C-RUI: Identification of Genes Regulating Cell Wall Integrity in Aspergillus nidulans

真菌生长和发育的一个重要决定因素是真菌细胞壁。然而，人们对墙的建造或发育修改的机制知之甚少。本研究的目的是在模式丝状真菌Nidulans中鉴定出一组新发现的基因(Cal基因)，其主要表型(对壁损害剂硫代氟化物超敏)与细胞壁结构缺陷的存在相关。推测，这些基因的功能等位基因在合成或运输壁组分、壁面成熟和修饰、或调节壁面代谢的信号转导途径中发挥作用。这项工作将分三个阶段进行。在第一阶段，将通过标准遗传方法增加可用的CAL突变体的数量(迄今已鉴定出6个CAL突变体，但筛选显示没有饱和迹象)，并将就对细胞壁化学、孢子萌发的时间和形态、菌丝形态、细胞质分裂(隔膜放置)和有丝分裂周期的影响，为所有突变体建立广泛的表型图谱。第二阶段的目的是通过1)用携带基因组(野生型)DNA的粘粒或质粒载体转化CAL突变体并对导致表型拯救的插入片段进行测序，2)通过转座子介导的每个候选开放阅读框的失活来识别互补基因，以及3)通过靶向基因替换来确定零突变的表型效应，从而识别几个Cal基因。在第三阶段，Cal基因产物将定位于细胞内，要么通过构建Cal基因与绿色荧光蛋白基因的融合，要么通过表位标记和免疫细胞化学(EM和LM)。许多真菌作为商业产品的生产者或动植物的病原体发挥着重要作用，我们对真菌生长和发育的基本机制的了解程度限制了我们操纵真菌的能力。这项研究将通过识别影响细胞壁完整性的新基因来增加我们对真菌生长和发育的知识。由于本科生将在这项研究的各个方面发挥积极的核心作用，该项目还将促进科学方面的本科生教育，并为有才华的年轻人提供与选择研究职业相关的经验。

An essential determinant of fungal growth and development is the fungal cell wall. Little, however, is known about the mechanisms by which the wall is constructed or developmentally modified. The goal of this research is to characterize a newly identified set of genes (Cal genes) in the model filamentous fungus Aspergillus nidulans, whose primary phenotype (hypersensitivity to the wall-compromising agent Calcofluor) has been correlated with the presence of structural defects in cell walls. Presumably, the functional alleles of these genes play roles in synthesizing or transporting wall constituents, in wall maturation and modification, or in signal transduction pathways that regulate wall metabolism. The work will proceed in three phases. In Phase I the number of available cal mutants will be increased by standard genetic methodology (six cal mutants have been identified to date, but the screening shows no signs of saturation), and extensive phenotypic profiles will be established for all mutants with regard to effects on cell wall chemistry, timing and morphology of spore germination, hyphal morphology, cytokinesis (septal placement), and the mitotic cycle. The objective of Phase II is to identify each of several Cal genes by 1) transforming cal mutants with cosmid or plasmid vectors carrying genomic (wild type) DNA and sequencing those inserts that cause phenotypic rescue, 2) identifying the complementing genes through transposon-mediated inactivation of each candidate open reading frame, and 3) determining the phenotypic effect of null mutations via targeted gene replacement. In Phase III, Cal gene products will be localized within the cell, either by construction of fusions between Cal genes and a green fluorescent protein gene or by epitope tagging and immunocytochemistry (EM and LM).Many fungi play important roles as producers of commercial products or as pathogens of plants and animals, and our ability to manipulate fungi to our advantage is limited by how well we understand the basic mechanisms of their growth and development. This research will increase our knowledge of fungal growth and development by identifying novel genes that affect cell wall integrity. Because undergraduate college students will play active, central roles in all aspects of this research, the project will also advance undergraduate education in the sciences and provide talented young people with experience relevant to making a choice to pursue research careers.