Genetics 2581b Final Review

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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)