The Role of Topoisomerases in Dosage Compensation of the Male X Chromosome of Drosophila melanogaster Open Access

Marcadis, Andrea Rachel (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/4t64gn408?locale=en
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Abstract

The Role of Topoisomerases in Dosage Compensation of the Male X Chromosome of
Drosophila melanogaster


By Andrea Rachel Marcadis


In fruit flies (Drosophila melanogaster), X chromosome dosage compensation is
achieved by twofold upregulation of transcription of the male X chromosome. This
increased level of transcription is the responsibility of the Male Specific Lethal (MSL)
complex, which functions by modifying the chromatin of dosage compensated genes,
enhancing the elongation step of transcription. Topoisomerases are enzymes that resolve
the torsional tension and topological hindrance that occur when DNA is being actively
transcribed. Because of their function in the elongation step of transcription, as well as
experimental evidence revealing topoisomerase interaction with the MSL complex, a role
for topoisomerases in the dosage compensation process was suspected. Using RNA
interference to knock down topoisomerases and a plasmid model system to reproduce
dosage compensation, this study reveals a role for topoisomerase II in Drosophila X
chromosome dosage compensation. This observation helps to further elucidate the
mechanism of action of the MSL complex.

Table of Contents

Background....................................................1

XY Sex Determination........................................1

Dosage Compensation in Drosophila....................2

Topoisomerases and Dosage Compensation..........6

Experimental Approach......................................7

Materials and Methods...................................8

S2 Cells...........................................................8

RNAi Knockdown of Topoisomerases....................8

Plasmids..........................................................9

Plasmid Transfection........................................10

Luciferase Assay..............................................10

Western Blot Analysis.......................................10

Results..........................................................11

Calculation of Relative Dosage Compensation.......11

1 + 4 Experiment.............................................12

4 + 4 Experiment.............................................14

3 + 4 Experiment.............................................16

Topoisomerase I...............................................18

Discussion.....................................................18

Future Work..................................................20

References....................................................22

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