BIOLOGICAL FUSIONS--CONJUGATION IN YEAST

生物融合——酵母中的接合

• 批准号: 2180227
• 负责人: GERALD R FINK
• 金额: $ 32.83万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2180227
• 关键词: Saccharomyces cerevisiae; biological signal transduction; cell fusion; chromosome aberrations; cytogenetics; electron microscopy; fungal genetics; gene mutation; genetic mapping; laboratory mouse; light microscopy; membrane fusion; microorganism conjugation; microorganism sexual dimorphism; molecular cloning; morphology; nucleic acid sequence; phosphorylation; polymerase chain reaction; transcription factor; video recording system

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.

这项建议的重点是遗传控制的形态发生在 真菌酿酒酵母。形状上最惊人的变化-- 将圆形酵母细胞转化为细长的丝状细胞 对人类疾病的重要影响。细长的细胞继续 分裂并保持连接，形成一串相连的细胞或 能穿透周围介质的假菌丝。这种二相性 从酵母菌转变为丝状菌，这是许多人类致病真菌的共同特征 (白色念珠菌、新生念珠菌和囊状念珠菌)现在可以通过 在酵母菌中应用先进的基因技术 但缺乏病原体。假菌丝形成所需的基因 形成PHD，将被克隆、测序并用于产生突变 这阻碍了酵母向丝状的转化。这个 假菌丝生长的途径将用两个 在实验室种群(S288C中的phd5、6、7)中发现自然发生的突变 与克隆的基因结合，当超过时会导致假菌丝形成 表达。野生型和PHD假菌丝的结构 突变株将用光学显微镜和电子显微镜进行检查。PHD4 导致塑料粘连的基因将被分子分析 和细胞生物技术，以及对内皮细胞的黏附， 因为粘连被认为在深层组织中起着重要作用 真菌入侵。第二个要分析的形态发生过程是 交配过程中的细胞融合。高分辨率时间推移显微镜酵母菌 共轭将被用来重建事件序列 核聚变过程。解开这条道路的关键将是 细胞融合突变体FUS 1、2、4、5、6和7及其所需基因的分析 对于核聚变，BIK1。BIK1包含不同的功能域， 微管联合的氨基末端和微管结合的羧基末端 核聚变。BIK1p的双重功能将通过使用 生化方法、BIK1的细胞学定位、BIK1的结合 BIK1p对微管的作用及其相关基因的特性 使用deltabik1(slb1、2和3)的合成致死。我们的工作重点是 两个外部信号的转导对激活 融合途径、交配信息素和Ca+2.FUS3(编码 信号转导和G1期停滞所需的蛋白激酶) 导致信号转导通路的结构性激活 信息素的缺乏将被用来确定 信号转导中FUS3p的磷酸化。实验是 旨在确定这些突变体是否过度磷酸化， 是这样的，FUS3p是如何被磷酸化和去磷酸化的。基因 它们共同介导信息素刺激的钙需求，聚合酶链式反应 与编码质膜(PMC1)和液泡钙的基因 泵(PMR1)将被用来重建钙在 在接合过程中发生的重要的膜重组。