Thesis advisor: Charles S. Hoffman / Transcription of the Schizosaccharomyces pombe fbp1 gene is subject to repression by environmental glucose that is detected through a cAMP signaling pathway. Previous research has identified several glucose insensitive transcription (git) genes that are required for glucose repression. One of these genes, git7, is required for cAMP signaling, but its exact function in the process is unknown. Analysis of this gene is complicated because it carries out additional essential functions that are unrelated to cAMP signaling. The git7-93 allele encodes a protein that possesses an18 amino acid duplication in the carboxy-terminus and only displays a defect in cAMP signaling, while two other mutant alleles that affect all Git7-dependent processes alter residues in the amino-terminus. My thesis work was composed of two projects designed to investigate the role of the Git7 carboxy-terminus in cAMP signaling and to identify more precisely the carboxy-terminal domain that is required for cAMP signaling. The first part of my study involved a genetic screen to isolate and characterize mutations that suppress the git7-93 allele, but fail to suppress a git7 mutant allele in which the amino-terminus of the protein is altered. Such allele-specific suppression may identify the direct target of Git7 function in the cAMP pathway. I have isolated a collection of 55 suppressor strains. Molecular and genetic analyses of these strains have reduced this number to nine candidates, which could have represented allele specific suppressors. However, the final genetic screen determined that all the candidates were extragenic suppressors. The second part of my study involved a screen for new mutant alleles of git7 that resemble git7-93 in that they confer a cAMP signaling defect, but do not confer cell wall or septation defects caused by the git7-27 or git7-235 alleles. This was carried out by performing a random mutagenesis on a cloned wild type git7 gene and then screening for plasmids that could suppress the cell wall or septation defects, but not the cAMP signaling defect, in a git7-235 host strain. Taken together, these two studies should help to identify the role that git7 plays in the cAMP signaling pathway Schizosaccharomyces pombe. / Thesis (BS) — Boston College, 2004. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

Thesis advisor: Charles Hoffman / The fission yeast Schizosaccharomyces pombe senses environmental glucose through a cAMP-signaling pathway. Elevated cAMP levels activate protein kinase A (PKA) to inhibit transcription of genes involved in sexual development and gluconeogenesis, including the fbp1⁺ gene, which encodes fructose-1,6-bisphosphatase. Glucose-mediated activation of PKA requires the function of nine git genes (git=glucose insensitive transcription), encoding adenylate cyclase, the PKA catalytic subunit and seven “upstream” proteins required for glucose-triggered adenylate cyclase activation. This thesis describes the cloning and characterization of the git10⁺ gene, which is identical to swo1⁺ and encodes the S. pombe Hsp90 chaperone protein. This discovery is consistent with the previous identification of the Git7 protein as a member of the Sgt1 Hsp90 co-chaperone family. Glucose repression of fbp1⁺ transcription is impaired by both hsp90⁻ and git7⁻ mutant alleles, as well as by chemical inhibition of Hsp90 activity and temperature stress. Unlike the swo1⁻ and git7⁻ ts mutant alleles, the git10-201 allele and git7-93 allele support cell growth at 37º and show no cytokinesis defect, while severely reducing glucose repression of an fbp1-lacZ reporter, suggesting a separation-of-function defect. A physical interaction between Git7 and Hsp90 in S. pombe was also detected and findings in this thesis suggest their involvement in the initial assembly of the cAMP complex. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.