Cloning, expression, and purification of the Drosophila melanogaster dosage compensation complex chromodomains and their Homo sapiens orthologues
Date
2009
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Degree Level
Masters
Abstract
Sexual differentiation is a fundamental characteristic of all eukaryotes, dictating sex-specific morphology, physiology and behavior. Diploid organisms with heteromorphic sex chromosomes (XX or XY) require regulatory compensation of the X chromosome to maintain correct levels of genetic expression between the sexes, a process termed sex-specific dosage compensation (SSDC). The fruit fly, Drosophila melanogaster dosage compensates by upregulating transcription of most X-linked genes two-fold. Associated with this two-fold up regulation is the male-specific lethal (MSL) complex, a RNA-protein complex comprised of at least five known proteins; MSL1, MSL2, MSL3, males absent on the first (MOF), and maleless (MLE) and two non-translated RNA molecules; roX1 (RNA on the X chromosome) and roX2. The complex modulates the chromatin structure of the male X chromosome via acetylation of H4K16. MOF and MSL3 both exhibit an N-terminal chromodomain, whose function is unclear. The MSL3 chromodomain has been suggested to bind H3K36Me3. Chromodomains are a paradigm of how a single structural fold has evolved in diverse proteins to bind distinct targets. Chromodomains are common to nuclear regulators, and bind diverse targets including histones, DNA, and RNA. They function as recognition motifs of histone post-translational modifications and facilitate the translation of the histone code into a distinct local chromatin structure via recruiting the appropriate chromatin modulating machinery.
The goal of this research is to determine the structure of the D. melanogaster MOF and MSL3 chromodomains by X-ray crystallographic and/or nuclear magnetic resonance techniques, to advance our understanding of the structural characteristics of these diverse domains. Here we report the cloning and reproducible expression and purification of the D. melanogaster MOF and MSL3 chromodomains and their Homo sapiens orthologues. The D. melanogaster MOF chromodomain, whose NMR structure was published during this research, has been crystallized. Attempts to solve the crystal structure by molecular replacement, multiple-wavelength anomalous dispersion, and single-wavelength isomorphous replacement are reported.
Description
Keywords
Dosage compensation, Drosophila melanogaster, Epigenetic regulation
Citation
Degree
Master of Science (M.Sc.)
Department
Biochemistry
Program
Biochemistry