Use of an Inducible Gene System to Study the Dynamics of the Drosophila Dosage Compensation Complex 公开

Jang, Peter (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/db78tc20t?locale=zh
Published

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

About this Honors Thesis

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Degree
Submission
Language
  • English
Research field
关键词
Committee Chair / Thesis Advisor
Committee Members
最新修改

Primary PDF

Supplemental Files