Nature Reviews Genetics

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Nature Reviews Genetics
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Yeasteukaryote model organism

• Eukaryote
• mitochondria,
• organelles,
• cell cycle, etc.
• Eukaryote Plus
• haploid, diploid,
• extra-chromosomal DNA.

Saccharomyces cerevisiae Bakers Yeast
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Yeast Genome Project

• Yeast Genome Project finished in 1996,
• 1.2 x 107 DNA base pairs,
• 16 chromosomes, 230 kb - 2, 352 kb,
• 6,000 Open Reading Frames (ORFs),
• Only 4 of the genes have introns,
• gt 70 of the genome is coding.

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Yeast Genome Projectvs. human genome

• 12.1 Mb Genomic DNA sequence (Human, 3,000 Mb)
• 70 coding sequence (Human, 1.8)
• Few Introns (Humans many)
• 6012 Genes (Human, 20-25,000)

About 70 of the genes found in humans, are found
in yeast.
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Known/Unknown(2001)

• 3,780 genes with some characterization
• 560 homologous with other organisms
• 1900 unknown

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Assigning Gene Function

• Geneticist gene sequence, expression, etc.
• Biochemist enzymatic function,etc.
• Cell Biologist cellular location, etc.
• - especially -
• Protein/DNA Interactions
• Protein/Protein Interactions
• Protein/Membrane Interactions
• etc.

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The Awesome Power of Yeast Genetics

• Homologous Recombination
• Transposons
• Life Cycle
• etc.

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Homologous Recombination

• the replacement of a gene with an exogenous gene
  through equal crossing over,

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Transposons
Someplace

• Transposons whole units of DNA that have the
  ability to insert themselves into DNA molecules,
• can carry other genes.

Inserts someplace else
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Hologous Recombination and Transposons

• Serve as shuttles to carry experimental DNA
  sequences into yeast,
• Regulatory sequences (promoters) drive the
  expression of,
• Mutant Genes for structure function analysis,
• Reporter Genes code for enzymes that signal
  their presence in specific cells,
• Epitope Tags proteins tagged with a foreign
  peptide sequence that binds to a specific
  antibody,
• etc.

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Reverse GeneticsFunctional Genomics
Function
Gene DNA Sequence
Phenotype Analysis
Gene Disruption
Development Physiology Cell Biology
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Fig. 1
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?

• Biology 470 WEB Page

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Transposon Down Sides
Up Sides?

• Insertions are essentially generated at random
• it is very difficult to mutagenize all genes
  within a genome by transposon mutagenesis alone,

• but really, transposon-specific biases in
  target-site selection,
• for reasons not fully understood, transposons
  such as Tn3 and Tn7 insert non-randomly into DNA.

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Site Directed Mutagenesis

• Systematic deletion of each ORF in the genome,
• homologous recombination replaces the gene with a
  selectable marker, and a DNA barcode,
• UPTAG,
• DOWNTAG.

Fig. 2
Whole set available1,500
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Fig. 1. Chemical genomic screening by using a
high-density cell array

• Of the 14 gene deletions that produce the
  rapamycin-enhanced phenotype, 13 genes have human
  homologs that showed gt30 identity (highly
  significant) at the protein level, and most of
  them encode mitochondrial proteins.
• Because mitochondrial dysfunction is known to
  underlie the pathogenesis of a wide range of
  neurodegenerative disordersour result suggests
  that rapamycin may be useful in preventing the
  progression of these diseases, including
  Alzheimer's, Parkinson's, and Huntington's
  diseases and brain aging.

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DNA Microarray

• DNA arrayed at high density on a solid substrate,
• In this experiment, DNA complementary to each
  ORFs UPTAG and DOWNTAG is arrayed in an ordered
  fashion.

http//www.bio.davidson.edu/courses/genomics/chip/
chip.html
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Homologous RecombinationUPTAG / DOWNTAG
Fig. 2a
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PCR Strategy
Big Primers
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Conditional Mutants
each strain has one gene KOd.
one strain each for gt5,900 genes.
Conditional Mutants mutants that have observable
phenotypes under a given set of growth conditions.
Fig. 2b
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DNA Protein Interactions Interactome 1
cont. next page
Fig 3.
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DNA Protein Interactions Interactome 1
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DNA Protein Interactions Interactome 1
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ProteomicsProtein-Protein Interactions

• Yeast Two Hybrid (Y2H),
• Protein Chips (not required),
• Mass Spectroscopy.

Signal Transduction Pathways, Heteromeric
Protein Complexes, Allosteric Interactions, etc.
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GAL4 Transcription Activatornative yeast
transcription factor
GAL4

• One Protein, Two Functional Domains
• BD Binding Domain,
• AD Activation Domain.

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Yeast Life Cycle
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Yeast Two Hybrid Vectors

• ...separate GAL4 Binding Domain and Activation
  Domain,
• ...create chimeric proteins, on expression
  vectors,
• Bait Gene fused to the Binding Domain Gene,
• Target (prey) Gene fused to the Activation Domain
  Gene.

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Yeast Two Hybrid Vectors
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• ...mate haploid cells, each expressing the
  recombinant proteins,
• one with bait,
• the other(s) with prey (target).

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No Interaction Bait/Prey

• ...bait binds DNA,
• ...prey does not associate with bait, or
  transcription machinery.

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Bait/Prey Interact

• ...bait binds DNA,
• ...prey associates with bait,
• ...activation domain is then in proximity to
  transcriptonal machinery,
• ...reporter gene turned on.

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Lots of Love Genetix

• High throughput screening,
• As many as 100,000 matings per day ,
• Automatic sample loading, reading and image
  analysis.

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Two Hybrid Analysis
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• Single Bait Strategy
• What interacts with the protein implicated in
  Huntingtons Disease?

PNAS 100(5)2712-2717
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Abstract

• Huntingtons disease (HD) is a neurodegenerative
  disease caused by polyglutamine (polyQ) expansion
  in the protein huntingtin (htt).
• Pathogenesis in HD seems to involve the formation
  of neuronal intranuclear inclusions and the
  abnormal regulation of transcription and signal
  transduction.
• To identify previously uncharacterized
  htt-interacting proteins in a simple model
  system, a yeast two-hybrid screen was used with a
  Caenorhabditis elegans protein expression
  library.

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Set-Up

• ...mate bait and prey cells, each expressing
  recombinant proteins,
• diploids that have restored GAL4 activated gene
  expression contain peptides that interact.

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Bait/Prey Interaction

• ...found a C. elegans protein (K08E3.3b) that
  interacts with N-terminal htt in two-hybrid tests.

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CIP4 in Human Brains

• A Normal, B ---gt D increasing Huntintons
  symptoms.
• Red Arrows represent CIP4 protein localization.
  Blue arrow points to brain lesions.

• A human homolog of the C. elegans K08E3.3b
  protein is the Cdc42-interacting protein 4
  (CIP4).
• Neuronal CIP4 immunoreactivity increased with
  neuropathological severity in the neostriatum of
  HD patients.

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CIP4 is Sufficient for HD Symptoms

• CIP4 protein was over expressed in rat brains.
  Cell death and Huntingtons Disease (HD)
  morphology resulted.

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The Skinnyand, how many species involved?

• Bait Human,
• Target (prey) C. elegans (roundworm),
• bait/target match found.
• C. elegans target gene has a human homolog cdc42
  interacting protein (CIP4),
• CIP4 found at high levels in HD patients brains,
• CIP4 sufficient to cause HD-like symptoms in rats.

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Y2H Weaknesses

• False Positives,
• some Baits are sticky, sticks to lots of
  Targets,
• some Targets are sticky, sticks to lots of
  Baits,
• fortuitous activation of marker promoter,
• usually assay for multiple markers,
• False Negatives,
• clone fidelity,
• protein conformation (especially membrane bound
  proteins),
• protein modifications (phosphorylation,
  glycosylation, etc.),
• Artifacts Y2H identified interactions require
  subsequent confirmation.

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Protein Arrays
Proteomics II
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Proteomics III

• Mass Spectrometry

Proteome Protein - Protein Interactions Protein
Complexes Peptide Sequencing etc.
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Mass Spectrometry

• Molecules to be analyzed, referred to as analytes
  are first ionized (usually in a vacuum),
• Newly charged (protonated) molecules are
  introduced into an electric and/or magnetic field
  in gas phase,
• Their path through the field is a function of the
  mass to charge ratio m/z,
• m/z of the ionized species can be used to deduce
  the mass of the analyte with high precision.

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Proteomics and Mass Spec
MALDI
Proteome Protein - Protein Protein
Complexes Peptide Sequencing etc.
ESI-MS
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Peptide Mass MappingMass Fingerprinting

• ...proteins are cleaved by proteolytic enzymes in
  a sequence specific manner,
• thus, each protein in a proteome has a unique
  peptide mass subset,
• these subsets can be computationally derived from
  protein databases, i.e via translated genomic DNA
  sequences,
• experimentally determined unknowns can be
  compared, via computers, to online databases for
  identification,
• ..scalable, multiple samples can be deposited at
  once, computers sort out the constituents.

One, and only one, 55.792 kD protein in the data
base w/ specific fragment pattern.
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Tandem Mass Spectroscopy(MS-MS)

• ...mass spectrometry can also be used to obtain
  sequence to identify peptides,
• treatment with sequence specific protelytic
  enzymes provides information on the terminal
  residues,
• the mass of the peptide fragment is determined,
• a short amino acid sequence from the peptide is
  obtained.

Often provides enough information to
unambiguously identify the entire protein when MS
data is compared to online databases.
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MS-MS

• MS 1 peptide fingerprinting is performed,
• peptides that have an appropriate mass for
  further study are isolated,
• MS 2 selected peptides are bombarded with argon
  gas, making random fractures in the peptide
  backbone, and mass spec is repeated,
• - the mass of each of these fragments is
  measured.

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Mass Difference Amino Acid Weight
693.37(EYL)1098.55

total peptide mass info

TQLYEYLQR
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Protein-Protein Interactions

• Interacting proteins are co-precipitated, and
  excised from 2-D Page gels
• gel slices are run through MS-MS,
• computers de-convolute slices with multiple
  proteins.

2-D Page
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Interaction Mapping

• Multiple proteins isolated in single gel slices
  are candidate interactors,
• Other experimental techniques are used to
  confirms interactions (including Y2H).

DNA Damage Repair Network
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Yeast Protein Interactome
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Questions

• Review

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Next

• Finish Up, Review
• Lectures online at my Course Materials Page.
• Read through pp. 579 of the Strategies Paper.