| One model for the molecular mechanism that generates circadian rhythmicity in Neurospora crassa involves a negative feedback loop in which transcription of the frequency (frq) gene is inhibited by FRQ protein. FRQ indirectly disrupts transcription of its own gene by binding to the proposed transcription factors, WHITE COLLAR 1 (WC1) and WHITE COLLAR 2 (WC2), which are necessary to activate frq transcription. However, several lines of evidence, including rhythmicity in frq and wc null strains, suggest factors operating outside the frq-wc oscillatory loop to generate rhythmicity in Neurospora.; The inherent complexity of the Neurospora clock necessitates the identification and characterization of other clock genes. The focus of this thesis is on the cloning and molecular analysis of the period-6 (prd-6) gene, which displays strong genetic interactions with several other clock genes, including frq. This work is organized into four Chapters. Chapter I introduces the reader to the general properties of circadian rhythms, the Neurospora system and the prevailing model for the clock in this organism. The section entitled “Many genetic loci contribute to circadian rhythms in Neurospora crassa” will appear as a chapter in the book Circadian Clocks in Eukaryotic Microbes, (Landes Bioscience Publishing, 2003).; In Chapter II, the cloning, sequencing and predicted protein function of prd-6 is described. The predicted prd-6 protein sequence codes for a homologue of hUPF1, a highly conserved RNA helicase involved in nonsense mediated decay and translation termination. The prd-6 protein sequence also indicates possible roles in protein turnover.; Chapter III presents the genetic isolation and molecular analysis of the null allele of prd-6 by repeat-induced point mutation (RIP). The phenotype of the null allele was found to be slightly more severe at lower temperature, but otherwise identical to the original prd-6 mutant allele.; In Chapter IV, an analysis of Neurospora clock gene expression is reported. Using wild type and prd-6 mutant strains, the expression profiles over a circadian time course were compared for the frq, prd-6 and ccg-7 genes. This study indicates prd-6 is involved not only in the regulation of its own expression but also that of the frq and ccg-7 genes. |
