9307299 Loros Virtually all eukaryotic organisms appropriately examined have been shown to possess the capacity for endogenous temporal control and organization. The cellular machinery that generates this ability is known collectively as the biological clock. Extensive research in the past has demonstrated the extent and significance of clock control of gene expression and enzyme activities, but in general little is known regarding how clocks control the metabolism of the cells in which they operate. One salient aspect of this regulation is clock control of mRNA abundance via circadian regulation of DNA transcription. This research is based on experiments in which we used subtractive hybridization to compare the populations of RNA present in Neurospora at two times of day. By this approach, two clock controlled genes (ccg 1 and ccg 2) were identified that are unequivocally regulated by the circadian clock through activation of transcription in the subjective morning. Transcripts arising from both genes are abundant, suggesting that they may be quite important for the organism, and the identity of ccg 2 has been demonstrated. Based on this past work, we are analyzing the function of the ccg 1 gene and its protein product in the cell, and we will continue to develop and implement a mutant selection scheme that can be used to identify novel genetic loci affecting clock function and suppressors of existing clock genes, by completing the development and implementation of an enrichment scheme for Neurospora circadian clock mutants that is based on the clock regulated expression of an antibiotic resistance gene. We will begin characterization, mapping and cloning of mutants. S pecific Aim 1 Identification of the product and analysis of the function of ccg 1 are beginning to determine the function of this gene in the cell. In addition to speaking to the identity and role of this potentially important gene, this information will be valuable in understanding the role of circadian regulation in general in the life of this organism. We will determine the developmental expression pattern of the ccg 1 gene over the course of Neurospora development. We will further examine what effects loss of Ccg 1 in our null strains has on the clock and biology of Neurospora. We will also examine gene dosage effects on growth and the circadian clock. If time allows, we hope to raise antibodies to Ccg 1 in order to further study the protein proct of this gene. These data, combined with the polypeptide sequence, should allow us to begin to make reasonable guesses as to the function of this extremely abundant, circadianly regulated gene product, and ultimately perhaps to the advantage of circadian control to the organism. Specific Aim 2 Implementation of an enrichment scheme for clock mutants. We are developing an enrichment/selection scheme for clock mutants by placing hygromycin phosphotransferase, encoded by the hph gene, under the control of the circadian clock so as to restrict the activity of the hygromycin resistance gene to a definable part of the circadian cycle in transformants. The development of this scheme, as a method for identification of genes involved in the mechanism in the circadian clock, is an ongoing project. h) 0*0*0* %%% These experiments are in pursuit of our long term goals: to develop the fungus Neurospora as a system where the pathways and mechanisms of clock regulation can be studied on a broad scale at the molecular level, to appreciate the role of daily rhythmicity in the life of the cell, and to understand the organization of the cell as a function of time. ***
