MBT510/GENET551 Berg

Paper for 11 Dec 2001

Stevenson, L. F., Kennedy, B. K., and Harlow, E. 2001. A large-scale overexpression screen in Saccharomyces cerevisiae identifies previously uncharacterized cell cycle genes. Proc. Natl. Acad. Sci. USA 98: 3946-3951.

All cells have evolved to use glucose as a carbon source, constitutively expressing the enzymes that break down this sugar. In contrast, expression of enzymes that act on other sugars is tightly regulated. These authors use this distinction to control the overexpression of cDNAs and then test for cell cycle defects. Homework: Write brief answers to the questions in bold.

Questions for Thought

1) What is the goal of these experiments? What is the logic for thinking more cdc genes exist? How do they approach the problem? Why think that overexpression will reveal new genes?

2) Why do the authors screen 180,000 clones when only ~6,000 genes exist in yeast? Have they found all the genes that affect the cell cycle? Explain.

3) The authors anticipate that "overexpression might confer less dramatic defects than traditional loss-of-function screens". Why would one expect such a result?

4) Generally, what sorts of genes were identified and how did they characterize them?

5) The authors analyze 36 hypothetical ORFs and found that most exhibited no phenotype when deleted (Table 3). Why did the authors recover these genes when they appear to have no role in regulating the cell cycle?

6) Seven instances of likely genetic redundancy were identified by BLAST searches comparing the hypothetical ORFs recovered in the screen and the entire yeast gene database. Some of the partner genes produced a cell cycle defect when overexpressed and others did not. Some produced cell cycle defects when both genes were deleted and others did not. Why didn't the authors recover all these genes in their screen? Why don't all double deletions show a phenotype?