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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.

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genetics 2581b final review

Genetics 2581b Final Review

Brought to you by Western SOS

lecture 18
Lecture 18
  • Transcriptome - Microarray
  • Interactome – ChIP on Chip, 2 Hybrid Analysis

Affinity Capture, Mass Spec

3. Proteome - ICAT

4. Phenome – Gene Knockouts

transcriptome microarrays
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
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
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
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
phenome gene knockout
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
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
Cell Cycle – Rao and Johnson

Looking for the M-phase promoting factor (MPF)

Fused interphase nuclie with M-phase – induces m-phase

Fused G1 nuclie with S-phase – induces S-phase

S-phase with G2 – doesn’t cause S phase again

Indicates cycle has checkpoints for proper order

cell cycle mutants in yeast
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
cyclin dependent kinases
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
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 genes
Oncogenes/Tumor repressor genes
  • Proto-oncogene: normal, wild-type gene that positively regulates cell proliferation
  • 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)
  • Tumor Suppressor genes: genes which negatively regulate cell proliferation eg. cell cycle regulators such as p16 (CKI) and RB
p53 guardian of the genome
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
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)