Date of Defense

4-22-2014

Date of Graduation

4-2014

Department

Biological Sciences

First Advisor

Donald Kane

Second Advisor

Douglas Coulter

Third Advisor

Tetiana Petrachkova

Abstract

The cell cycle is essential for proper growth and development in all organisms. Many diseases, including cancer result from defects in control of the cell cycle. Here we characterize the zebrafish recessive cell cycle mutant zombie (zom).The chromosomes of zombie mutants arrest in the metaphase-anaphase transition of mitosis, suggesting a mutation in the cell cycle machinery of the embryo. Zom was mapped to a small interval in lineage group 2, which included CDC20 locus. CDC20 is an activator of the Anaphase Promoting Complex, and the role of this complex is to separate chromosomes during the metaphase-anaphase transition. Sequencing the mutant showed that a transversion occurred in exon 7 of the CDC20 gene producing a nonsense mutation. In situ analysis shows there are fewer CDC20 mRNA transcripts in the mutant indicating nonsense -mediated degradation of mRNA. This is a common feature of premature termination. Thus, zom is likely a complete loss of function of CDC20. In support of this, a mutation in the CDC20 homolog in the D. melanogaster also causes arrested mitosis. Antisense splice blocking oligonucleotides were used to knock down CDC20 gene function in an attempt to recapitulate the mutant phenotype. However, we are yet unable to reproduce the mutant phenotype perhaps because the resulting mRNA product is only altered by several nucleotides. Currently, we are using CDC20 mRNA and CRISPRs, to attempt rescue of the mutant phenotype and knockout of the CDC20 gene, respectively. Identifying the gene mutated in zom will enable us to start to unravel the complex control of the cell cycle during animal development.

Access Setting

Honors Thesis-Open Access

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