Download FunctGenomics - Nematode bioinformatics. Analysis tools and data

Functional Genomics
•Functional genomic datasets
•Biological networks
•Integrating genomic datasets
BIO520 Bioinformatics
Jim Lund
Functional genomics
• Genome scale experiments to
understand the function of all the
proteins--what they do and how they
interact.
• Many different experimental designs
– Different kinds of information generated.
• Each has experimental limitations
– Coverage: full genome, limited?
– False positives.
– False negatives.
The Two-Hybrid System
for identifying protein/protein binding pairs
• Two hybrid proteins are generated with
transcription factor domains
• Both fusions are expressed in a yeast cell
that carries a reporter gene whose
expression is under the control of binding
sites for the DNA-binding domain
Activation
Domain
Bait
Protein
Prey
Protein
Binding
Domain
Reporter Gene
The Two-Hybrid System
• Interaction of bait and prey proteins
localizes the activation domain to the
reporter gene, thus activating transcription.
• Since the reporter gene typically codes for a
survival factor, yeast colonies will grow only
when an interaction occurs.
Activation
Domain
Prey
Protein
Bait
Protein
Binding
Domain
Reporter Gene
Interactions shown as a network
Networks
• When methods of detecting functional
linkages are applied to all the proteins of an
organism, network of interacting, functionally
linked proteins can be traced.
• As methods improve for detecting protein
linkages, it seems likely that most of the
proteins will be included in the network.
What do you miss?
• Tertiary interactions
• Regulated interactions
– Subcellular localization dependent
– Cofactor dependent (eg. Hormoneregulated)
• Low-affinity (Kd>10-6)
Cellular Location
• Immunolocalization
– FUSION PROTEINS
YFG
GFP
• Prediction
– Membrane vs non-membrane
• improved by homology
• WHICH MEMBRANE
– Nuclear vs cytoplasmic
Drosophila Fusion Project
(FlyTrap)
• Exon GFP vector
– Inserts fairly
randomly.
• Fluorescent sort
thousands of
embryos.
– Find embryos with an
insertion that
produces GFP
expression.
• Image
– Capture and analyze
images
• Curate by hand.
• Computer image
analysis and
classification.
Developmental Localization
Mouse genomic gene expression
• Allen Brain Atlas (ABA) is an interactive, genome-wide
image database of gene expression in the mouse and
human brain. 17,000 mouse gene expression patterns,
cortex expression for 2,000 human genes.
Allen Brain Atlas
3D mouse gene expression project
Single gene expression database for the
mouse research community. Integrated in
the Mouse Genome Database (MGD) at
the Jackson Laboratory.
10,302 expression entries
WT1 expression (red) on a section
of the E9 (Theiler Stage 14) embryo
from the Edinburgh Mouse Atlas.
The gut epithelium is shown in
yellow and the neural tube in a blue
overlay. WT1 is expressed in the
presumptive mesothelium of the
coelom and in the intermediate
mesoderm (ventral to the somites).
Methods for discovering
protein function
•Automated Binding Assays
•High Throughput Enzyme Assays
Genome-wide Knockouts
• Yeast Genome
– Recombination
strategy
• Mouse Genome
• More in Functional
Genomics!!!
Essential vs Non-essential
• Transcription similar
– >99% essential genes transcribed
• Transcript level 70% higher
– >90% non-essential transcribed
• Genome locations similar
– Not clustered
– Essential genes rarely near telomeres
Why only 20% essential?
• Redundant
– 8.5% of non-essential had CLOSE
homolog in genome (P<10-150)
• Essential in another condition
• Marginal Benefit
Resources
YEAST
MOUSE
• Saccharomyces
Genome Deletion
Project
• Mouse Phenome Database
– http://wwwsequence.stanford.edu/
group/yeast_deletion_p
roject/deletions3.html
– http://phenome.jax.org/pubcgi/phenome/mpdcgi?rtn=docs/h
ome
• Knockout Mouse Project
– http://www.knockoutmouse.org/
Genome-Scale
Biochemical Assay
• Protein arraysbiochemically
active
Databases
• Relationships between
genes/proteins.
• How are different types of
experimental data integrated?
– Schema
• Data quality
– Who curates?
– Who revises?
Proteome Projects
• SwissProt (ExPasy)
– http://expasy.org/ch2d/
• Saccharomyces Genome Database
(SGD) Gene Function Information
– 2-hybrid, functional assignments, pathways.
– http://www.yeastgenome.org/SearchContents.shtml
• Yale TRIPLES
– Database of TRansposon-Insertion
Phenotypes, Localization, and Expression in
Saccharomyces.
• 2-hybrid databases
– http://proteome.wayne.edu/YTHwebsites.html
Pathway and interaction databases
• KEGG (http://www.genome.jp/kegg/)
– Metabolic and signaling pathways
• PUMA (http://compbio.mcs.anl.gov/puma2/cgi-bin/index.cgi)
– Metabolic and signaling pathways
• DIP (http://dip.doe-mbi.ucla.edu/)
– Protein-protein interactions
• BIND (http://bind.ca/)
– Molecular and genetic interactions
KEGG pathway map
HISTIDINE METABOLISM
Pentose phosphate cycle
5P-D-1-ribulosylformimine
3.5.1.-
Phosphoribosyl-AMP
PRPP
3.6.1.3
1
2.4.2.1
7
3.5.4.1
9
PhosphoribulosylFormiminoAICAR-P
2.6.1.-
5.3.1.1
6
4.2.1.1
9
2.4.2.-
PhosphoribosylFormimino-AICAR-P
Phosphoriboxyl-ATP
Imidazoleacetole P
ImidazoleGlicerol-3P
3.1.3.1
5
2.6.1.9
L-Histidinol-P
1.1.1.2
3
5P Ribosyl-5-amino 4Imidazole carboxamide
(AICAR)
Aneserine
2.1.1.2
2
Purine metabolism
Carnosine
3.4.13.
3
N-Formyl-Lspartate
3.5.3.5
Imidazolone
acetate
3.5.2.-
1.1413
5
Imidazole4-acetate
Imidazole
acetaldehyde
1.2.1.3
1.4.3.6
3.4.13.2
0
Histamine
L-Hisyidinal
4.1.1.2
2
4.1.1.2
8
1-MethylL-histidine
3.4.13.
5
6.3.2.1
1
L-Hisyidinal
2.1.1.6.3.2.1
1
1.1.1.2
3
6.1.1
Hercyn
L-Histidine
Integrating pathway and expression data
The list of genes
being activated
or inactivated or
that are
unaffected when
comparing two
samples becomes
more informative
if the genes can
be mapped onto
maps from which
functions can be
deduced.