טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentHoltzman Tzvi
SubjectMechanisms of Regulation of the Cyclin Genes CLN3 and PCL5
in the Yeast S. cerevisiae
DepartmentDepartment of Medicine
Supervisor Professor Daniel Kornitzer


Abstract

Cellular regulatory pathways integrate a multitude of inputs to generate a pathway-specific output. In the case of unicellular organisms, the inputs often relate to the availability and quality of food sources. Two pathways were investigated here: the Cln3-dependent pathway determining entry into the cell cycle, and the Pcl5-dependent pathway which represses the activity of the major amino acid starvation-regulated transcription factor Gcn4. Regarding Cln3, the possible role of a putative transcription factor, Mak32, in linking carbon source quality to CLN3 transcription was investigated. Although Mak32 appeared to increase the affinity of the cell-cycle transcription factor Mcm1 for the CLN3 promoter, no conclusive evidence for a role for Mak32 in CLN3 regulation was found. Regarding Pcl5, the activity of which leads to Gcn4 degradation, we found that the PCL5 gene is transcriptionally induced by Gcn4. Thus, activation by Gcn4 of its inhibitor Pcl5 results in a negative feedback loop, ensuring that Gcn4 activity is held in check. Furthermore, analysis of the PCL5 transcript uncovered an extensive 5’ untranslated region, which includes two short open reading frames (ORFs). The presence of these upstream ORFs leads to 10-fold reduction on PCL5 translation, compared to a mutant lacking these ORFs. Such a transcript configuration is usually indicative of translational control. Indeed, we found that under low-glucose conditions, or in the presence of Rapamycin (an inhibitor of the starvation-sensitive Tor pathway), PCL5 translation is increased. We suggest that this glucose-dependent regulation of Pcl5 ensures that under conditions of amino acid starvation, amino acid synthesis pathways are not unnecessarily activated when carbon and energy sources are scarce as well.