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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.

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functional genomics

Functional Genomics

Functional genomic datasets

Biological networks

Integrating genomic datasets

BIO520 Bioinformatics Jim Lund

functional genomics1
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
The Two-Hybrid Systemfor 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

Prey

Protein

Bait

Protein

Binding

Domain

Reporter Gene

the two hybrid system
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

Reporter mRNA

Bait

Protein

Reporter mRNA

Reporter mRNA

Reporter mRNA

Binding

Domain

Reporter mRNA

Reporter Gene

networks
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
What do you miss?
  • Tertiary interactions
  • Regulated interactions
    • Subcellular localization dependent
    • Cofactor dependent (eg. Hormone-regulated)
  • Low-affinity (Kd>10-6)
cellular location

YFG

GFP

Cellular Location
  • Immunolocalization
    • FUSION PROTEINS
  • Prediction
    • Membrane vs non-membrane
      • improved by homology
      • WHICH MEMBRANE
    • Nuclear vs cytoplasmic
drosophila fusion project flytrap
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.
mouse genomic gene expression
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.
3d mouse gene expression project
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
Methods for discovering protein function

Gene Knockouts

  • Automated Binding Assays
  • High Throughput Enzyme Assays

Protein Assay

genome wide knockouts
Genome-wide Knockouts
  • Yeast Genome
    • Recombination strategy
  • Mouse Genome
  • More in Functional Genomics!!!
essential vs non essential
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
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

Saccharomyces Genome Deletion Project

http://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html

MOUSE

Mouse Phenome Database

http://phenome.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/home

Knockout Mouse Project

http://www.knockoutmouse.org/

Resources
genome scale biochemical assay
Genome-Scale Biochemical Assay
  • Protein arrays-biochemically active
databases
Databases
  • Relationships between genes/proteins.
  • How are different types of experimental data integrated?
    • Schema
  • Data quality
    • Who curates?
    • Who revises?
proteome projects
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
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
KEGG pathway map

HISTIDINE METABOLISM

Pentose phosphate cycle

5P-D-1-ribulosyl-

formimine

3.5.1.-

Phosphoribulosyl-

Formimino-AICAR-P

2.6.1.-

Imidazole-

acetole P

Phosphoribosyl-AMP

L-Hisyidinal

3.6.1.31

3.5.4.19

5.3.1.16

4.2.1.19

3.1.3.15

2.4.2.17

2.4.2.-

2.6.1.9

PRPP

Phosphoribosyl-

Formimino-AICAR-P

Imidazole-

Glicerol-3P

Phosphoriboxyl-ATP

L-Histidinol-P

1.1.1.23

5P Ribosyl-5-amino 4-

Imidazole carboxamide

(AICAR)

1-Methyl-

L-histidine

L-Hisyidinal

3.4.13.5

Aneserine

6.3.2.11

2.1.1.-

2.1.1.22

Purine metabolism

6.3.2.11

Carnosine

1.1.1.23

3.4.13.3

3.4.13.20

6.1.1

N-Formyl-L-

aspartate

Imidazolone

acetate

Imidazole-

4-acetate

Imidazole

acetaldehyde

Histamine

Hercyn

4.1.1.22

1.14135

3.5.3.5

3.5.2.-

1.2.1.3

1.4.3.6

4.1.1.28

L-Histidine

integrating pathway and expression data
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.