Use of an Inducible Gene System to Study the Dynamics of the Drosophila Dosage Compensation Complex Open Access
Jang, Peter (2012)
Abstract
Abstract
Use of an Inducible Gene System to Study the Dynamics of the Drosophila Dosage Compensation Complex
In Drosophila, males have one transcribing X chromosome while females have two transcribing X chromosomes. The males and females need to transcribe equal amounts of the genes on the X chromosome and achieve dosage compensation. The Male Specific Lethal (MSL) complex is responsible for making this happen. The MSL complex acetylates histones, specifically H4K16, which loosens the chromatin and increases the accessibility of the X chromosome, allowing hypertranscription in males. A mystery lies in how the MSL complex is recruited to active genes and spreads on the X chromosome. Two ideas exist: one, the MSL complex could start at the promoter region and move towards the 3' end of the gene; two, the MSL complex moves in an opportunistic manner to the transcribed region where it modifies the histones between the passages of RNA Polymerase II (Pol II) molecules (Lucchesi, 2009). To figure out this aspect of the MSL complex, an inactive gene that can be activated and that recruits the MSL complex needs to be examined. Previous work by Boehm with Drosophila melanogaster used chromatin immunoprecipitation (ChIP) on the inactive, activatable hsp70 (heat-shock protein 70) gene to examine the movements of different proteins before and after transcription is initiated. A hsp gene would be suitable for similar studies with the MSL complex but none exists on the X chromosome. Such a gene is on the XR chromosome of D. pseudoobscura. The purpose of this study is to demonstrate that after ensuring its inactive state the hsp28 gene on the XR chromosome of D. pseudoobscura can be activated and is able to recruit the MSL complex, so that it can be used to clarify where the MSL complex is recruited and how it enhances transcription.
by
Peter Jang
Adviser: Dr. John C. Lucchesi, Ph. D.
Table of Contents
TABLE OF CONTENTS
Introduction…………………………………………………………………...…...1
Materials and
Methods…………………………………………………………9
Results…………………………………………………………………........………..20
Figure 1: Chromosome orcein staining of the distal XR chromosome
of non-heat shocked
and heat-shocked
larvae....................................................................................................20
Figure 2: Chromosome orcein staining of the proximal XR
chromosome of non-heat
shocked and heat-shocked
larvae......................................................................................21
Figure 3: FISH of
hsp28......................................................................................................21
Figure 4: FISH of
hsp83......................................................................................................22
Figure 5: Conservation of MSL2 between D.
melanogaster and D.
pseudoobscura..............23
Figure 6: Conservation of MLE between D. melanogaster
and D. pseudoobscura…..............24
Figure 7: Immuno-FISH of MSL2 protein and hsp28
gene...................................................25
Figure 8: Immuno-FISH of HSF protein and hsp83
gene.....................................................26
Figure 9: MSL2 and Ser-2P immunostaining of non-heat
shocked………………….......................29
Figure 10: MSL2 and Ser-2P immunostaining of 2.5 min
HS……………….….......................….....30
Figure 11: MSL2 and Ser-2P immunostaining of 20 min
HS………………….........................….…..31
Figure 12: HSF immunostaining of non-heat
shocked………………………...............................…….32
Figure 13: HSF immunostaining of 2.5 minutes heat
shocked……………….....................…......…32
Figure 14: HSF immunostaining of 20 minutes heat
shocked…………….….….........................….33
Figure 15: Gene expression levels of hsp
genes……………………………………….............................…34
Figure 16: Dosage compensation of heat shocked hsp
genes……………….……..........................34
Figure 17: Dosage compensation of non-heat shocked
hsp
genes………….…...................…..…34
Figure 18: Chromosome orcein staining of the XR chromosome of
non-heat shocked,
dissected under, cold shocked, and heat-shocked
larvae…………………….….......................…...36
Discussion……………………………………………………………..………….37
Future
Direction……………………………………………………………....42
References…………………………………………………………………....….43
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