Date Thesis Awarded
Honors Thesis -- Access Restricted On-Campus Only
Bachelors of Science (BS)
Diane C. Shakes
Mark H. Forsyth
Matthew J. Wawersik
Robert A. Orwoll
Mitosis is a fundamental process shared by all eukaryotes for partitioning DNA into two identical nuclei. In a series of phases, DNA must be properly condensed, aligned, and segregated to give each daughter cell an exact copy of the parental genome. As a failure in mitosis is almost invariably lethal, a plethora of evolutionary conserved molecules exist to regulate and facilitate each step of the process. Among these essential cellular components are histones, nuclear proteins that wind DNA into tightly packed structures known as nucleosomes. Histones are ubiquitously associated with DNA in eukaryotes, and play roles in gene regulation and imprinting in higher organisms. In mitosis, these crucial proteins are responsible for condensing the disordered chromatin into distinct chromosomes, as well as attaching to the kinetochores and microtubules that govern segregation. In this study, we present our mapping and analysis of the temperature sensitive C. elegans mutant (ax941) previously described by Mark Astoria (2006) as mutant 1115. The ax941 mutant was originally isolated by the Seydoux lab in a screen for maternal effect lethal mutations. The maternal effect refers to the preloading of protein products into the embryo by the mother, so whereas mutant males are able to fertilize wildtype hermaphrodites, the fertility of hermaphrodite mutants cannot be restored in crosses with wildtype males. Affected embryos undergo aberrant mitoses, with a range of defects including aberrant DNA segregation during anaphase and delayed telophase often followed by cytokinesis failure. Meiotic divisions are phenotypically normal, but the first mitotic division typically shows errors, with the phenotype fully manifesting by the second or third mitotic divisions. Previous SNP mapping and three point mapping had placed ax941 on chromosome I but failed to determine the molecular identity of the mutant. We have subsequently performed additional SNP mapping, deficiency complementation, and RNA interference experiments in order to determine the gene responsible. Our analysis suggests that the ax941 mutant gene corresponds to one of two neighboring histone proteins, his-67 and his-68, that form core parts of the nucleosome during early embryogenesis. We propose that since each cell division depletes the quantity of histones available per cell, later cell divisions are unable to properly condense the DNA into distinct chromosomes. Unable to resolve or untangle the interwoven DNA strands, mitosis slows down and a multitude of cellular defects result, producing the variety of phenotypes associated with this mutant. Preliminary sequencing of the two histone genes reveals a potential mutation in the his-67 gene that changes an alanine residue to a proline residue, a non-conserved change predicted to disrupt the otherwise highly conserved histone structure.
Chi, Stephen, "Mapping a Mutation: Characterization of the ax941 Mutant" (2010). Undergraduate Honors Theses. William & Mary. Paper 746.
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