Analysis of the Git7 protein function in the Schizosachharomyces pombe cAMP pathway

Abstract

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.