In this thesis I describe a functional genomics resource in which each yeast gene, with its native promoter and 3’UTR, is cloned on a uniquely barcoded low-copy vector. We refer to this resource as the Molecular Barcoded Yeast ORF (MoBY-ORF) library 1.0. Each gene carried by MoBY-ORF 1.0 should mimic its native expression and thus is best suited for complementation cloning. The vector backbone of MoBY-ORF 1.0 is compatible with the mating-assisted genetically integrated cloning (MAGIC) system for recombination cloning in bacterial cells, which allows the transfer of the ORF fragment and its barcoded cassette to other vector backbones. Taking advantage of the MAGIC system, we created a multi-copy version of the library, which we refer to as MoBY-ORF 2.0. I used MoBY-ORF 1.0 to map drug resistant mutants by complementation cloning with a barcode microarray readout. I investigated several drugs with known targets in my proof-of-principle experiments and showed the feasibility of this method. I identified a single mutation that causes resistance to two different natural products, theopalauamide and stichloroside. By doing so, I was able to link these two chemicals to their cellular target, ergosterol. In fact, theopalauamide represents a new class of sterol binding chemical. I also describe the use of MoBY-ORF 2.0 to clone dosage suppressors of conditional temperature-sensitive mutants. By doing so, and combing our own data with published literature, we showed that dosage suppression interactions often overlap with protein-protein interactions and negative genetic interactions but not positive interactions; however the majority of dosage suppression interactions are unique and thus they represent an unique edge on a global functional interaction map. We also describe the first genome-wide dosage suppressor interaction map of budding yeast.

In this thesis I describe a functional genomics resource in which each yeast gene, with its native promoter and 3’UTR, is cloned on a uniquely barcoded low-copy vector. We refer to this resource as the Molecular Barcoded Yeast ORF (MoBY-ORF) library 1.0. Each gene carried by MoBY-ORF 1.0 should mimic its native expression and thus is best suited for complementation cloning. The vector backbone of MoBY-ORF 1.0 is compatible with the mating-assisted genetically integrated cloning (MAGIC) system for recombination cloning in bacterial cells, which allows the transfer of the ORF fragment and its barcoded cassette to other vector backbones. Taking advantage of the MAGIC system, we created a multi-copy version of the library, which we refer to as MoBY-ORF 2.0. I used MoBY-ORF 1.0 to map drug resistant mutants by complementation cloning with a barcode microarray readout. I investigated several drugs with known targets in my proof-of-principle experiments and showed the feasibility of this method. I identified a single mutation that causes resistance to two different natural products, theopalauamide and stichloroside. By doing so, I was able to link these two chemicals to their cellular target, ergosterol. In fact, theopalauamide represents a new class of sterol binding chemical. I also describe the use of MoBY-ORF 2.0 to clone dosage suppressors of conditional temperature-sensitive mutants. By doing so, and combing our own data with published literature, we showed that dosage suppression interactions often overlap with protein-protein interactions and negative genetic interactions but not positive interactions; however the majority of dosage suppression interactions are unique and thus they represent an unique edge on a global functional interaction map. We also describe the first genome-wide dosage suppressor interaction map of budding yeast.

Thesis (Ph. D.)--University of California, (Davis), 1952. / Degree granted in Plant Pathology. Includes bibliographical references (leaves 69-71). Also available via the World Wide Web. (Restricted to UC campuses).

The Silent Information Regulator (SIR) complex is responsible for the formation of silent chromatin domains in Saccharomyces cerevisiae, and consists of the NAD-dependent histone deacetylase Sir2, and histone binding proteins Sir3 and Sir4. The current model of silent chromatin assembly proposes that histone deacetylation by Sir2 is required to promote recruitment of Sir3 and Sir4, and assembly of full SIR complexes on chromatin. However, recent work has suggested unique roles for the histone binding proteins Sir3 and Sir4 in this process. Here we present data suggesting that Sir3 is primarily responsible for mediating the spreading of silent chromatin from sites of nucleation, while regulation of Sir4 abundance controls the rate of silencing establishment. We have also investigated a potential novel dimerization domain in Sir3, which may represent a conserved function in vertebrates. Investigations into the regulation of silent chromatin assembly in budding yeast will facilitate our understanding of the mechanisms that control heterochromatin-mediated gene repression in higher organisms.

Increasing gene dosage provides a powerful means of probing gene function, as it tends to cause a gain-of-function effect due to increased gene activity. In the budding yeast, Saccharomyces cerevisiae, systematic gene overexpression studies have shown that in wild-type cells, overexpression of a small subset of genes results in an overt phenotype. However, examining the effects of gene overexpression in sensitized cells containing mutations in known genes is a powerful means for identifying functionally relevant genetic interactions. When a query mutant phenotype is rescued by gene overexpression, the genetic interaction is termed dosage suppression. I comprehensively investigated dosage suppression genetic interactions in yeast using three approaches. First, using one of two novel plasmid libraries cloned by two colleagues and myself, I systematically performed dosage suppression screens and identified over 130 novel dosage suppression genetic interactions for more than 25 essential yeast genes. The plasmid libraries, called the molecular barcoded yeast ORF (MoBY-ORF) 1.0 and 2.0, are designed to streamline dosage analysis by being compatible with high-throughput genomics technologies that can monitor plasmid representation, including barcode microarrays and next-generation sequencing methods. Second, I describe a detailed analysis of the novel dosage suppression interactions, as well as of literature-curated interactions, and show that the gene pairs exhibiting dosage suppression are often functionally related and can overlap with physical as well as negative genetic interactions. Third, I performed a systematic categorization of dosage suppression genetic interactions in yeast and show that the majority of the dosage suppression interactions can be assigned to one of four general mechanistic classifications. With this comprehensive analysis, I conclude that systematically identifying dosage suppression genetic interactions will allow for their integration into other genetic and physical interaction networks and should provide new insight into the global wiring diagram of the cell.

Increasing gene dosage provides a powerful means of probing gene function, as it tends to cause a gain-of-function effect due to increased gene activity. In the budding yeast, Saccharomyces cerevisiae, systematic gene overexpression studies have shown that in wild-type cells, overexpression of a small subset of genes results in an overt phenotype. However, examining the effects of gene overexpression in sensitized cells containing mutations in known genes is a powerful means for identifying functionally relevant genetic interactions. When a query mutant phenotype is rescued by gene overexpression, the genetic interaction is termed dosage suppression. I comprehensively investigated dosage suppression genetic interactions in yeast using three approaches. First, using one of two novel plasmid libraries cloned by two colleagues and myself, I systematically performed dosage suppression screens and identified over 130 novel dosage suppression genetic interactions for more than 25 essential yeast genes. The plasmid libraries, called the molecular barcoded yeast ORF (MoBY-ORF) 1.0 and 2.0, are designed to streamline dosage analysis by being compatible with high-throughput genomics technologies that can monitor plasmid representation, including barcode microarrays and next-generation sequencing methods. Second, I describe a detailed analysis of the novel dosage suppression interactions, as well as of literature-curated interactions, and show that the gene pairs exhibiting dosage suppression are often functionally related and can overlap with physical as well as negative genetic interactions. Third, I performed a systematic categorization of dosage suppression genetic interactions in yeast and show that the majority of the dosage suppression interactions can be assigned to one of four general mechanistic classifications. With this comprehensive analysis, I conclude that systematically identifying dosage suppression genetic interactions will allow for their integration into other genetic and physical interaction networks and should provide new insight into the global wiring diagram of the cell.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Thesis (M.S.)--University of Michigan, 1976.

Thesis (M.S.)--University of Wisconsin--Madison, 1971. / Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The cell cycle of the budding yeast, Saccharomyces cerevisiae, is regulated by a complex network of chemical reactions controlling the activity of the cyclin-dependent kinases (CDKs), a family of protein kinases that drive the major events of the cell cycle. A previous mathematical model by Chen et al. (2000) described a molecular mechanism for the Start transition (passage from G1 phase to S/M phase) in budding yeast. In this thesis, my main goal is to extend Chen's model to include new information about the mechanism controlling Finish (passage from S/M phase to G1 phase). Using laws of biochemical kinetics, I transcribed the hypothetical molecular mechanism into a set of differential equations. Simulations of the wild-type cell cycle and the phenotypes of more than 60 mutants provide a thorough understanding of how budding yeast cells exit mitosis. / Master of Science