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Term Report

Term Report. Next week. Protein-protein interaction Yeast two-hybrid ( Nature 2000 ) TAP-tag + MS ( Nature 2002 ) FLAG-tag + MS ( Nature 2002 ) Pooling strategy ( Science 1999 ). 1,2, 3,4 , …… . …… , 8,9,10. Now what ?. 576 strains, 20 growth conditions Data, data, data … .

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Term Report

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  1. Term Report

  2. Next week • Protein-protein interaction • Yeast two-hybrid (Nature 2000) • TAP-tag + MS (Nature 2002) • FLAG-tag + MS (Nature 2002) • Pooling strategy (Science 1999)

  3. 1,2,3,4,…….……,8,9,10 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

  4. http://bioinfo.mbb.yale.edu/genome/phenotypes/ Data analysis Growth conditions • Transformants clustering • Graphical representation Transformants

  5. Data clusters • 20 Growth conditions

  6. Double cluster • Horizontal cluster: transformants • Vertical cluster: growth conditions • Identify assays for functionally related proteins

  7. Cell wall biogenesis and maintenance DNA metabolism Growth condition cluster • Clustering growth conditions that result in similar phenotypes • More effective screening functionally related proteins

  8. 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?

  9. PCR-based Gene Deletion • Deletion strategy http://www-sequence.stanford.edu/group/yeast_deletion_project/PCR_strategy.html

  10. 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

  11. 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

  12. Normal growth (expression) Grow slow (reduced expression) Grow fast (enhanced expression) 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

  13. Correlation of growth rate • Where is the wild type ? In rich medium In minimal medium r = 0.97 r = 0.94

  14. 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.

  15. mTn method Pros: Cons: Bar code method Pros: Cons: Comparison

  16. Completed genome • Unicellular eukaryotes • Budding yeast, Saccharomyces cerevisiae • Multicellular eukaryotes • Nnematode, Caenorhabditis elegans • Fruit fly, Drosophila melanogaster

  17. RNAi • RNA interference by Andy Fire, 1998 • RNAi transiently inhibits the activity of a target gene with a dsRNA

  18. E. Coli with Gene A B C RNAi and C. elegans • C. elegans eats E. coli expressing specific dsRNA • Observe phenotypes of adult and embryo development

  19. Functional Distribution • Genes on chromosome I of C. elegans Fraser et al., 2000 Nature 408, 325-330.

  20. 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

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