Transcript INTEGRATION FROM PROTEINS TO ORGANS: THE PHYSIOME

Post-genomics
Post-genomics
Functional genomics
(A) Identifying genes from the sequence
(B) Gene expression profiling
(transcriptome)
(C) Model systems
Proteomics
Systems biology
(A) Hunting genes from the sequence
2 broad approaches
1) Ab initio method (computational)
2) Experimental method
1) Ab initio method (computational)
Scanning ORFs (open reading frames)
AAC
TAA
ATG
5’- ATGACGCATGATCGAGGAT –3’
3’ – TACTGCGTACTAGCTCCTA –5’
CTA
CCT
TCC
Ab initio method (computational)
 initiation or termination codons
 Codon bias found in specific species
Not all codons used at same frequency
e.g.human leucine mainly coded by CTG and
rarely by TTA or CTA
 Exon-intron boundaries (splice sites)
5’-AG GTAAGT-3’ hit and miss affair
 Upstream control sequences – e.g
conserved motifs in transcription factor
binding regions
 CpG islands
2) experimental method
Experimental evaluation based on the use of
transcribed RNA to locate exons and
entire genes from DNA fragment.
experimental method
Some strategies
 Hybridisation approaches – Northern
Blots, cDNA capture / cDNA select, Zoo
blots
 Transcript mapping: RT-PCR, exon
trapping etc
In this method, known DNA databases are
searched to find out whether the test
sequence is similar to any other known
genes, suggesting an evolutionary
relationship.
Northern Blot
Zoo Blot
(B) Gene expression profiling
Transcriptome
complete
collection
of
transcribed
elements of the genome (global mRNA
profiling)
transcriptome maps will provide clues on
•
•
•
•
•
Regions of transcription
Transcription factor binding sites
Sites of chromatin modification
Sites of DNA methylation
Chromosomal origins of replication
COMPUTATIONAL APPROACH
Homology searches (BLAST searches)
- Orthologous genes (homologues in
different organisms with common
ancestor) – comparative genomics
- Paralogous genes (genes in the
same organism, e.g. multigene
families)
- orphan genes / families
The transcriptome
Analysis can be done by either
Microarray technology
SAGE (serial analysis of gene expression)
technology
(a) Schematic drawing of a DNA chip.
Microarray
(chip)
Microarrays
Segment of
a chip
A
G
G
A
C
G
T
Spot containing copies
of a single DNA
molecule
(b) The analysis of the hybridization process identifies
genes that respond in specific ways.
cCNA from
untreated
cells
Cell samples are stabilized
and fluorescent labels
are added.
T A
C G
C G
T A
G C
C G
A T
DNA
bases
Part of one
DNA strand
Pair of
complementary
bases
C G
C G
C G
G A
G C
A T
T A
chip DNA
T A T A
T A T A
A T A T
A T A T
G C G C
C G C G
G C G C
Examples
of reactions
(c) Computer analysis of the binding of complementary
sequences can identify genes that respond to drug
treatment.
Gene that strongly increased
activity in treated cells
Gene that strongly decreased
activity in treated cells
Gene that was equally active
in treated and untreated cells
Gene that was inactive
in both groups
cDNA
from
treated
cells
MODEL SYSTEMS
gene inactivation methods
(knockouts, RNAi, sitedirected mutagenesis,
transposon tagging, genetic
footprinting etc)
Gene overexpression methods
(knock-ins, transgenics,
reporter genes)
RNAi
RNAi mimics loss-offunction mutations
Non-inheritable
Lack of reproducibility
How does RNAi work?
http://www.nature.com/focus/rnai/animations/
index.html
MODEL SYSTEMS
Gene overexpression methods (knock-ins,
transgenics, reporter genes etc)
Proteomics
Analysis of protein expression
Protein structure
Protein-protein interactions
Nature (2003) March 13: Insight articles from pg 194
Proteomics
Proteome projects - co-ordinated by the HUPO
(Human Protein Organisation)
Involve protein biochemistry on a highthroughput scale
Problems
limited and variable sample material,
sample degradation,
abundance,
post-translational modifications,
huge tissue, developmental and temporal
specificity as well as disease and drug
influences.
Nature (2003) March 13: Insight articles from pgs 191-197.
Approaches in proteomics
High throughput approach
1) Mass- spectrometry
2) Array based proteomics
3) Structural proteomics
Nature (2003) March 13: Insight articles from pgs 191-197.
High throughput approaches in proteomics
1) Mass spectrometry-based proteomics:
relies on the discovery of protein
ionisation techniques.
used for
 protein identification and
quantification,
 profiling,
 protein interactions and
 modifications.
Nature (2003) March 13: Insight articles from pgs 191-197
Identification of proteins in complex
mixtures
two dimensional gels and mass spectrometry
two dimensional gels
19_09.jpg
Mass spectrometry (MS)
Nature (2003) March 13: Insight articles from pgs 191-197
Principle of MS
ionizer source: converts analyte to gaseous ions
mass analyser: measures mass-to-charge ratio
(m/z)
detector: registers the number of ions at each m/z
Types of ionizer sources
Electrospray ionisation (ESI)
matrix-assisted laser desortion/ionisation
(MALDI)
MALDI-MS - simple peptide mixtures whereas
ESI-MS - for complex samples.
Nature (2003) March 13: Insight articles from pgs 191-197.
2) Array-based proteomics
Based on the cloning and amplification of
identified ORFs into
homologous (ideally used for bacterial and
yeast proteins) or sometimes
heterologous systems (insect cells which
result in post-translational
modifications similar to mammalian
cells).
A fusion tag (short peptide or protein
domain that is linked to each protein
member e.g. GST) is incorporated
into the plasmid construct.
Nature (2003) March 13: Insight articles from pgs 191-197.
Array based proteomics….
a. Protein expression and purification
b. Protein activity: Analysis can be done using
biochemical genomics or
functional protein microarrays.
c. Protein interaction analysis
two-hybrid analysis (yeast 2-hybrid),
FRET (Fluorescence resonance energy
transfer),
phage display etc
d. Protein localisation:
immunolocalisation of epitope-tagged
products.
E.g the use of GFP or luciferase tags
Nature (2003) March 13: Insight articles from pgs 191-197.
Array based proteomics….
Protein chips
Antibody chips – arrayed antibodies
Antigen chips – arrayed antigens
Functional arrays – arrayed proteins
Protein capture chips – arrayed capture
agents that interact with proteins e.g.
BIAcore
Solution arrays – nanoparticles
Nature (2003) March 13: Insight articles from pgs 191-197.
3) Structural proteomics
19_14.jpg
3) Structural proteomics
19_14.jpg
Identification of proteinprotein interactions
affinity capture/mass
spectrometry
Fig. 10. 31
Identification of protein-protein interactions
Phage display
Fig. 10.32
Systems Biology – the global study of multiple
components of biological systems and their
interactions
• New approach to studying biological
systems has made possible
– Sequencing genomes
– High-throughput platform development
– Development of powerful
computational tools
– The use of model organisms
– Comparative genomics
Six steps in systems approach
• Formulate computer based model for the system
• Discovery science to define as many of the
system’s elements as possible
• Perturb the system genetically or
environmentally
• Integrating levels of information form
perturbations
• Formulate hypothesis to explain disparities
between model and experimental data
• Refine the model after integrating data
19_20.jpg
Systems biology approach to studying how Halobacterium
NRC-1 transcriptome responds to uv radiation
Nitin S. Baliga et al. Genome Res. 2004; 14: 1025-1035
The systems biology approach to studying
how yeast turns genes on and off in the
utilization of galactose
Fig. 10.34
Fig. 10.35
Human Genome Project has changed
the potential for predictive/preventive
medicine
• Provided access to DNA polymorphisms underlying
human variability
– Makes possible identification of genes
predisposing to disease
– Understanding of defective genes in context of
biological systems
– Circumvent limitations of defective genes
• Novel drugs
• Environmental controls
• Approaches such as stem-cell transplants or
gene therapy
Challenges for the future – ‘physiome’
Nature Reviews Molecular Cell Biology 4; 237-243 (2003)
General Reading
– Chapter 19- HMG3 by Strachan and Read
Reference
- Nature (13 March 2003). Proteomics insight
articles from Vol. 422, No. 6928 pgs 191197