Genetic Analysis of Morphologic Differentiation in Saccharomyces Cerevisiae

这个项目是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基因相同但形态不同的细胞，这表明形态分化是发育的一个组成部分。关于调控形态分化的分子机制的信息很少。这个问题将通过确定影响酿酒酵母子囊菌中已定义的形态分化途径的基因位点来解决。目前正在使用一种筛选方法来识别这一群体中可能编码分化反应调控成分的基因。这些基因将通过分子克隆方法进行表征，它们的作用位点将按照一个假定的决定分化状态的事件层次进行排序。该项目的第一个具体目标是确定酿酒葡萄球菌基因组中正常控制细胞二态性所需的其他位点。利用细胞和集落形态的视觉特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产物的一级结构。这一目标将通过从基因组库中选择质粒来实现，这些质粒是基于它们恢复酵母菌样生长的能力来达到特定的假菌丝突变体。每个克隆基因在确定形态分化中的作用将通过位点特异性诱变和基因替代方法得到证实。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，如果是的话，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元素之间的上位关系来实现。完成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。我们将发现控制形态分化的特定蛋白质，它们的主要结构很可能表明这种调节是通过生化活动完成的。关于这些分子在分化途径中作用的相关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在生化水平上描述形态发育的控制，并检查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. ***