The Varkud satellite (VS) plasmid is found in the mitochondria of some strains of Neurospora, and exhibits properties that may allow it to be developed as a genetic transformation vector to study mitochondrial molecular biology. An ideal transformation vector would not confer a phenotype. The overall goal of my thesis was to examine interactions of VS with its Neurospora host to identify possible phenotypes, using biochemical and proteomic approaches. Biochemical experiments provided evidence consistent with the plasmid transcript, VS RNA, being present as a ribonucleoprotein particle that can be separated from ribosomal subunits by sucrose density gradient centrifugation; however, no VS-specific proteins were identified under the purification conditions examined. During the analyses of proteomic data I obtained new insights into the consequences of the statistical methods commonly used to normalize quantitative 2D gel data. However, irrespective of the method used, the fraction of the proteome amenable to 2D gel-based proteomics revealed, at most, subtle effects of VS on the abundance of a few proteins. I also observed no differences in growth rate between strains differing by the presence or absence of VS when grown in the presence of inhibitors and stressors affecting a wide range of mitochondrial and other cellular functions. Overall, despite VS RNA being as abundant as the large and small mitochondrial ribosomal RNAs, my genetic, biochemical and proteomic investigations of the effect of VS on its host strain provides evidence that VS is a phenotypically neutral element.

Circadian clocks are present in most eukaryotes and some prokaryotes and control rhythms in behavior, physiology and gene expression. One well-characterized circadian clock is that of Neurospora crassa. In addition to the well-described N. crassa FRQ/WCC oscillator, several lines of evidence have implied the presence of other oscillators which may have important functions in the N. crassa circadian clock system. However, the molecular details are only known for the core FRQ/WCC oscillator. The light mutant oscillator (LMO) was identified by two mutations (LM-1 and LM-2) and shown to control developmental rhythms in constant light (LL), conditions in which the FRQ/WCC oscillator is not functional. The objective of this project was to determine whether the developmental rhythms driven by the LMO are circadian, whether the components of the LMO communicate with components of the FRQ/WCC oscillator, and to begin to define the molecular nature of the LMO. First, the conditions for growth of the LM-1 mutant strain that reveals the best circadian rhythm of development in LL were found. Second, the LMO was determined to display the three properties required of a circadian oscillator. Third, the LMO was shown to function independently of the FRQ/WCC oscillator to control developmental rhythms in LL. However, evidence suggests that the FRQ/WCC oscillator and the LMO communicate with each other. Finally, using Cleaved Amplified Polymorphic Sequence (CAPS) markers, the LM-1 mutation was genetically mapped to the right arm of linkage group I within a 1069 kb region. Together, these results provide a start towards understanding of the complexity of oscillators that form a circadian clock in organisms.

Circadian rhythms in biological processes occur in a wide range of organisms and are generated by endogenous oscillators. In Neurospora crassa, the FRQ-oscillator (comprised of FRQ, WC-1 and WC-2) is essential for rhythms in asexual sporulation and gene expression. How this oscillator signals to the cell to control rhythmicity is unknown. Furthermore, under certain growth conditions, rhythms are observed in FRQ-null strains, indicating the presence of one or more FRQ-less oscillators (FLOs). Interestingly, while circadian rhythms are observed in the related Aspergillus spp., they lack the frq gene, leading to the hypothesis that a FLO is responsible for rhythms in Aspergillus. Thus, Aspergillus provides a useful organism to investigate the components of the FLO. To investigate how an oscillator controls circadian output, we characterized the role of N. crassa NRC-2. The nrc-2 gene is under control of the clock and encodes a putative serine-threonine protein kinase. In a NRC-2-null strain cultured in low glucose conditions, FRQ-oscillator-dependent outputs are arrhythmic, but are rhythmic in high glucose. Our data suggests a model whereby NRC-2 relays metabolic information to the FRQ-oscillator to control rhythmic output. To understand the role of FLO(s) in the N. crassa circadian system, we examined regulation of the ccg-16 gene. We show that ccg-16 transcript rhythmicity is FRQ-independent, but WC-1-dependent. Furthermore, in contrast to current models for the FRQ-oscillator, we observed that rhythms in WC-1 protein accumulation persist in the absence of FRQ. These data support a new model involving two oscillators that are coupled through the WC-1 protein and that regulate different outputs. One approach to identify components of the FLO involved characterizing circadian rhythms in Aspergillus spp, which lacks FRQ. We find that A. flavus and A. nidulans, display circadian rhythms in sporulation and gene expression, respectively. Together, these findings provide a foundation for the identification of FLO components in both Aspergillus and N. crassa, that will ultimately lead to an understanding of how a multi-oscillator system can generate and coordinate circadian rhythmicity.

Thesis (Ph.D.)--York University, 2008. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 165-180). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR39028

Thesis (Ph. D.)--Illinois State University, 1968. / Title from title page screen, viewed Aug. 17, 2004. Dissertation Committee: H.E. Brockman (chair), J.L. Frehn, C.W. Hardiman, A.E. Liberta, J.E. Perham, D.D. Pittman. Includes bibliographical references (leaves 61-65). Also available in print.

Thesis (Ph. D.)--University of Oregon, 2003. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 91-96). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

Not available / text

The Neurospora crassa arg-2 gene encodes the small subunit of carbamoyl phosphate synthetase, the first enzyme in fungal arginine (Arg) biosynthesis. The arginine attenuator peptide (AAP), specified by an upstream open reading frame (uORF), stalls ribosomes at its termination codon in response Arg to control the translation of arg-2. In project 1, the effect of AAP and Arg on ribosome peptidyl transferase center (PTC) activity was analyzed in N. crassa and wheat germ cell-free translation extracts using the transfer of nascent AAP to puromycin as an assay. The results show that inhibition of PTC activity by the AAP and Arg is the basis for the AAP’s function. The mode of PTC inhibition appears unusual because neither a specific amino acid nor a specific nascent peptide chain length was required for AAP to function. In eukaryotic translation initiation, the stringency of start codon selection impacts initiation efficiencies at AUG codons in different contexts and at near-cognate codons (NCCs) that differ from AUG by a single nucleotide. In project 2, a codon-optimized firefly luciferase reporter was used to examine the stringency of start codon selection in N. crassa. In vivo and in vitro results indicated that the hierarchy of initiation in N. crassa is similar to that in human cells. The preferred context was more important for efficient initiation from NCCs than from AUG. In project 3, the use of NCCs was also specifically examined for the N. crassa cpc-1 gene. cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying a transcription activator, which drives the primary transcriptional response to amino acid starvation. In vitro studies showed that uORF1 and uORF2 in cpc-1 are functionally analogous to uORF1 and uORF4 in GCN4. uORF1 promotes reinitiation at downstream start codons. uORF2 inhibits translation from the main cpc-1 start codon. Four NCCs in the CPC1 reading frame and upstream of uORF2 can also be used for translation initiation. In summary, we explored uORF-mediated translational regulation and the use of NCCs as initiation codons. Taken together, these studies establish N. crassa as a model system to examine mechanisms contributing to translational control including initiation and termination.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1955. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Bibliography: leaves [41]-44.