Craig Rogers, Department of Chemistry and Biochemistry

Abstract

In the yeast strain Saccharomyces cerevlslae, the gene CDC34 encodes for a ubiquitln ligase necessary for the transition from GI to the S phase of the cell division cycle. CDC34 is one of several cell division cycle (CDC) genes that act as checkpoints, or regulators, of the cell division cycle In yeast. The Cdc34 protein attaches ubiquitin molecules onto proteins to target them for degradation. The amino acid sequence of the Cdc34 protein Is highly conserved among other ubiqultin conjugating enzymes. Proposed targets for ubiquination and degradation by the Cdc34 protein Include cell cycle regulatory proteins, such as Far!, Gcn4, Sic!, and tumor suppressor proteins. The human form of the CDC34 gene was recently isolated. This project was part of a larger study conducted by Dr. Sharon Pion (Baylor College of Medicine) to test the hypothesis that the human CDC34 gene is an essential regulator of the Gl-S checkpoint, as it is in yeast. In order to determine the influence of human CDC34 on the cell cycle, mammalian cells with conditional Cdc34 activity must be constructed. Plasmid-based, conditional human cdc34 alleles can be used to rescue cdc34 activity In homozygous null embryonic stem cells to determine whether CDC341s an essential regulator of the cell cycle. My specific assignment in the overall project was to create temperature sensitive mutations of the human CDC34 gene to be used in the plasmid rescue experiment. I performed site-directed mutagenesis on human CDC34 in order to match known temperature sensitive mutations of yeast cdc34. My hypothesis is that temperature sensitive mutations of yeast cdc34 alleles will also be temperature sensitive for the human CDC34 if the mutations correspond to conserved amino acid residues of the two proteins.

After failing to create the desired mutations with a BioRad mutagenesis procedure and by PCR mutagenesis, a point mutation named A62V was obtained using an Amersham mutagenesis procedure. If the mutation is temperature sensitive, it can be isolated by a yeast assay that selects for human CDC34 genes that complement a yeast cdc34 null mutation In a temperature sensitive manner. The mutant can then be used to construct mammalian cells with conditional Cdc34 activity. The complementation tests with the A62V mutation have not been completed by Baylor College of Medicine. The null embryonic conditional cells would be a springboard for future experiments to determine the influence of CDC34 on tumor suppressor gene and oncogene products. Knowledge of the mechanism whereby CDC34 regulates cell division could be beneficial in improving cancer treatment.