Semester

Fall

Date of Graduation

2011

Document Type

Thesis

Degree Type

MS

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Clifton Bishop.

Abstract

The objective of these projects was to generate two fluorescently based systems within Drosophila melanogaster for the advancement of molecular research. The first project was aimed to create fluorescent phosphomimetic constructs of m8, a regulator in eye formation. The second project was to create a fluorescent visual marker for heterochromatic gene silencing in Drosophila. Proper formation of the eye requires that only one cell from a group of equipotent cells assumes a neural fate (the R8 cell). The cells surrounding the R8 cell are prevented from assuming this outcome through a process known as lateral inhibition. Lateral inhibition uses the regulation of proteins from the Enhancer of split complex E(spl)C, to down regulate the neural protein, Atonal. The functionality of a specific E(spl) protein, M8, is regulated by phosphorylation by CK2 at a specific site within the phosphorylation domain of M8. It was the goal of this project to create fluorescently labeled phosphomimetic variants of M8 with aspartic acid and alanine substitutions at key phosphorylation sites (the CK2 phosphorylation site and all four phosphorylation sites within the M8 phosphorylation domain). These constructs were used to generate preliminary data in a Drosophila Expression System. The initial data indicated that M8S 159A showed statistical differences between the control and the treatment group, while M8, M8S159D, and M8SDDDD, showed no statistical difference between treatments and controls. The location of these constructs in Schneider 2 cells suggested that M8 and M8S159A proteins were at times located in the nucleus, while the other phosphomimetic variants were restricted to the cytoplasm. This project generated a solid foundation of data collection methods and gave some insight into the location of M8 that can give rise to future studies within this system.;The second project was aimed at creating a fluorescent marker for an epigenetic phenomenon known as Position Effect Variegation (PEV) within Drosophila. PEV is caused by the relocation of a gene close to, if not adjacent to heterochromatin. When in close proximity, the heterochromatin can spread to silence the gene in some cells, rendering the gene silent; while failing to spread in others, allowing for expression resulting in a variegated phenotype within Drosophila. Male flies with a miniwhite gene adjacent to Gal4/UAS driven Yellow Fluorescent Protein were mutated in hopes of causing a translocation or inversion which would bring the fluorescent marker in close proximity to heterochromatin to observe epigenetic silencing. Although no variegated flies were found, this tool would serve as an effective marker to visually observe, using different UAS drivers within Drosophila, where PEV is occurring spatially and at what developmental time points within the fly as well. Such a variegating mutant would provide a molecular tool for future research on how CK2 and other modifiers of PEV act within the cascade of epigenetic changes within Drosophila.

Download

Share

COinS