Overexpression of CDC7 with the removal of DBF4 in Saccharomyces cerevisiae
Mentor:Wendy Dixon, Doctorate of Microbiology, Cal Poly Pomona
Two essential proteins associated with the S phase are Dbf4 and Cdc7, which come together to form an active complex. We were interested in seeing how the Cdc7 protein is effected by the removal of the DBF4 gene and ultimately the Dbf 4 protein from yeast cells. We used a Saccharomyces cerevisiae dbf4 temperature sensitive (ts) mutant with the CDC7 gene on a high expression plasmid. Normally a S. cerevisiae dbf4 ts mutant will arrest with a large bud at the restrictive temperature. However, CDC7 gene complements the dbf4 ts mutant. For this reason, we had to work out a procedure to get the dbf4 ts mutant to arrest as a large bud even when the Cdc7 protein was overexpressed in the cell. In preparation, we needed a healthy supply of yeast, achieved by growing in Ura-/Glu- solution and transferred to a Ura-/Raf- solution two days later. CDC7 was induced by addition of galactose and eventually the yeast were incubated in a water bath at 37ºC for 4-18 hours. Our results showed that at 18 hours of incubation, there was significantly more large and small buds than unbudded cells. Thus, we determined that a dbf4 ts arrest can be obtained even with overexpression of CDC7 and the conditions required for the arrest. For both control and arrested yeast, a workup procedure was then preformed to fix the cells and stain the genome with DAPI. For these studies, we are using a green fluorescent tagged CDC7 so that its location can be observed by microscopy. Microscopy data is still being collected and analyzed. We will present our results from the microscopy at the meeting. Thus we can overcome complementation and observe Cdc7 without Dbf4. In a broader scope, we will be able understand how factors influence the start of S phase.