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 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

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

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

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 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 x–y position of proteins on stained gel uniquely identifies the proteins BasicAcidic High MW Low MW

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 –Some proteins inactive Covalent attachment –Some proteins inactive Affinity –Orientation of protein precisely controlled Diffusion Adsorption/ Absorption Covalent 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 Fusion protein Chromosome PCR product COOH NH 2 Homologous recombination GFP HIS3MX6 ORF1 ORF2 protein GFP

Location of proteins revealed 75% of yeast proteome localized –> 40% of proteins in cytoplasm 67% of proteins were previously unlocalized Localizations correlate with transcriptional modules A protein localized to the nucleus nucleus cytoplasm

FlyTrap Screen for Protein Localization ale.edu/

Patterns of protein localization