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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 In previous unpublished studies, zombie was mapped to Chromosome 2 and was fine mapped to the vicinity of cdc20, suggesting mutant cdc20 as a candidate gene.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.
Johnston, Peyton, "Is the zebrafish zombie mutant caused by a mutation in CDC20?" (2015). Honors Theses. 2628.
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