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The circadian proteome

Reference	BB/C006941/1
Principal Investigator / Supervisor	Professor Charalambos Kyriacou
Co-Investigators / Co-Supervisors	Professor Kathryn Lilley
Institution	University of Leicester
Department	Genetics
Funding type	Research
Value (£)	994,763
Status	Completed
Type	Research Grant
Start date	13/06/2005
End date	12/10/2010
Duration	64 months

Abstract

We will analyse the neuronal circadian proteome of two model organisms, Drosophila, and the mouse, using appropriate brain tissues with the newly developed DiGe and ICAT systems. We have performed preliminary work with both mouse SCN and fly brains to show that this is technically feasible. We shall isolate proteins that show circadian cycles in abundance, or in their posttranslational modifications, in both organisms, and identify them using Mass Spectrometry. We shall use cutting edge proteomic technology to increase the resolution of our analysis such as using iTRAQ tags, and we shall compare cycling proteomes in both organisms with the corresponding cycling transcriptomes. This will provide an estimate of what proportion of the proteome cycles because of transcriptional or posttranscriptional rhythms. We shall also examine which proteins are specifically regulated by light, focusing particularly on rapid posttranslational events such as phosphorylation. We will enhance our analysis by using techniques to fractionate the different cellular compartments and use co-IP to define new proteins that associate with the transcriptional complex of clock proteins at different phases of the circadian cycle. Novel proteins will be studied using a variety of methods. We shall examine their expression patterns in fly and mouse brains, and also jump the species barriers by finding, say, the homologues of a mouse, in the fly, and vice-verse. The fly provides an excellent in vivo system for overexpressing or knocking-down these proteins in clock cells using the GAL4 UAS system, and for examining the effects on circadian behaviour. For proteins that associate which clock complexes, we shall use transcriptional assays to examine their properties, as well as validating their putative physical interactions with known clock proteins using the yeast two hybrid system. Functional analysis will also be performed with human cell lines by knocking down or overexpressing these genes and observing the effects on the molecular cycles in these cells. SCN organotypic slices that carry cycling clock gene reporters will also be used in this way and transfected with a gene-gun. Finally we shall use CHIP analysis and genome array analysis to locate the promoters that are controlled by these circadian complexes

Summary

unavailable

Committee	Closed Committee - Animal Sciences (AS)
Research Topics	Microbiology, Neuroscience and Behaviour
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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