Date of Award

12-2012

Degree Name

Master of Science

Department

Biological Sciences

First Advisor

Dr. Donald A. Kane

Second Advisor

Dr. Silvia Rossbach

Third Advisor

Dr. Pamela Hoppe

Access Setting

Masters Thesis-Open Access

Abstract

The early arrest mutant ogre, isolated in the Tübingen screen, has a block in cytokinesis (Kane et al., 1996). Here I use genetic and molecular techniques to demonstrate that the ogre phenotype is caused by a lesion in the gene encoding Racgap1. I performed fine mapping studies using microsatellite markers to a 0.15 cM region containing the ogre locus. DNA sequence data revealed a nonsense mutation in the Racgap1 gene of ogre mutants, and in situ hybridization experiments showed a loss of racgap1 mRNA at later stages of development, likely by nonsense mediated decay. This evidence supports the abolishment of Racgap1’s GTPase activating (GAP) function which is required for the inactivation of RhoA, a necessary step for the completion of cytokinesis. This failure in cytokinesis drastically affects some neural, erythrocyte and podocyte populations resulting in cell death most likely by apoptosis. Injection of wild-type racgap1 mRNA in ogre resulted in a partial rescue of the morphological, neural, and cellular phenotypes. These data provide definitive evidence that ogre is Racgap1 and indicate that just as in the fly, and the worm, GAP function of Racgap1 is necessary for cytokinesis in zebrafish.

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