Role of Topoisomerase I in Drosophila melanogaster's Mechanism of Dosage Compensation Open Access

Luk, Kevin Man-Hin (2013)

Permanent URL: https://etd.library.emory.edu/concern/etds/8s45q910p?locale=en
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Abstract

Dosage compensation is the equalization of X-linked gene products between male and females. In male Drosophila melanogaster, the single X-chromosome is hyper-transcribed twofold and is mediated by the Male Specific Lethal (MSL) complex. This twofold increase in expression of the male X chromosome is achieved by an enhanced rate of transcription elongation. Topoisomerases are a family of enzymes that maintain nucleic acid topology during cellular processes, such as transcription. Based on topoisomerases' role in transcription and experimental evidence revealing topoisomerase II's role in Drosophila dosage compensation, an investigation of topoisomerase I's role in dosage compensation was warranted. Using RNA interference, a plasmid model system that reproduces dosage compensation, and qRT-PCR of endogenous genes, this study reveals the ability of the MSL complex to mediate dosage compensation in male Drosophila even in the absence of topoisomerase I. This observation further elucidates the relationship of topoisomerase enzymes and MSL complex mediated dosage compensation.

Table of Contents

Table of Contents
1. General Background...1

Differences in X-Linked Gene Products...1
The MSL Complex and Dosage Compensation...2
Nucleic Acid Topology and Gene Transcription...4
Topoisomerase I & II...5

2. Purpose...7
3. Experimental design...8
4. Methods and Materials...9

S2 Cells & Culturing...9
RNA Interference (RNAi)...9
Plasmids...9
Plasmid Transfection...10
Luciferase Assay...10
Reverse Transcription, Real Time PCR (qRT-PCR)...11
Immunoprecipitation...12
Western Blot...13
Coomassie Staining...13
Topoisomerase I Antigen Synthesis...13
Antigenicity Profile...14

5. Data & Results...15
6. Discussion...31
7. Future Work...32
8. References...33

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