, and switch phenotypes will be determined by Northern analysis. If not already analyzed, the ability of the gene to complement a ram2 strain of S. cerevisiae will be determined. A candidal fusion protein will be produced and used to elicit antibodies. These antibodies will be used to monitor protein levels. If time permits, the gene will be disrupted.

描述：该项目建议克隆白色念珠菌RAM2基因 指定原苯酶的常见亚基。 几种方法是 考虑到优先级的方法是将PCR与退化一起使用 基于其他蛋白质的数据库序列的寡核苷酸 有机体。 如果此方法不成功，则将使用其他方法 依次直到取得成功为止。 按照这些方法为：1） 在酿酒酵母中与白色念珠菌基因组文库中的互补 适当的向量； 2）制备酿酒酵母融合的抗体 蛋白质，用候选提取物确认反应性并筛选 白色念珠菌表达式库； 3）用 酿酒酵母基因。 AIM 2具有多个组件来表征RAM2。 包括启动子在内的基因将进行测序。 如果原始克隆确实 不产生整个基因，整个序列将从A中分离出来 基因组文库或较大的克隆分离，基因将为 亚克隆。 染色体位置和等位基因的数量将是 由染色体和南部印迹确定。 基因的表达 在不同的菌株中，在不同的生长，形态和转换下 表型将通过北方分析确定。 如果还没有 分析了基因补充S.的RAM2菌株的能力。 将确定酿酒酵母。 将产生候选融合蛋白 并用于引起抗体。 这些抗体将用于监测 蛋白质水平。 如果时间允许，该基因将被破坏。