Date of Defense

12-11-2015

Date of Graduation

12-2015

Department

Biological Sciences

First Advisor

Donald Kane

Second Advisor

Rachel Warga

Third Advisor

Pamela Hoppe

Abstract

The zombie mutant was identified as an early arrest mutant, stopping in development around the 10-somite stage (14 hours of development). Further inspection revealed that this mutant was a cell cycle mutant and cells in the mutant arrest during metaphase as early as the 5-somite stage (11.6 hours of development). A similar phenotype is seen in the Drosophila melanogaster cell cycle gene fizzy, known to be to be a homolog of the Saccharomyces cerevisiae gene, cell division cycle 20 (cdc20). CDC20 is an activator protein of the anaphase promoting complex/cyclosome (APC/C), an ubiquitin E3 ligase that is responsible for cell cycle progression. In previous unpublished studies, zombie was mapped to Chromosome 2 and was fine mapped to the vicinity of cdc20, suggesting mutant cdc20 as a candidate gene. However, failure to rescue the zombie mutant phenotype using Xenopus cdc20mRNA was unsuccessful. Here, we investigate whether cdc20 is the gene mutated in zombie via two experimental assays: rescuing the zombie mutant phenotype with wild-type zebrafish cdc20mRNA and mutagenizing the wild-type cdc20 chromosome to produce the zombie mutant phenotype by using the CRISPR/Cas 9 system. Antibody staining for phosphorylated histone H3, showed an increase in mitotic cells in zombie mutants compared to wild-type siblings as early as the 5-somite stage. Unfortunately, CDC20 injected zombie embryos showed no phenotypic rescue of the mutant phenotype. In addition, mutagenesis of the wild-type cdc20 chromosome showed a phenocopy of the zombie mutant phenotype, convincing us that zombie is a mutation of CDC20.

Access Setting

Honors Thesis-Open Access

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