Christina Doerr
Olivet Nazarene University
Kankakee , IL
Faculty Mentor: Dr. Gail Waring

The eggshell of Drosophila melanogaster is a complex multilayered structure. Proteins are secreted from somatically derived follicle cells into the extracellular space between the follicle cells and the oocyte. These proteins organize specifically over time to compose two major morphologically distinct layers. The vitelline membrane (VM) forms during stages 9 and 10 of egg chamber development, while the endochorion is formed during stages 12 to 14. Although many eggshell proteins have been identified, little is known about their assembly and interactions. The aim of this project is to gain some insight on the chemical nature of the interactions involving three eggshell proteins - sV23, s36, and dec-1. In order to determine what is necessary to release these proteins from the eggshell, egg chambers were exposed to different solubulizing conditions and pelleted at 15,000 x g. The supernatant and pellet fraction were then analyzed by Western Blot analysis.

S36 is a chorion protein produced during early eggshell development. Although, the majority of it is found in the vitelline membrane at stage 12, by stage 14 it is all localized in the chorion. Western blot analysis in this experiment has shown that strong denaturants such as urea are required to release s36 into the supernatant. Detergents were found to be ineffective unless accompanied by high temperatures. This suggests that hydrogen bonds may be prominent in retaining s36 in the eggshell.

The dec-1 gene synthesizes three pro-proteins. The most abundant of these is fc106. This protein is further cleaved during eggshell development. The C-terminal derivatives, s80 and s60, are studied in this experiment. There is one cysteine present in the C-terminus of the fc106 protein. It was found that although s80 and s60 are soluble in urea at early stages, this solubility decreases over time. At high temperatures, Sodium Dodecyl Sulfate (SDS) is able to displace s80 and s60 into the supernatant at all stages. However with Triton, more s80 and s60 is found in the supernatant upon the addition of BME. This indicates limited disulfide linkages at all stages and a decrease in the strength of the hydrogen bonds involving s60 at stage 14. SV23 is localized in the vitelline membrane at all stages of development. Western blot analysis has shown that the sV23 protein undergoes processing during oogenesis. There are three conserved cysteines present in sV23. The results in this experiment show that although noncovalent interactions are prominent during early stages of eggshell development, disulfide linkages become a prominent force holding sV23 in the eggshell by stage 14.