Molecular Genetic Analysis of Conidiation in Neurospora

9405001亚诺夫斯基无性孢子形成，即分生孢子形成，是真菌传播和生存以及用于其他目的的一种常见策略。粗糙脉孢菌的分生孢子是一个诱人的发育过程，我们正在分子水平上对其进行分析。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。粗枝菌是一种特性良好的单倍体真菌，在遗传分析中得到了广泛的应用。我们将确定和表征在分生孢子形成过程中负责多重信号接收、顺序基因表达和昼夜节律调节的顺式元件、反式作用因子和调节事件，以及在各种突变体中被阻断的阶段。我们已经分离出在分生孢子形成过程中转录激活的基因，其中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。这些基因的调控区域将被用作监管调查的目标。位于这些基因之前的上游区域的许多共同位置的重要性将被检验。调控位点将通过突变分析、条带移位分析和包括启动子域交换分析在内的其他程序来确定。首先，我们将利用两个重要的发现。首先，在菌丝生长过程中，在con10基因之前的一段400bpDNA片段似乎负责抑制该基因的表达。其次，我们指定的RCO-1(用于分生孢子形成的调节)基因的突变会导致几个分生孢子基因在菌丝中的高水平表达，并阻碍分生孢子的正常发育。因此，现在可以研究与菌丝抑制有关的部位和因素。我们已经克隆了RCO-1，并发现它编码酵母Tup1的同源物，Tup1是一种主要的调节蛋白，介导了与各种过程相关的基因的抑制。我们将在体内和体外鉴定Rco-1，并试图确定与Rco‘S存在相关的usptream位点(S)。Tup1似乎不是一种DNA结合蛋白。我们将尝试克隆酵母基因SSN6的对应基因，因为SSN6与Tup1在异源二聚体中起作用。我们突变的另一个RCO基因可能编码这个同源基因。我们设计了一种有效的克隆附子基因的策略。我们将应用这一策略来克隆几个已知的基因，如ffffy，ffffyid，acon-2和acon-3，当这些基因突变时，会阻止分生孢子的形成。我们将使用不同的选择来分离其他类别的RCO突变体。我们已经准备了菌株，将用于鉴定负责光调节和分生孢子基因表达的昼夜节律控制的基因和位点。%这些研究应该能够识别调节控制分生孢子发育的各种信号的接收的调节分子，并使我们能够确定在这一过程中负责顺序基因表达和光依赖的昼夜节律控制的调节机制(S)。***

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. ***