Integrated Genomic and Proteomic Analyses of a Systematically Perturbed Metabolic Network

1. Integrated Genomic and Proteomic Analyses of a Systematically Perturbed Metabolic Network Science, Vol 292, Issue 5518, 929-934 , 4 May 2001

2. Availabilities: • Fully sequenced genome • DNA microarray for measuring the mRNA expression • Globally and quantitatively measuring protein expression • Two-hybrid system

3. Integrating diverse data • Assimilating data(types) into biological model • Predict cellular behavior • Signal transduction web

4. 4 distinct steps: • (step I) Define all of the genes in the genome and the subset of genes, proteins, and other small molecules constituting the pathwayof interest. If possible, define an initial model of the molecularinteractions governing pathway function, drawn from previous geneticand biochemical research.

5. Permease transporters epimerase galactokinase phosphoglucomutase uridylyltransferase

6. Gal Gal GAL3p GAL80p GAL80p GAL4p GAL6(LAP3) GAL4p

7. 4 distinct steps: • (step II) Perturb each pathway component through a series of genetic (e.g., gene deletions or overexpressions) or environmental(e.g., changes in growth conditions or temperature) manipulations.Detect and quantify the corresponding global cellular responseto each perturbation with technologies for large-scale mRNA- andprotein-expression measurement.

8. 6200 nuclear yeast genes for DNA microarrays • 997 genes identified by mRNA levels significantly difference • 16 cluster genes pooled by similar mRNA expression

12. ICAT (isotope-coded affinity tag) • Wt+gal (hot) wt-gal (cold) • Tandem mass spectrometry (MS/MS) • Trypsin, multidimensional chromatography • To examine differences in protein abundance • 289 genes product identified

14. 30 proteins displayed clear changes correlated with their mRNA counterpart • 15 proteins corresponding mRNA without significant change • Posttranscriptional regulation • Ribosomal-protein • 3~5 increase in mRNA but not protein products

15. 4 distinct steps: • (step III) Integrate the observed mRNA and protein responses with the current, pathway-specific model and with the global networkof protein-protein, protein-DNA, and other known physical interactions.

16. 2709 published list protein-protein interaction • 317 proteinDNA interactions recorde in the transcription-factor databases • 348 genes and 362 associated interactions in fig 4A.

18. Limited interaction correlation to every other affected genes by unknown interaction • Gal4p to identify putative interactions • Upstream Gal4p binding site • Cluster 1-3: 7 known, 9 unknown • YMR318C

19. 4 distinct steps: • (step III) Integrate the observed mRNA and protein responses with the current, pathway-specific model and with the global networkof protein-protein, protein-DNA, and other known physical interactions.

21. Neighbor and Drawtree program based on Euclidean distance • 997 significantly affected genes were analyzed by log10 mRNA expression rations

23. 4 distinct steps: • (step iv) Formulate new hypotheses to explain observations not predicted by the model. Design additional perturbation experimentsto test these, and iteratively repeat steps (ii), (iii), and (iv).

24. Gal7 and gal10 

26. Prove by gal1 gal10  strain

27. gal80 -gal • Gal4 gal80 double deletion • Gal2 gal80 double deletion