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Genomics I:
The Transcriptome
RNA Expression Analysis
Determining genomewide RNA
expression levels
Real-time PCR
• Sensitive means of
measuring RNA
abundance
• Not genomewide: used to
verify microarray results
• TaqMan method uses
fluorescently tagged
primers
• Fluorescent tag released
by Taq polymerase
Real-time PCR readout
• The readout of a realtime PCR reaction is
a set of curves
• The curves indicate
the PCR cycle at
which fluorescence is
detected
• Each cycle is twice
the amount of the
previous cycle
Genomic analysis of gene
expression
• Methods capable of giving a “snapshot” of RNA
expression of all genes
• Can be used as diagnostic profile
– Example: cancer diagnosis
• Can show how RNA levels change during
development, after exposure to stimulus, during
cell cycle, etc.
• Provides large amounts of data
• Can help us start to understand how whole
systems function
Meta-analysis of Microarray Data
Genomics II:
The Proteome
Using high-throughput methods to
identify proteins and to
understand their function
• Definition of proteomics
• Protein profiling
Contents
– 2-D gel electrophoresis
– Protein chips
• Protein-protein interactions
– Yeast two-hybrid method
– Protein chips
– TAP tagging/Mass spectrometry
• Biochemical genomics
• Using proteomics to uncover
transcriptional networks
What is proteomics?
• An organism’s proteome
– A catalog of all proteins
• Expressed throughout life
• Expressed under all conditions
• The goals of proteomics
– To catalog all proteins
– To understand their functions
– To understand how they interact with each
other
The challenges of proteomics
• Splice variants create an enormous
diversity of proteins
– ~25,000 genes in humans give rise to
200,000 to 2,000,000 different proteins
– Splice variants may have very diverse
functions
• Proteins expressed in an organism will
vary according to age, health, tissue, and
environmental stimuli
• Proteomics requires a broader range of
technologies than genomics
Diversity of function in splice
variants
• Example: the calcitonin gene
– Gene variant #1
• Protein: calcitonin
• Function: increases calcium uptake in bones
– Gene variant #2
• Protein: calcitonin gene-related polypeptide
• Function: causes blood vessels to dilate
Posttranslational modifications
• Proteolytic cleavage
– Fragmenting protein
• Addition of chemical groups
Chemical modifications
– Phosphorylation: activation and inactivation of
enzymes
– Acetylation: protein stability, used in histones
– Methylation: regulation of gene expression
– Acylation: membrane tethering, targeting
– Glycosylation: cell–cell recognition, signaling
– GPI anchor: membrane tethering
– Hydroxyproline: protein stability, ligand interactions
– Sulfation: protein–protein and ligand interactions
– Disulfide-bond formation: protein stability
– Deamidation: protein–protein and ligand interactions
– Pyroglutamic acid: protein stability
– Ubiquitination: destruction signal
– Nitration of tyrosine: inflammation
Protein Profiling:
Practical applications
• Comparison of protein expression in
diseased and normal tissues
– Likely to reveal new drug targets
• Today ~500 drug targets
• Estimates of possible drug targets: 10,000–20,000
• Protein expression signatures associated
with drug toxicity
– To make clinical trials more efficient
– To make drug treatments more effective
2-D gel electrophoresis
• x–y position of
proteins on stained
gel uniquely identifies
the proteins
Basic
Low MW
– pH gradient along first
axis neutralizes
charged proteins at
different places
– pH constant on a
second axis where
proteins are separated
by weight
Acidic
High MW
• Polyacrylamide gel
• Voltage across both
axes
Differential in gel
electrophoresis
• Label protein samples
from control and
experimental tissues
– Fluorescent dye #1 for
control
– Fluorescent dye #2 for
experimental sample
• Mix protein samples
together
• Identify identical
proteins from different
samples by dye color
with
benzoic
acid
Cy3
without
benzoic
acid
Cy5
Caveats associated with 2-D
gels
• Poor performance of 2-D gels for the
following:
– Very large proteins
– Very small proteins
– Less abundant proteins
– Membrane-bound proteins
• Presumably, the most promising drug targets
Protein chips
• Thousands of
proteins analyzed
simultaneously
• Wide variety of
assays
– Antibody–antigen
– Enzyme–substrate
– Protein–small
molecule
– Protein–nucleic acid
– Protein–protein
– Protein–lipid
Yeast proteins detected
using antibodies
Fabricating protein chips
• Protein substrates
– Polyacrylamide or
agarose gels
– Glass
– Nanowells
• Proteins deposited on
chip surface by robots
Protein attachment strategies
• Diffusion
– Protein suspended in
random orientation,
but presumably active
• Adsorption/Absorption
Diffusion
Adsorption/
Absorption
– Some proteins inactive
• Covalent attachment
Covalent
– Some proteins inactive
• Affinity
– Orientation of protein
precisely controlled
Affinity
Difficulties in designing protein
chips
• Unique process is necessary for
constructing each probe element
• Challenging to produce and purify each
protein on chip
• Proteins can be hydrophobic or hydrophilic
– Difficult to design a chip that can detect both
Subcellular localization of the yeast
proteome
• Complete genome sequences allow each
ORF to be precisely tagged with a reporter
molecule
• Tagged ORF proteins indicate subcellular
localization
– Useful for the following:
• Correlating to regulatory modules
• Verifying data on protein–protein interactions
• Annotating genome sequence
Attaching a GFP tag to an ORF
GFP
PCR product
HIS3MX6
Homologous
recombination
Chromosome
Fusion protein
ORF1
NH2
protein
ORF2
GFP COOH
Location of proteins revealed
• 75% of yeast
proteome localized
cytoplasm
nucleus
– > 40% of proteins in
cytoplasm
• 67% of proteins were
previously unlocalized
• Localizations
correlate with
transcriptional
modules
A protein localized
to the nucleus
FlyTrap
Screen for
Protein
Localization
http://flytrap.med.y
ale.edu/
Patterns of protein localization