FISSION YEAST PRE-MRNA SPLICING FACTORS

裂殖酵母前 mRNA 剪接因子

• 批准号: 3306992
• 负责人: Judith Ann Potashkin
• 金额: $ 12.97万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3306992
• 关键词: Escherichia coli; RNA splicing; Schizosaccharomyces pombe; computer assisted sequence analysis; fungal genetics; gene expression; genetic regulatory element; laboratory rabbit; molecular cloning; northern blottings; nucleic acid reconstitution; nucleic acid sequence; precursor mRNA; recombinant DNA; temperature sensitive mutant

The dynamic nature of the spliceosome stimulates our interest in understanding its role in gene expression. The long term objectives of the proposed research are to identify factors that are required for pre- mRNA splicing and to determine their role in splicing. The mechanism of pre-mRNA splicing will be studied in the fission yeast Schizosaccharomyces pombe. Yeast offer powerful classical and molecular genetic and biochemical approaches to the study of splicing. Fission yeast are a particularly informative model for studying universal splicing factors because they have many characteristics with regards to gene structure and splicing apparatus that are similar to metazoans. To initiate our studies, we have recently identified temperature sensitive fission yeast mutants that are defective in pre-mRNA splicing. The wild type genes defective in these mutants will be cloned by rescue of the temperature sensitive growth phenotype and sequenced. A combined genetic and biochemical approach will be taken to analyze the gene products and determine their function. Bacterially expressed gene products will be used in reconstitution studies and to raise antibodies that will be used to define functional regions of the nucleus and to aid in the identification of factors that interact with the original splicing gene products. This involves searching for multicopy number suppressors of dominant negative mutants. The collection of temperature-sensitive mutants will also be expanded and screened for mutants that accumulate pre-mRNAs and the U6 precursor. Since U6 itself is essential for splicing, the second assay has the potential for identifying novel U6- specific splicing factors that are possible regulatory elements. Through this unique combination of genetics, biochemistry and structural analyses the pre-mRNA splicing complex will be elucidated. Characterization of the splicing complex is the initial step in understanding the pre-mRNA splicing mechanism. The antibodies and mutants obtained from these studies will provide the foundation for future studies in which the conservation of the splicing mechanism may be investigated in detail.

剪接体的动态性质激发了我们的兴趣 了解其在基因表达中的作用。 的长期目标 拟议的研究旨在确定预实施所需的因素 mRNA 剪接并确定其在剪接中的作用。 其机制为 将在裂殖酵母中研究前 mRNA 剪接 粟酒裂殖酵母。 酵母提供强大的经典和分子 研究剪接的遗传和生化方法。 裂变 酵母是研究普遍性的特别有用的模型 剪接因子，因为它们具有许多特性 基因结构和剪接装置与后生动物相似。 为了开始我们的研究，我们最近确定了温度 前体 mRNA 剪接有缺陷的敏感裂殖酵母突变体。 这些突变体中存在缺陷的野生型基因将通过拯救被克隆 温度敏感的生长表型并测序。 一个组合 将采用遗传和生化方法来分析基因 产品并确定其功能。 细菌表达基因 产品将用于重构研究并产生抗体 将用于定义细胞核的功能区域并帮助 识别与原始剪接相互作用的因素 基因产物。 这涉及寻找多拷贝数抑制子 显性失活突变体。 温度敏感的集合 突变体也将被扩大并筛选积累的突变体 前 mRNA 和 U6 前体。 由于 U6 本身对于 剪接，第二个测定有可能鉴定新型 U6- 可能的调控元件的特定剪接因子。 通过遗传学、生物化学和结构学的独特结合 分析前mRNA剪接复合物将得到阐明。 剪接复合物的表征是第一步 了解前体 mRNA 剪接机制。 抗体和 从这些研究中获得的突变体将为 未来的研究可能会保护剪接机制 进行详细调查。