Download Li, 2004

A map of the interactome network
of the metazoan C. elegans.
Li S, Armstrong CM, Bertin N, Ge H, Milstein S, Boxem M, Vidalain PO, Han JD, Chesneau A, Hao T, Goldberg DS, Li N,
Martinez M, Rual JF, Lamesch P, Xu L, Tewari M, Wong SL, Zhang LV, Berriz GF, Jacotot L, Vaglio P, Reboul J,
Hirozane-Kishikawa T, Li Q, Gabel HW, Elewa A, Baumgartner B, Rose DJ, Yu H, Bosak S, Sequerra R, Fraser A,
Mango SE, Saxton WM, Strome S, Van Den Heuvel S, Piano F, Vandenhaute J, Sardet C, Gerstein M, Doucette-Stamm L,
Gunsalus KC, Harper JW, Cusick ME, Roth FP, Hill DE, Vidal M.
Science. 2004 Jan 23;303(5657):540-3
MEDGEN 505
Jan Stöpel
Goal
How do protein-protein interactions relate
to multicellularity?
Approach
1. Select candidate genes
2. Use high-throughput Y2H to screen for interactions
3. Verify coverage
4. Verify accuracy of interaction
5. Build network
6. …future use
Candidate genes by BLAST
ancient
S. cerevisae
C. elegans
A. thaliana
D.melanogaster
H. sapiens
worm
multicellular (3024)
Making Y2H constructs
Used Invitrogen Gateway® system
utilizes  phage recombination
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
• fast
• automated
• allows large scale
tagging and plasmid shuffling
Yeast 2 hybrid
If proteins interact,
don’t interact,
reporter
reporter
gene isgene
switched
remains
on inactive
and strain can grow
not grow
on selective
on selective
media
media
QuickTime™
QuickTime™and
andaa
TIFF
TIFF(Uncompressed)
(Uncompressed)decompressor
decompressor
are
areneeded
neededtotosee
seethis
thispicture.
picture.
lost 105 candidates due to autoactivation or toxicity
Screen
1873 Gal4-BD constructs
Vs.
AD-wormcDNA
contains ~11000 ORFs
AD-yfg multiple junctions
AD-ORFeome
contains ~11000 ORFs
AD-yfg single junction
Screen
858
1299
1892
Results
#
interactions
∑
DB-X/AD-Y
Core-1/ Core2
858/1299
2135
502/1039
NonCore
1892
1892
531/1395
ADworm/
AD-ORF
2783/
1505
4027
239
(overlap)
Coverage
coverage efficiency: ~
10%
Literature search for known and verified interactions of any
ORF used as bait.
108 interactions
AD-wormcDNA+ AD-ORFeome
DB-X (literature)
(8+2)/108
Verification
using GST-bait and Myc-prey fusion
in Myc immunoprecipitation
accuracy
82%
59%
35%
Building a network
sources:
HT-Y2H
literature search
interlogs
scaffolds
(4027)
(108)
(949)
(?)
Interlogs: in silico approach to identify conserved interactions
Y2H data set from S.cerevisiae BLASTed against C.elegans proteome
scaffolds: Y2H data sets for biological processes of C.elegans
protein degradation, vulval development, DNA damage response, germline formation
Evolution is using old and
new
748
1314
836
Transcriptome vs Interactome