Download Modern methods in biology

Modern methods in biology
From reductionism to systems biology
Nature, 25 April, 1953
Nature, 15 February, 2001
Zoi Lygerou, University of Patras, Greece
Mark Perutz (Nobel price 1962)
Human Genome Project
US Department of Energy
U.S. Department of Energy Genome Program's Genome Management Information System (GMIS)
James Watson
Francis Collins
Sequencing Centers
The Welcome Trust Sanger Institute (UK)
National Human Genome Research Institute
Whitehead Institute for Biomedical Research (USA)
Washington University Genome Sequencing Center
Baylor College of Medicine Human Genome Sequencing Center
RIKEN Genomic Sciences Center (Japan)
Genoscope and CNRS (France)
Genomes to Life Program Roadmap, April 2001, DOE/SC-0036, U.S. Department of Energy Office of Science,
http://genomicsgtl.energy.gov/
From reductionism to
The …omics approach
A holistic view into cells and organisms…
Gene
mRNA transcripts
Protein
Metabolites
Genome
Transcriptome
Proteome
Metabolome
Interactions
Cell
Phenotype
Biological time
Reaction
Interactome
Cellome
Phenome
Chronome
Reactome
Systems biology
Genomics
Transcriptomics
Proteomics
Metabolomics
(but also Metabonomics….)
Interactomics
Cellomics
Phenomics
Chronomics
Reactomics
Fluxomics
Pharmacogenomics
New approaches – novel methodologies
High-throughput research
Quantitative data
Computer assisted data analysis,
safekeeping and distribution
Bioinformatics
Databases
Modeling
The virtual cell
Interdisciplinarity
The race for the sequence: of patents and men
Private companies join the race…
And introduce new norms…
Celera
Graig Venter
In the US,
an estimated one fifth of human genes are
patented…
Systems biology
"The whole is more than the sum of its parts”
Aristotle
Studies a biological system as a whole
rather than focusing on its components
Integration rather than reduction
Requires high-throughput and/or quantitative data on
component interactions and interdependencies
employs theoretical analysis and modeling
Is highly interdisciplinary
New approaches – novel methodologies
Microarrays (Chips)
DNA microarrays
Protein micorarrays
Tissue microarrays
Chemical compound microarrays
DNA microarrays
Gene expression profiling
Data analysis
Image analysis
Image
Statistical analysis
Clustering
relationships
Heatmap
Biological
Hypothesis
Comparative genomic hybridization
Chip-CGH
Genome arrays (full genome: tiling arrays, or part of)
Answer the question: how many copies of each region
of the genome are present in my cells of interest?
Pathological situations (cancer)
During a physiological process (replication)
Chip-CGH to identify replication origins genome-wide
Where does replication start from across the genome?
How often does it start from each site?
[1] C. Heichinger, J. Penkett, J. Bahler and P. Nurse. Genome-wide characterization of fission
yeast DNA replication origins, EMBO, 25(21), 2006.
1. Isolate genomic DNA
a. from cells in S-phase
3. Analyse data to identify
regions over-rerepresented in a
replicating cell
b. from non-replicating cells
2. Hybridize to full genome tiling array
Origin mapping
[1] C. Heichinger, J. Penkett, J. Bahler and P. Nurse. Genome-wide characterization of fission
yeast DNA replication origins, EMBO, 25(21), 2006.
Proteomics
The study of all proteins in a cell, organ or organism
under specific conditions
The proteome is a dynamic not a static entity
Protein
DNA
1. Functional proteomics:
What is the function of each product of the 30,000 human genes
- where do protein localize
- what do they interact with
- how are they modified
Interactomics
2. Expression proteomics
What differences in protein expression levels accompany certain
Aspects of a cell’s physiology? (normal or pathological)
3. Structural proteomics:
What is the 3D structure of proteins and multi-protein machines?
4. Clinical proteomics:
Applications of proteomics to the clinical practice
in the early (or prosymptomatic) diagnosis
in choice of treatment (personalized)
in patient follow-up
The interactome…
Expression proteomics
Separation, quantification and identification
of large numbers of proteins from biological specimens
2D gel electophoresis
Mass Spec analysis
Large scale perturbations of gene function
The mouse gene knock-out project
EMBL Moterotondo
A facility with 35,000 mice
Not far from here…
Functional imaging
in live cells, organs and organisms
Green Fluorescent Protein: an autofluorescent protein
from the jelly fish Aequorea victoria
GFP and its variants permit observation of proteins within
living cells…
or organisms
And allow protein function and interactions to be assessed
FRAP (Fluorescence Recovery After Photobleaching)
Protein mobility within the nucleus
Pre-bleach
t=0
t=0.078sec
t=0.390sec t=0.780sec
t=1.560sec
t=3.12sec
t=6.24sec
ntensity
Norm.rel.intens
Norm.rel.I
1.2
1
0.8
GFPnls
Cdt1GFP
Cdt1del1-140nls
0.6
0.4
0.2
0
0
5
10
15
20
25
Time(sec)
A read-out of protein interactions
Modelling of molecule movement and simulation
enables identification of parameters
such as kon, koff and diffusion coefficient
Assessing protein-protein interactions in live cells
by Fluorescence Resonance Energy Transfer (FRET)
and Fluorescence Lifetime Imaging Microscopy (FLIM)
Α
Α
Β
Excitation
Emission
RFP
GFP
ET
R
F
Μ
Δφ
GFP
G
Μ
RFP
Phase lifetime
Cdt1GFP
1.6
GemdhcRed
GemΔ90-120
Mod. lifetime
2.8 1.8
Δφ
3.0
Phase lifetime
Cdt1
Geminin binds to Cdt1 throughout the nucleus in live cells
Cdt1GFP
+GeminindhcRed
Merged
+GemΔ90-120
Merged
Mod. lifetime
Cdt1GFP
Assessing relative Kd in vivo
Peter Verveer,
P. Bastiaens lab
MPI Dortmund
Quantification of protein-protein interactions in single live cell
In vivo binding isotherms
Relative binding affinities in cells
Quantitative Information
Modelling of Biological Systems
"Now this is not the end.
It is not even the beginning of the end.
But it is, perhaps, the end of the beginning."
Winston Churchill, 1942
Further reading
For an overview of the human genome project…
http://www.ornl.gov/sci/techresources/Human_Genome/project/info.shtml
Animations on Microarrays
and Mass Spec…
http://multimedia.mcb.harvard.edu/
Plus the full, narrated version of the Inner Life of a Cell!
More on functional imaging
Van Roessel P., Brand AH
Imaging into the future: visualizing gene expression and protein interactions
with fluorescent proteins. Nat Cell Biol. 2002;4(1):E15-20. Review