1 Term Report Term Report
Dec 30, 2015
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Term ReportTerm ReportTerm ReportTerm Report
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Next week
• Protein-protein interaction– 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 ?
• 576 strains, 20 growth conditions• Data, data, data….• Look for extremes ?• Cluster
– 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
1,2,3,4,…….……,8,9,10
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Data analysis
• Transformants clustering
• Graphical representation
Growth conditions
Transformants
http://bioinfo.mbb.yale.edu/genome/phenotypes/
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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
DNA metabolism
Cell wall biogenesis and maintenance
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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
http://www-sequence.stanford.edu/group/yeast_deletion_project/PCR_strategy.html
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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) homologous to 5’ to the Kan gene
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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
– 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
In rich medium
• Hybridization intensity = growth rate– Normal growth = 1– Grown fast (abundant) > 1– Grown slow (fewer) < 1
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
– 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