Genomics I: The Transcriptome

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Genomics IThe Transcriptome
RNA Expression Analysis
Determining genomewide RNA expression levels
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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

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Real-time PCR readout

• The readout of a real-time 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

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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

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Meta-analysis of Microarray Data
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Genomics IIThe Proteome

• Using high-throughput methods to identify
  proteins and to understand their function

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Contents

• Definition of proteomics
• Protein profiling
• 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

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What is proteomics?

• An organisms 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

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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

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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

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Posttranslational modifications

• Proteolytic cleavage
• Fragmenting protein
• Addition of chemical groups

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Chemical modifications

• Phosphorylation activation and inactivation of
  enzymes
• Acetylation protein stability, used in histones
• Methylation regulation of gene expression
• Acylation membrane tethering, targeting
• Glycosylation cellcell recognition, signaling
• GPI anchor membrane tethering
• Hydroxyproline protein stability, ligand
  interactions
• Sulfation proteinprotein and ligand
  interactions
• Disulfide-bond formation protein stability
• Deamidation proteinprotein and ligand
  interactions
• Pyroglutamic acid protein stability
• Ubiquitination destruction signal
• Nitration of tyrosine inflammation

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Protein ProfilingPractical 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 10,00020,000
• Protein expression signatures associated with
  drug toxicity
• To make clinical trials more efficient
• To make drug treatments more effective

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2-D gel electrophoresis

• Polyacrylamide gel
• Voltage across both axes
• pH gradient along first axis neutralizes charged
  proteins at different places
• pH constant on a second axis where proteins are
  separated by weight
• xy position of proteins on stained gel uniquely
  identifies the proteins

Basic
Acidic
High MW
Low MW
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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
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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

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Protein chips

• Thousands of proteins analyzed simultaneously
• Wide variety of assays
• Antibodyantigen
• Enzymesubstrate
• Proteinsmall molecule
• Proteinnucleic acid
• Proteinprotein
• Proteinlipid

Yeast proteins detected using antibodies
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Fabricating protein chips

• Protein substrates
• Polyacrylamide or agarose gels
• Glass
• Nanowells
• Proteins deposited on chip surface by robots

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Protein attachment strategies

• Diffusion
• Protein suspended in random orientation, but
  presumably active
• Adsorption/Absorption
• Some proteins inactive
• Covalent attachment
• Some proteins inactive
• Affinity
• Orientation of protein precisely controlled

Diffusion
Adsorption/ Absorption
Covalent
Affinity
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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

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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 proteinprotein interactions
• Annotating genome sequence

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Attaching a GFP tag to an ORF
GFP
HIS3MX6
PCR product
Homologous recombination
Chromosome
ORF1
ORF2
protein
COOH
NH2
GFP
Fusion protein
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Location of proteins revealed
cytoplasm

• 75 of yeast proteome localized
• gt 40 of proteins in cytoplasm
• 67 of proteins were previously unlocalized
• Localizations correlate with transcriptional
  modules

nucleus
A protein localized to the nucleus
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FlyTrap Screen for Protein Localization

• http//flytrap.med.yale.edu/

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Patterns of protein localization