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Integrated Genomic and Proteomic Analyses of a Systematically Perturbed Metabolic Network. Science , Vol 292, Issue 5518, 929-934 , 4 May 2001. Availabilities:. Fully sequenced genome DNA microarray for measuring the mRNA expression Globally and quantitatively measuring protein expression - PowerPoint PPT Presentation
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Integrated Genomic and Proteomic Analyses of a Systematically Perturbed Metabolic Network
Science, Vol 292, Issue 5518, 929-934 , 4 May 2001
Availabilities:
Fully sequenced genome DNA microarray for measuring the
mRNA expression Globally and quantitatively measuring
protein expression Two-hybrid system
Integrating diverse data
Assimilating data(types) into biological model
Predict cellular behavior Signal transduction web
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 pathway of interest. If possible, define an initial model of the molecular interactions governing pathway function, drawn from previous genetic and biochemical research.
galactokinase
Permeasetransporters
epimerase
uridylyltransferase
phosphoglucomutase
GAL4p
Gal
GAL3pGAL80p
Gal
GAL80pGAL4p
GAL6(LAP3)
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 response to each perturbation with technologies for large-scale mRNA- and
protein-expression measurement.
6200 nuclear yeast genes for DNA microarrays
997 genes identified by mRNA levels significantly difference
16 cluster genes pooled by similar mRNA expression
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
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
4 distinct steps:
(step III) Integrate the observed mRNA and protein responses with the current, pathway-specific model and with the global network of protein-protein, protein-DNA, and other known physical interactions.
2709 published list protein-protein interaction
317 proteinDNA interactions recorde in the transcription-factor databases
348 genes and 362 associated interactions in fig 4A.
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
4 distinct steps:
(step III) Integrate the observed mRNA and protein responses with the current, pathway-specific model and with the global network of protein-protein, protein-DNA, and other known physical interactions.
Neighbor and Drawtree program based on Euclidean distance
997 significantly affected genes were analyzed by log10 mRNA expression rations
4 distinct steps:
(step iv) Formulate new hypotheses to explain observations not predicted by the model. Design additional perturbation experiments to test these, and iteratively repeat steps (ii), (iii), and (iv).
Gal7 and gal10
Prove by gal1 gal10 strain
gal80 -gal
•Gal4 gal80 double deletion
•Gal2 gal80 double deletion