FISSION YEAST PRE-MRNA SPLICING FACTORS

剪接体的动态性质激发了我们对 了解它在基因表达中的作用。的长期目标 拟议的研究是为了确定预先确定的因素 并确定它们在剪接中的作用。它的作用机制 将在分裂酵母中研究前-mRNA剪接 裂殖酵母Pombe.酵母提供强大的经典和分子 研究剪接的遗传学和生物化学方法。裂变 酵母菌是研究通用性的一个特别有用的模型 剪接因子，因为它们在以下方面具有许多特征 类似后生动物的基因结构和剪接装置。 为了启动我们的研究，我们最近确定了温度 在前mRNA剪接中有缺陷的敏感裂解酵母突变体。 这些突变体中存在缺陷的野生型基因将被救助性克隆 对温度敏感的生长表型进行了鉴定和测序。合并后的 将采用遗传和生化方法对基因进行分析 并确定其功能。细菌表达基因 产品将用于重建研究和提高抗体 它将被用来定义核的功能区，并帮助 在鉴定与原始剪接相互作用的因子时 基因产品。这涉及到搜索多拷贝数抑制器 显性负性突变体。温度敏感度的采集 突变株也将被扩展和筛选积累的突变株。 前信使核糖核酸和U6前体。因为U6本身对于 剪接，第二种检测方法有可能鉴定新的U6- 可能是调控因素的特定剪接因子。 通过这种遗传学、生物化学和结构的独特结合 对前mRNA剪接复合体的分析将得到阐明。 剪接复合体的特性是 了解Pre-mRNA剪接机制。抗体和 从这些研究中获得的突变体将为 在未来的研究中，剪接机制的保守可能 被详细调查。

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.