Download Proteomics

Term Report
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Next week
• Protein-protein interaction
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Yeast two-hybrid (Nature 2000)
TAP-tag + MS (Nature 2002)
FLAG-tag + MS (Nature 2002)
Pooling strategy (Science 1999)
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Now what ?
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576 strains, 20 growth conditions
Data, data, data….
Look for extremes ?
Cluster
1,2,3,4,…….……,8,9,10
– Group together similar patterns
– Mathematical description
• Co-expression
• Standard correlation coefficient
– Graphical representation
• Original experimental observation
• Color, dark and light
• Visualize and understand the relationships intuitively
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Data analysis
http://bioinfo.mbb.yale.edu/genome/phenotypes/
Growth conditions
Transformants
• Transformants
clustering
• Graphical
representation
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Data clusters
• 20 Growth conditions
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Double cluster
• Horizontal cluster: transformants
• Vertical cluster: growth conditions
• Identify assays for functionally related proteins
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Growth condition cluster
• Clustering growth conditions that result in similar phenotypes
• More effective screening functionally related proteins
Cell wall biogenesis
and maintenance
DNA metabolism
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Discovery Questions
• What advantage is there to clustering the
phenotypes in this manner?
• Some of the genes identified in this analysis had
no known function. How can clustering these data
help us predict possible functions?
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PCR-based Gene Deletion
• Deletion strategy
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http://www-sequence.stanford.edu/group/yeast_deletion_project/PCR_strategy.html
Primers
• 8~10 different primers/ORF
• High-throughput primer synthesis
– Primer-picking scripts
– Input:
• ORF data + UPTAG list
– Output:
• Primer sequences
• Automated Multiplex Oligonucleotide Synthesizer
• Homology to ORF upstream
• Common tag priming site (U1)
• UPTAG (20 bases)
• Common tag priming site (U2)
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homologous to 5’ to the Kan gene
Functional assays
• 6925 deletion strains of yeast constructed…
• 2026 ORF (1/3 of the genome)
• Screen for genes essential for viability
– Spores from heterozygous strains on YPD media at 30oC
• 356 haploid deletants could not be recovered
• 1620 ORFs not essential for viability in yeast
– Construct one additional homozygous and two haploid deletants
Essential gene: tall bar
Nonessential: short bar
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Competitive growth assays
• How to characterize the genes nonessential for viability?
• Pooled functional assay
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558 homozygous deletion strains were pooled
Grow in rich and minimal media for ~60 generations
Remove aliquots from the two pools at various time points
Tags were amplified and hybridized to DNA array
Red: grown for 0 hr
Green: grown for 6 hr
Normal growth (expression)
Grow slow (reduced expression)
Grow fast (enhanced expression)
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Correlation of growth rate
• Where is the wild type ?
In rich medium
r = 0.97
In minimal medium
r = 0.94
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Normalized growth rate
• Hybridization intensity = growth rate
– Normal growth = 1
– Grown fast (abundant) > 1
– Grown slow (fewer) < 1
In rich medium
p. 170, Q22:
Predict what might happen if
only the slowest growing strains
were incubated together.
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Comparison
• mTn method
• Pros:
• Cons:
• Bar code method
• Pros:
• Cons:
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Completed genome
• Unicellular eukaryotes
– Budding yeast, Saccharomyces cerevisiae
• Multicellular eukaryotes
– Nnematode, Caenorhabditis elegans
– Fruit fly, Drosophila melanogaster
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RNAi
• RNA interference by Andy Fire, 1998
• RNAi transiently inhibits the activity of a
target gene with a dsRNA
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RNAi and C. elegans
• C. elegans eats E. coli expressing specific dsRNA
• Observe phenotypes of adult and embryo
development
E. Coli with Gene A
B
C
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Functional Distribution
• Genes on chromosome I of C. elegans
Fraser et al., 2000 Nature 408, 325-330.
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Comparisons
• The British group
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Chromosome I
Bacterial expressed dsRNA
By feeding
Viability, and observable phenotypes
• The German group
– Chromosome III (cell division process)
– PCR amplified, in vitro transcription ssRNA, annealed to
generate dsRNA
– By microinjection
• RNAi strategy
– Pros
– Cons
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