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所需的因素， mRNA剪接，并确定其在剪接中的作用。 的机理 将在裂殖酵母中研究前mRNA剪接 裂殖酵母 酵母提供强大的经典和分子 基因和生物化学方法来研究剪接。 裂变 酵母是一种特别有用的模型， 剪接因子，因为它们具有许多特性， 与后生动物相似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 敏感的分裂酵母突变体，其在前mRNA剪接中有缺陷。 这些突变体中有缺陷的野生型基因将通过拯救克隆 的温度敏感生长表型并测序。 组合 将采用遗传学和生物化学方法分析该基因 产品并决定其功能。 细菌表达基因 产品将用于复溶研究， 它将被用来定义细胞核的功能区域， 在识别与原始剪接相互作用的因子中， 基因产物 这涉及到搜索多拷贝数抑制器 显性负突变体的数量 温度传感器的收集 突变体也将扩大和筛选的突变体，积累 pre-mRNA和U6前体。 由于U6本身是必不可少的 剪接，第二种测定法有可能鉴定新型U6- 特异性剪接因子是可能的调节元件。 通过这种独特的遗传学、生物化学和结构学的结合， 分析前mRNA剪接复合物将被阐明。 剪接复合物的表征是 了解前体mRNA剪接机制。 的抗体和 从这些研究中获得的突变体将为 未来的研究中，剪接机制的保守性可能 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.