1. Protein Interaction Maps and Model Organisms
Guang-Chao Chen
IBC, Academia Sinica

2. Genome sizes of humans and other organisms
Organism estimated size estimated gene average gene chromosome number density number
Homo sapiens (human) million bases ~30, gene per 100,000 bases
Rattus norvegicus (rat) million bases ~30, gene per 100,000 bases
Mus musculus (mouse) million bases ~30, gene per 100,000 bases
Drosophila melanogaster 180 million bases , gene per 9,000 bases
(fruit fly)
Arabidopsis thaliana million bases , gene per 4000 bases
(plant)
Caenorhabditis elegans million bases , gene per 5000 bases
(roundworm)
Saccharomyces cerevisia 12 million bases gene per 2000 bases
(yeast)
Escherichia coli (bacteria) 4.7 million bases gene per 1400 bases

3. Network Biology

4. Conventional approaches:
one or a few proteins
Functional genomic approaches:
tens of thousands of proteins
-microarrays (gene expression profile)
-large scale gene knockout (RNA-mediated interference)
-large scale protein localization (GFP)
-protein interaction maps

5. Protein interactions are crucial in many aspects
of biological function.

6. Genetic vs physical interaction maps

7. Two directions for proteomics in protein interaction mapping.
Gene (DNA)
Transcription
(RNA)
Protein
Genome
Transcriptome
Proteome
Forward
genetics
Reverse
genetics
Reverse
proteomics
Classical
proteomics

9. The life cycle of Saccharomyces cerevisiae

10. The Nobel Prize in Physiology or Medicine 2001
"for their discoveries of key regulators of the cell cycle"
Leland H. Hartwell R. Timothy Hunt Sir Paul M. Nurse

11. Yeast mutants with cell-cycle defects
Wild-type
Haploid meiosis/ pat1
cdc2
cdc28
Nat Rev Genet. 2001

12. Suppressor mechanisms
Nat Rev Genet. 2001

13. Cloning suppressors
ts-
ts+
Nat Rev Genet. 2001

14. A novel genetic system to detect protein–protein interactions
Nature 340, (20 July 1989); doi: /340245a0
   
A novel genetic system to detect protein–protein interactions
Stanley Fields & Ok-kyu Song

15. High-throughput two hybrid screen and interactome mapping

16. Stringent Y2H screening strategy

17. False-positives and false-negatives in Y2H

18. I. Validation of Y2H by orthogonal assays
Classification and validation of potential interactions.
I. Validation of Y2H by orthogonal assays
II. Interologues
III. Lirature

19. (EGF)
(Ras)
Y2H

20. Clustering analysis suggests the existence of a multiprotein complex
The synthetic multivulva (synMuv) genes in C. elegans

21. Boulton et al. (2002) Science

23. The Nobel Prize in Physiology or Medicine 1995
"for their discoveries concerning the genetic control of
early embryonic development"
Edward B. Lewis Christiane Eric F. Wieschaus
Nüsslein-Volhard

24. Second-site enhancers and suppressors

25. GAL4-dependent ectopic expression of a gene
(the GAL4-UAS system)
expression of
engrailed (en)
Brand and Perrimon 1993 Development

26. Clonal (mosaic) analysis
Nat Rev Genet. 2002

29. Global views of the protein-interaction map

30. Global views of the protein-interaction map

31. Local pathway views

32. Quality control in high-throughput protein interaction networks
- Existing protein complex
Reproducibility of interactions
Evolutionary conservation
- Functional classification
- Subcellular localization
- Gene expression

33. The awesome power of comparative interactomics

37. Limitations of the yeast two-hybrid system
interaction in the nucleus
not suitable for membrane proteins and large-size proteins
may not undergo posttranslational modification

38. Analysis of protein-protein interactions in vivo
B2H
Integral membrane protein
adenylate cyclase
- galactosidase
lactamase
GFP
luciferase
Miller (2005) PNAS
Karimova (2005) J Bacteriol.

39. Posttranslational modification Cytoplasmic interaction
- effective phosphorylation
- cytotoxicity
Prey fused with a
myristylation sequence
Guo (2004) Nature Biotechnol
Aronheim (2004) Mol Cell Biol

40. Epitope phage display

41. Protein-peptide interactions with random phage display

42. Zebrafish embryonic development
Haffter et al Development

43. The zebrafish toolbox Forward genetics Reverse genetics
- Chemical mutagenesis
- Insertional mutagenesis
Reverse genetics
- Morpholinos
- Tilling (Targeting Induced Local Lesions IN Genomes)
Expression profilling
- Whole-embryo in situ hybridization
- Gene chip
- Spotted microarrays

44. Chemical screens in whole, mutant zebrafish
gridlock
Small molecules that suppress gridlock circulation defects
Nat Rev Drug Discov. 2005

45. Zebrafish as a system for small-molecule screens
small molecular library
embryos/female
96 or 384-well assay plates
examine phenotypes (automated readout)
search library database
Nat Rev Drug Discov. 2005

46. RNA interference (RNAi) as a platform for
dissecting the function of independent genes
High-throughput genetics:
large-scale RNAi libraries and large number of cell-based assays

47. High-throughput RNAi screens by cell imaging.
Kiger et al J Biol.

48. Possible fates of mammalian cells in vitro
Hela neuronal cells T epithelial cells

49. Short hairpin RNA (shRNA)

50. Genome-wide screens in mammalian cells (RNAi)

51. Direct loss-of –function versus modifier screens
Nat Rev Genet. 2006