Genetic Analysis of Morphologic Differentiation in Saccharomyces Cerevisiae

Myers 9319028该项目正在研究控制形态发育的分子机制。 多细胞真核生物包含许多类型的遗传上相同但形态上不同的细胞，表明形态分化是发育的一个组成部分。 关于形态分化调控的分子机制的信息很少。 这个问题将通过鉴定影响子囊菌酿酒酵母中一个确定的形态分化途径的基因位点来解决。 一个屏幕正在被用来识别基因在这一组可能编码的调节成分的分化反应。 这些基因将通过分子克隆方法来表征，并且它们的作用位点将以确定分化状态的事件的假定层级来排序。 该项目的第一个具体目标是确定S.酿酒酵母基因组所需的正常控制细胞二型性。 使用细胞和集落形态的视觉表征作为可评分表型的遗传分析将实现这一目标。 第二个目标是通过分子克隆分离选定的基因，并通过核苷酸序列分析确定相应蛋白质产物的一级结构。 这一目标将通过从基因组文库中选择质粒来实现，所述质粒基于它们恢复酵母样生长至特定假菌丝突变体的能力。 每个克隆基因在形态分化的决定中的作用将通过位点特异性诱变和基因置换方法来证实。 第三个目标是确定基因是否在控制分化反应的线性途径中起作用，如果是，则确定它们的相对作用位点。 这一目标将通过确定刺激或抑制假菌丝生长的遗传因素之间的上位性关系来实现。 实现这些目标将为控制细胞形状发育的分子机制提供重要的新见解。 将确定控制形态分化的特定蛋白质，并且它们的一级结构最有可能提示完成这种调节的生化活性。 关于这些分子在分化途径中的相对作用位点的信息将提示特定因子的功能。 未来的研究可以利用这些信息来表征在生物化学水平上对形态发育的控制，并研究类似的机制是否在进化中广泛保守。 ***

Myers 9319028 This project is investigating the molecular mechanisms controlling morphologic development. Multicellular eucaryotes contain many types of genetically identical yet morphologically distinct cells, indicating morphologic differentiation is an integral aspect of development. Little information is available regarding the molecular mechanisms by which morphologic differentiation is regulated. This question will be addressed through the identification of gene loci that influence a defined morphologic differentiation pathway in the ascomycete Saccharomyces cerevisiae. A screen is being used to recognize genes within this group likely to code for regulatory components of the differentiation response. The genes will be characterize by molecular cloning methods, and their sites of action will be ordered in a presumed hierarchy of events that determine the differentiation state. The first specific objective of this project is to identify additional loci in the S. cerevisiae genome required for normal control of cellular dimorphism. Genetic analysis using visual characterization of cell and colony morphology as a scorable phenotype will accomplish this goal. The second objective is to isolate selected genes by molecular cloning, and to determine the primary structure of the corresponding protein products by nucleotide sequence analysis. This goal will be met by selecting plasmids from a genomic library based on their ability to restore yeast-like growth to a specific pseudohyphal mutant. The role of each cloned gene in determination of morphologic differentiation will be confirmed by site-specific mutagenesis and gene replacement methods. The third objective is to determine whether the genes act in a linear pathway controlling the differentiation response and, if so, to determine their relative sites of action. This goal will be accomplished by determining epistasis relationships between genetic elements that either stimulate or repress pseudohyphal growth. %%% Accomplishing these objectives will provide significant new insights into the molecular mechanisms that control development of cell shape. Specific proteins will be identified that control morphologic differentiation, and their primary structures most likely will suggest biochemical activities by which this regulation is accomplished. Information regarding the relative sites of action of these molecules in a differentiation pathway will suggest the function of specific factors. Future investigations could then utilize this information to characterize control of morphologic development at the biochemical level, and to examine whether similar mechanisms are broadly conserved in evolution. ***