Protein protein interactions

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Protein protein interactions
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Introduction
Protein b unknown
Protein a known
Interaction
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Introduction
Protein b Function b
Protein a Function a
Interaction
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Old world vs. new world
MAINLY IN THIS LECTURE
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Yeast two hybrid system Simple transcription
Transcription Complex
transcription
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Yeast two hybrid system Hybrid proteins
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Yeast two hybrid system Hybrid proteins
Transcription Complex
transcription
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Yeast two hybrid system plasmids
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Yeast two hybrid system plasmids
Yeast
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Yeast array producing the array
A comprehensive analysis of protein-protein
interactions in S. cerevisiae P. Uetz et al..

• ORF Open Reading Frames

• Produce the yeasts 6000 ORFs

• 2 colonies of each transformation are
• inserted to the array

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Yeast array producing the array
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Yeast array using the array

• Selection of 192 easy proteins

MATING creating diploids
SELECTION OF LIVING COLONIES BASED ON HIS3
PRODUCTION
DETECTING THE ARRAYS PROTEIN ACCORDING TO
ITS POSITION
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Yeast array - results
BEFORE (Pcf11)
AFTER
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Yeast array - Results over-view

• 2 undependable assays were preformed for each of
  the 192 proteins.
• 87 out of 192 proteins were detected as involved
  in protein-protein interactions (passed the 2
  assays)

• total of 281 interactions were detected

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Activation Domain library

• Production of an AD library

MATING (haploids to diploids)
Transferring to a selection plate
Detecting the ORFs using PCR
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Activation Domain library - results

• 817 out of 6000 proteins were detected as
  involved in protein-protein interactions

• total of 692 interactions were detected

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Array vs. library - Comparison
LIBRARY ARRAY
Rather quick Takes a lot of time Time
Huge number (6000X6000) Plenty (192X6000) Amount of checked interactions
1.8 3.3 Average number of Interactions for an interacting protein
14 48 Number of interactions of 12 proteins that were detected in both methods
CONCLUSIONS - THE METHODS QUALITIES
QUICK, SIMPLER, CHEAPER
SENSETIVE, BETTER RESULTS
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Protein arrays producing the array
Protein chips from concept to practice Young-Sam
Lee et al..

• Producing the yeasts 6000 ORFs using plasmids
• Attaching histidine anchors to every protein
• Attaching the proteins to an array

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Protein arrays using the array

• Pouring a protein onto the array

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Mass Spectrometry of purified complexes

• The complex is isolated using the tag

• Each protein is identified by Mass Spectrometry

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Mass Spectrometry of purified complexes
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Synthetic Lethal Mutations
Whats lethal mutation?

• Examining the creature carrying them

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Synthetic Lethal Mutations
Hypothesis these proteins are in interactions
METHOD 1 Synthetic Lethal Mutations

• Create an artificial DNA containing 2 genes with
  conditional mutations

• Change the conditions and detecting dead
  creatures

METHOD 2 Synthetic Lethal Mutations Arrays

• Pour different yeasts carrying different
  mutations

MATING creating diploids
STIMULATING THE CREATION OF SPORES SELECTION
FINDING THE DEAD
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Computational methods

• Mentioned in this seminar, mainly for
  understanding proteins
• Functions and using to detect interactions

Correlated mRNA expression

• Measuring mRNA levels under a variety of
  cellular conditions

• Grouping the genes that have similar
  transcriptional responses

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Computational methods Genome analysis IN SILICO
Prokaryotic's operons

• Genes that are consistently in the same operon,
  in the same order but in different and distanced
  creatures

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Computational methods Genome analysis
Phylogenetic profile

• Interacting proteins have a tendency to be
  either present ot absent
• together from fully sequenced genomes

Gene fusion
(ROSETTA STONE)

• One gene in creature A some genes in creature B

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Computational methods Genome analysis
Otology relationships are not so clear, not
always reliable