genetics 2581b final review
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Genetics 2581b Final Review. Brought to you by Western SOS. Lecture 18. KEY CONCEPTS: Transcriptome - Microarray Interactome – ChIP on Chip, 2 Hy brid Analysis Affinity Capture, Mass Spec 3. Proteome - ICAT 4. Phenome – Gene Knockouts. Transcriptome - Microarrays. - PowerPoint PPT PresentationTRANSCRIPT
Genetics 2581b Final Review
Brought to you by Western SOS
Lecture 18
• KEY CONCEPTS:1.Transcriptome - Microarray2.Interactome – ChIP on Chip, 2 Hybrid Analysis
Affinity Capture, Mass Spec
3. Proteome - ICAT4. Phenome – Gene Knockouts
Transcriptome - Microarrays
• A comparative analysis between two tissues or conditions
• A glass slide coated with ORFs• Cy3 and Cy5 stain each sample which is added to
slide
Interactome (DNA-Protein)ChIP on Chip
• Transcription factors bound covalently to DNA
• DNA then sheared• Sample is split in half, antibody to
specific protein added to one – other is control
• Complexes specific to protein of interest isolated
• Red dye incorporated into DNA• Spotted on intergenic array (probes
representing cis-control elements)
Interactome – (Protein- Protein)Two Hybrid Assay
• Genetically engineered strains of yeast often used• Determine unknown proteins that interact with a
protein of interest• Protein of interest 1 is bound to a bait domain• Second protein of interest bound to an activation
domain• If two proteins of interest interact bait and activation
also interact• Activates gene in yeast which is used to select for
interaction
Affinity capture and Mass Spectrometry
• Measure mass of proteins by determining migration rates of ionized form through an electric field
• Protein cleaved and ionized with laser• Run through vacuum and results analyzed by
computer which identifies proteins by comparing with database
• Further characterization also possible, run through collision cell and analyze hydrolyzed fragments
Isotope-coded Affinity Tag Approach
Phenome – Gene knockout
• Replace wild copy of a gene within yeast genome with selectable marker via homologous recombination
• Localizome – genetically engineer GFP onto sequence of interest and view using flourescence microscopy
Lecture 19
• Cancer – disruption in balance between cell proliferation and cell death
• DNA replication and repair are not perfect –c mutations in cell cycle occur
• Cancers are clonal descendents of a single cell• Is the result of multiple genetic leasions
Cell Cycle – Rao and Johnson
Looking for the M-phase promoting factor (MPF)Fused interphase nuclie with M-phase – induces
m-phaseFused G1 nuclie with S-phase – induces S-phaseS-phase with G2 – doesn’t cause S phase again
Indicates cycle has checkpoints for proper order
Cell Cycle Mutants in yeast• A mutations causes block in DNA division –
lethal• Mutations are conditional (will not go at
restrictive temp)• Cdc2 identified as inducer of mitosis• Associates with cyclin to form MPF
MPF
Cdc2Cyclin B
Microtubule- associated proteins
Histone H1
Lamins
Spindle assembly
Chromosome condensation
Nuclear envelope breakdown
Cyclin B
Ubiquitin molecules Proteasome
Cdc2
Cyclin dependent kinases
• One cdc2 and many cyclins – cdc2 phosphorylates many substrates throughout the cell which are determined by the bound cyclin
• Cell cycle controls are highly conserved – human cdc2 rescues mutant yeast cdc2
Lecture 20 • Rb-E2F pathways• A growth factor
inhibits p16 or p15 (cyclin dependent kinase inhibitor) allowing cycle to progress
• When CKIs not inhibited Rb is not phosphorylated
Oncogenes/Tumor repressor genes1. Proto-oncogene: normal, wild-type gene that positively
regulates cell proliferation
2. Oncogene: gene carrying a mutation (gain-of-function) in a positive regulator of cell proliferation, e.g. cell cycle regulators such as
• cyclin D (gene amplification), • cdk4 (insensitive to CKI inhibition)
3. Tumor Suppressor genes: genes which negatively regulate cell proliferation eg. cell cycle regulators such as p16 (CKI) and RB
Cell Cycle Checkpoints
p53 – Guardian of the Genome• Induces genes that negatively regulates cell cycle
and positively regulate apoptosis• Found mutated in half of human cancers• DNA damage activates p53
Applications of Expression Profiling Using Microarray Technology
• 1) Differentiate between different classes of cancer ie ALL and AML
• 2) Determining Resistance of tumors to different drugs
• 3) Predicting rate and extent of cancer progression (prognosis)