Lecture 5:
Challenges in the postgenomic era
The tiger leg leaf frog
Photo: Zig Leszccynski
Image: courtesy Rainforest Alliance
Are genome projects the end?
Genomics & Genetics Resources
Human Genome
Project:
Copy
Number
Variation:
To access the Human Genome Project
(HGP ) research programme resources
here at the WTS I
The CNV project examines
copy number changes in
the population
Cancer Genome
Project:
Genomic
Microarrays:
Human
Epigenome
Project (HEP):
Understanding malaria
from basic biology to
practical applications
Provides an epigenetic resourc e of
genome-wide DNA methylation
reference profiles in human tissues and
cell lines.
Pathogen Arrays: MICER:
Comparative genomics and expression Mutagenic Insertion and
profiling of small genomes
Chromos ome Engineering
Resource
Molecular
ENCODE:
Cytogenetics:
Identifying somatically acquired
Detailed analysis of
sequence variants/mutations and hence genomic copy number
changes in tumours
genes critical in the development of
human cancers
Immunogenomics: Microarray
Facility:
Genetics and epigenetics of the
immune subgenome and their roles in
evolution and dis ease
Molecular
Cytogenetics:
Development and use of
high-density arrays for
expression profiling
Molecular
Genetics &
Proteomics:
Chromos ome organisation and
structure in human disease
Uses of human
chromos ome 22 as a
model system for genomic
Malaria:
Molecular
DECIPHER:
Genetics &
Proteomics:
Encyclopedia Of DNA Elements project DatabasE of
sets out to
identify
all functional
Chromos omal Imbalance
Chromos
ome
organisation
and
elements in
in human
the human
genome
and Phenotype in
structure
disease
sequence
Humans
Ensembl
Uses of using
human
Resources
chromos ome 22 as a
model system for genomic
analysis
Mouse Genomics: Genetrap:
Investigations of the function of genes
using the mouse as model genetic
system
Sanger Institute Gene
Trap Resource
Fission Yeast
Functional
Genomics:
Atlas of
Gene
Expression:
Research focuses on genome-wide
gene expression profiles and
regulatory networks using DNA
microarrays and molecular genetics
Describing different cell
types in adult/embryonic
tissues alongside a
description of ex pression
level/localisation of protein
products
http://www.sanger.ac.uk/PostGenomics/
Post-genomics
Functional genomics
(A) Identifying genes from the sequence
(B) Gene expression profiling
(transcriptomics)
(C) Model systems
Proteomics
Systems biology
(A) Hunting genes from the sequence
2 broad approaches
1) Ab initio method (computational)
 Codon analysis
 Regulatory regions (TF binding sites, CpG
islands etc)
 Exon-intron boundaries
2) Experimental method
 Hybridisation approaches – Northern Blots,
cDNA capture / cDNA select, Zoo blots
 Transcript mapping: RT-PCR, exon trapping
etc
Northern Blot
Zoo Blot
(B) Gene expression profiling
Transcriptome
complete
collection
of
transcribed
elements of the genome (global mRNA
profiling)
transcriptome maps provide clues on
•
•
•
•
•
Regions of transcription
Transcription factor binding sites
Sites of chromatin modification
Sites of DNA methylation
Chromosomal origins of replication
The transcriptome
Analysed by DNA Microarrays
Advantages:
high-throughput information
Gene expression profile of
the cell/tissue
problems
false –positives
data analysis
Cost
Microarrays….
(C) 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 and function
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:
Nobel prize in Chemistry (2002)
John B. Fenn
Koichi Tanaka
Kurt Wüthrich
"for the development of methods for identification and structure
analyses of biological macromolecules"
"for their development of soft desorption ionisation methods for mass
spectrometric analyses of biological macromolecules"
"for his development of nuclear magnetic resonance spectroscopy for
determining the three-dimensional structure of biological
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
8HDF / MTHF?
19_14.jpg
FAD
Modelling of a novel photolyase based on sequence
Winnie Wu
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 approaches to studying biological systems
– Sequencing genomes
– High-throughput platform development
– Development of powerful computational
tools
– The use of model organisms
– Comparative genomics
19_20.jpg
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 from
perturbations
• Formulate hypothesis to explain disparities
between model and experimental data
• Refine the model after integrating data
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
Challenges for the future – ‘
Genomics – CNV’s in medicine
‘Physiome’
Translational medicine
• General Reading
– Chapter 19- HMG3 by Strachan and Read
Reference
• Science 9 Feb 2007 Vol. 315. no. 5813, pp. 848 – 853
(CNV report)
• Nature (13 March 2003). Proteomics insight articles
from Vol. 422, No. 6928 pgs 191-197
• Crit Rev Biotechnol. 2007 Apr-Jun;27(2):63-75 (good
current review on challenges in transcriptomics
/proteomics)
Resource:
http://www.sanger.ac.uk/PostGenomics/