Determining SPE-16 interacting proteins during Caenorhabditis elegans spermatogenesis Open Access

Reiter, Katherine Hays (2009)

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The spe-16 gene affects how sperm develop and move in the nematode Caenorhabditis elegans. My lab has cloned spe-16 and found that it encodes an ubiquitin E3 ligase, homologous to Mind bomb, which is found in arthropods and vertebrates. Protein ubiquitination is used to tag proteins for removal from cells and also to direct their trafficking within the cell. This suggests that SPE-16 affects sperm development and movement by controlling ubiquitination of one or more substrate proteins. The goal of this work was to define proteins that are potential substrates for SPE-16. spe-16 mutants are temperature sensitive so that hermaphrodites and males produce viable sperm at 20°C, but defective sperm when grown at 25°C. Consequently, large numbers of homozygous mutants can be propagated at 20°C and subsequently shifted to 25°C so that the mutant phenotype can be examined. My overall goal was to discover SPE-16 substrates and two experimental approaches were taken. First, I used a genetic suppressor approach where the goal was to explore whether spe-16 self-sterile mutants could be restored to self-fertility. Such suppressors could define gene products that interact with SPE-16, however, none were recovered despite extensive screening of three spe-16 mutants. Next, I took a biochemical approach that included 1D and 2D gel electrophoresis, Western blots, and ubiquitin assays. These approaches suggest that spe-16 mutants affect the distribution and abundance of several proteins, but further research is needed to fully evaluate these potential substrates.

Table of Contents

Chapter I. Introduction

A. Overview of Caenorhabditis elegans
B. Spermatogenesis
C. Spermiogenesis
D. Mutations affecting spermatogenesis
E. Introduction to spe-16
F. Introduction to Notch signaling
G. Introduction to ubiquitin pathways
Chapter II. Genetic Suppression of spe-16 mutants

A. Introduction
B. Materials and methods
C. Results
D. Discussion

Chapter III. Biochemical analysis
A. Introduction

B. Materials and methods

C. Results
D. Discussion

Chapter IV. Future experiments

Chapter V. Conclusions

Tables and Figures

Literature Cited

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