BIOLOGICAL FUSIONS--CONJUGATION IN YEAST

生物融合——酵母中的接合

基本信息

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
项目类别：
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

项目摘要

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.

该提议的重点是对形态发生的遗传控制酿酒酵母真菌。形状最惊人的变化 - 将圆形酵母细胞转换为长长的丝状形式对人类疾病的重要意义。长细胞继续划分并剩余的附着，形成一系列连接的单元或可以穿透周围培养基的假氢。这个二态从酵母到细丝的转变，许多真菌致病人类常见（C.Albicans，d.neoformans和H. capsulatum）现在可以被揭开应用糖疗中可用的复杂遗传技术但缺乏病原体。伪氢所需的基因形成博士将被克隆，测序并用于创建突变这样可以阻止酵母转换为丝状形式。这使用两者都将重建假氢生长的途径在实验室库存中发现的自然发生突变（S288C中的PhD5,6,7）与克隆的基因的结合，在过度表达。野生型和博士学位的假氢结构将通过光和电子显微镜检查突变体。 PHD4 引起塑性依从性的基因将通过分子进行分析和细胞生物学技术以及依从性内皮细胞，由于依从性被认为在深层组织中起重要作用真菌的入侵。要分析的第二个形态发生过程是交配过程中的细胞融合。高分辨率的时衰变显微镜酵母共轭将用于重建事件序列融合过程。揭开这一途径的关键将是细胞融合突变体FUS 1,2,4,5,6和7的分析以及所需的基因用于核融合，Bik1。 Bik1包含不同的功能域，微管关联的氨基素和羧基苯甲酸核融合。 Bik1p的双重函数将通过使用生化方法，Bik1的细胞学定位，结合 Bik1p至微管和基因的表征与Deltabik1（SLBL，2和3）合成致死。我们的工作专注于两个外部信号的转导关键是激活融合途径，交配信息素和Ca +2。 FUS3中的突变（编码蛋白质激酶需要信号转导和G1停滞）这导致信号转导途径的本构激活缺乏信息素将用于确定 FUS3P信号转导中FUS3P的磷酸化。实验是旨在确定这些突变体是否是高磷酸化的，并且因此，FUS3P如何变得磷酸化和去磷酸化。基因介导信息素刺激Ca+2的要求，PCR，一起用编码质膜（PMC1）和液泡钙的基因泵（PMR1）将用于重建钙在在共轭过程中进行的重要膜重组。