Molecular Genetic Analysis of Conidiation in Neurospora

脉孢菌分生孢子的分子遗传学分析

• 批准号: 9405001
• 负责人: Charles Yanofsky
• 金额: $ 50万
• 依托单位: Stanford University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-15 至 2000-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9405001&HistoricalAwards=false
• 关键词: Molecular; Genetic; Analysis; Conidiation; Neurospora

9405001 Yanofsky Asexual spore formation, conidiation, is a common strategy used by fungi for their dispersal and survival, and for other purposes. Conidiation in Neurospora crassa is an attractive developmental process that we are analyzing at the molecular level. Our major objective is to determine the molecular mechanisms responsible for multiple signal reception and sequential gene expression. N. crassa is a well-characterized, haploid fungus that has been used extensively in genetic analyses. We will identify and characterize the cis elements, trans acting factors, and regulatory events that are responsible for multiple-signal reception, sequential gene expression, and circadian rhythm regulation, during conidiation, and the stages blocked in various mutants. We have isolated genes that are transcriptionally activated during conidiation, including two that are involved in carotenoid biosynthesis. Some of these genes are also activated by carbon or nitrogen starvation, or by light. One gene we are studying encodes the major coat protein of the conidium. The regulatory regions of these genes will be used as targets for reguatory investigations. The significance of the many common sites located in the upstream regions preceding these genes will be examined. Regulatory sites will be identified by mutational analysis, band shift assays, and other procedures, including promoter domain swap analyses. Initially we will exploit two important findings. First, a 400 bp DNA segment of the region preceding the gene con-10 appears to be responsible for repression of this gene's expression during mycelial growth. Second, mutations at a locus we have designated rco-1 (for regulation of conidiation) result in high level mycelial expression of several conidiation genes, and block normal conidial development. Thus sites and factors involved in mycelial repression are now available for investigation. We have cloned rco-1 and have found that it encodes a homolog of TUP1 of yeast, a master regula tory protein that mediates repression of genes concerned with a variety of processes. We will characterize rco-1 in vivo and in vitro, and attempt to determine the usptream site(s) that respond to RCO1's presence. TUP1 does not appear to be a DNA-binding protein. We will attempt to clone N. crassa's counterpart of the yeast gene SSN6, since SSN6 functions in a heterodimer with TUP1. One of the other rco genes we have mutated may encode this homolog. We have devised an effective strategy for cloning aconidial genes. We will apply this strategy to clone several known genes such as fluffy, fluffyoid, acon-2 and acon-3, which, when mutated, block conidiation. We will isolate other classes of rco mutants using different selections. We have prepared strains that will be used to identify genes and sites that are responsible for light regulation and circadian control of conidiation gene expression. %%% These investigations should permit identification of regulatory molecules that mediate reception of the various signals that control conidia development, and should allow us to determine the regulatory mechanism(s) responsible for sequential gene expression and light-dependent circadian control during this process. ***

9405001 Yanofsky无性孢子形成，分生孢子，是真菌用于其传播和存活以及用于其他目的的常见策略。 粗糙脉孢菌的分生孢子是一个有吸引力的发育过程，我们正在分子水平上进行分析。 我们的主要目标是确定负责多重信号接收和顺序基因表达的分子机制。N. crassa是一种特征良好的单倍体真菌，已广泛用于遗传分析。 我们将确定和表征的顺式元件，反式作用因子，并负责多信号接收，顺序基因表达和昼夜节律调节，在分生孢子形成过程中的调控事件，以及在各种突变体中阻断的阶段。 我们已经分离出在分生孢子形成过程中转录激活的基因，包括两个参与类胡萝卜素生物合成的基因。 这些基因中的一些也被碳或氮饥饿或光激活。 我们正在研究的一个基因编码分生孢子的主要外壳蛋白。 这些基因的调控区将被用作调控研究的靶点。 位于这些基因之前的上游区域的许多共同位点的意义将被检查。 将通过突变分析、条带转移试验和其他程序（包括启动子结构域交换分析）鉴定调控位点。 首先，我们将探讨两个重要的发现。 首先，基因con-10之前区域的400 bp DNA片段似乎负责菌丝生长期间该基因表达的抑制。 其次，我们指定为rco-1（用于调节分生孢子形成）的基因座的突变导致几种分生孢子形成基因的菌丝体高水平表达，并阻止分生孢子的正常发育。 因此，现在可以对菌丝体阻遏中涉及的位点和因素进行研究。 我们已经克隆了rco-1，并发现它编码酵母TUP 1的同源物，TUP 1是一种主调节蛋白，介导与多种过程相关的基因的抑制。 我们将在体内和体外表征rco-1，并试图确定响应RCO 1存在的usptream位点。 TUP 1似乎不是DNA结合蛋白。 我们将尝试克隆N。crassa的酵母基因SSN 6的对应物，因为SSN 6在与TUP 1的异源二聚体中起作用。 我们突变的另一个rco基因可能编码这种同源物。 我们设计了一种克隆乌头类基因的有效策略。 我们将应用这一策略克隆几个已知的基因，如fluffy，fluffyoid，acon-2和acon-3，当突变时，阻止分生孢子。 我们将使用不同的选择分离其他类型的rco突变体。 我们已经制备了菌株，将用于确定基因和网站，负责光调节和昼夜控制分生孢子基因表达。 这些调查应允许鉴定调节分子，介导控制分生孢子发育的各种信号的接收，并应使我们能够确定在此过程中负责顺序基因表达和光依赖性昼夜节律控制的调节机制。 ***