Transcript Proteomics
Term Report 1 Next week • Protein-protein interaction – – – – Yeast two-hybrid (Nature 2000) TAP-tag + MS (Nature 2002) FLAG-tag + MS (Nature 2002) Pooling strategy (Science 1999) 2 Now what ? • • • • 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 3 Data analysis http://bioinfo.mbb.yale.edu/genome/phenotypes/ Growth conditions Transformants • Transformants clustering • Graphical representation 4 Data clusters • 20 Growth conditions 5 Double cluster • Horizontal cluster: transformants • Vertical cluster: growth conditions • Identify assays for functionally related proteins 6 Growth condition cluster • Clustering growth conditions that result in similar phenotypes • More effective screening functionally related proteins Cell wall biogenesis and maintenance DNA metabolism 7 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? 8 PCR-based Gene Deletion • Deletion strategy 9 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) 10 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 11 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) 12 Correlation of growth rate • Where is the wild type ? In rich medium r = 0.97 In minimal medium r = 0.94 13 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. 14 Comparison • mTn method • Pros: • Cons: • Bar code method • Pros: • Cons: 15 Completed genome • Unicellular eukaryotes – Budding yeast, Saccharomyces cerevisiae • Multicellular eukaryotes – Nnematode, Caenorhabditis elegans – Fruit fly, Drosophila melanogaster 16 RNAi • RNA interference by Andy Fire, 1998 • RNAi transiently inhibits the activity of a target gene with a dsRNA 17 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 18 Functional Distribution • Genes on chromosome I of C. elegans Fraser et al., 2000 Nature 408, 325-330. 19 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 20