BIOLOGICAL FUSIONS--CONJUGATION IN YEAST

这项建议的重点是遗传控制的形态发生在 真菌酿酒酵母 最引人注目的形状变化-- 将圆形酵母细胞转化为细长丝状形式， 对人类疾病的重要影响。 细长的细胞继续 分裂并保持附着，形成一串相连的细胞， 能穿透周围介质的假菌丝。 这种二态性 从酵母菌转变为丝状菌，常见于许多人类致病真菌 （白色念珠菌、新型念珠菌和荚膜梭菌）现在可以通过以下方法来解开： 应用酵母菌中先进的遗传技术 但缺乏病原体 假菌丝所需的基因 形成PHD，将被克隆，测序，并用于创建突变 阻止酵母转化为丝状形式。 的 假菌丝生长的途径将重建使用这两个 在实验室储备中发现的天然突变（S288 C中的phd 5、6、7）， 与克隆基因的组合，当超过时引起假菌丝形成 表达。 野生型和PHD假菌丝的结构 将通过光学和电子显微镜检查突变体。 PHD4 基因，导致粘附塑料，将分析分子 和细胞生物学技术以及粘附于内皮细胞， 由于粘附被认为在深部组织中起重要作用 真菌入侵。 要分析的第二个形态发生过程是 交配过程中的细胞融合 高分辨率时间推移显微镜酵母 共轭将被用来重建事件的顺序， 聚变过程 解开这条途径的关键是 细胞融合突变体fus 1、2、4、5、6和7的分析和所需基因 用于核聚变BIK 1。 BIK 1包含不同的功能结构域， 氨基末端用于微管结合，羧基末端用于微管结合。 核聚变 BIK 1 p的双重功能将通过使用 生物化学方法，BIK 1的细胞学定位， BIK 1 p与微管的关系以及相关基因的表征 用deltabik 1（slb 1，2和3）合成致死。 我们的工作重点是 两个外部信号的转导是激活 融合途径、交配信息素和Ca +2。 FUS 3突变（编码 信号转导和G1期阻滞所需的蛋白激酶） 导致细胞内信号转导通路的组成性激活， 信息素的缺乏将被用来确定的作用 FUS 3 p在信号转导中的磷酸化。 实验 旨在确定这些突变体是否过度磷酸化， FUS 3 p如何磷酸化和去磷酸化。 基因 它们共同介导信息素刺激的Ca+2需求，PCR， 与编码质膜（PMC 1）和液泡钙的基因 泵（PMR 1）将被用来重建钙的作用， 在接合过程中发生的重要的膜重组。

This proposal focuses on the genetic control of morphogenesis in the fungus Saccharomyces cerevisiae. The most striking change in shape--the conversion of a round yeast cell to a long narrow filamentous form has important implications for human disease. The long thin cells continue to divide and, remaining attached, form a chain of connected cells or pseudohypha that can penetrate the surrounding medium. This dimorphic shift from yeast to filament, common to many fungi pathogenic for humans (C.albicans, D.neoformans, and H.capsulatum) can now be unraveled by applying the sophisticated genetic techniques available in Saccharomyces but lacking in its pathogens. The genes required for pseudohyphal formation PHD, will be cloned, sequenced and used to create mutations that block the conversion of the yeast to the filamentous form. The pathway for pseudohyphal growth will be reconstructed using both the naturally occurring mutations found in lab stocks (phd5,6,7 in S288C) in combination with cloned genes that cause pseudohyphal formation when over expressed. The structure of the pseudohypha in wild type and the phd mutants will be examined both by light and electron microscopy. The PHD4 gene, which caused adherence to plastic, will be analyzed by molecular and cell biological techniques and for adherence to endothelial cells, since adherence is thought to play an important role in deep tissue invasion by fungi. The second morphogenetic process to be analyzed is cell fusion during mating. High resolution time lapse microscopy yeast conjugation will be used to reconstruct the sequence of events in the fusion process. Key to the unraveling of this pathway will be the analysis of cell fusion mutants fus 1,2,4,5,6,and 7 and a gene required for nuclear fusion, BIK1. BIK1 contains distinct functional domains, the aminoterminus for microtubule association and the carboxyterminus for nuclear fusion. The dual functions of BIK1p will be dissected by using biochemical methods, cytological localization of BIK1, the binding of BIK1p to microtubules and the characterization of genes that are synthetic lethals with deltabik1 (slbl, 2 and 3). Our work focuses on the transduction of two external signals key to the activation of the fusion pathway, mating pheromone and Ca +2. Mutations in FUS3 (encoding a protein kinase required both for signal transduction and G1 arrest) that cause constitutive activation of the signal transduction pathway in the absence of pheromone will be used to determine the role of phosphorylation of FUS3p in signal transduction. Experiments are designed to determine whether these mutants are hyperphosphorylated and, is so, how FUS3p becomes phosphorylated and dephosphorylated. Genes which mediate the pheromone stimulated Ca+2 requirement, PCR, together with the genes encoding the plasma membrane (PMC1) and vacuolar calcium pumps (PMR1) will be used to reconstruct the role of calcium in the important membrane reorganizations that take place during conjugation.