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. ***
