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