Title: Regulatoin of the inv1+ gene in Schizosaccharomyces pombe
Description: Glucose is the most abundant and preferred carbon source for many organisms. Many genes required for alternative carbon source utilization are often repressed by glucose. This dissertation describes our analysis of one such glucose-repressed gene, the inv1 + gene of the fission yeast Schizosaccharomyces pombe. We have analyzed both trans- acting factors and cis- regulatory elements to characterize the regulation of inv1 + in response to glucose levels. First, to identify trans-acting factors that affect the inv1 + gene in S. pombe, we have taken both a candidate gene approach based on regulators of two related glucose-repressed genes, S. cerevisiae SUC2 and S. pombe fbp1 + and performed a screen of the S. pombe deletion set. From the candidate gene approach, we learned that similarities and clear differences exist between regulators of SUC2, fbp1 + and inv1 + . From the deletion set screen, we have identified eukaryotic translation initiation factor 3j (eIF3j) as a factor required to achieve normal inv1 + steady state transcript levels. Our results suggest that reduced translation of inv1 + results in reduced mRNA stability in low glucose conditions, as deletions of genes encoding other factors involved in translation also cause a mild reduction in inv1 + mRNA levels. We also analyzed the inv1 + transcript and found that inv1 + has a long 5' UTR of 872 bases that contributes to the steady state level of the inv1 + mRNA.

To identify cis-regulatory elements that regulate inv1 + , we have made a systematic set of deletions of the upstream regulatory region of the gene. Surprisingly, we found that there are at least three redundant regulatory regions spread over approximately 1.5 kb upstream of inv1 + , with the most distant region actually residing within the next open reading frame. Furthermore, each of these three regions is able to confer glucose repression at a heterologous gene. From these studies, we have determined that glucose-mediated regulation of inv1 + is complex, with both a transcriptional and post-transcriptional component, suggesting the possibility that there can be many pathways leading to inv1 + regulation.