Transcript blumberg-lab.bio.uci.edu

Presented by Evelia Ayala and Muaeen Obadi
Interactome
•Interactome: the set of protein-protein
interactions that occur in a cell
•Molecular interactions mediate
biological functions that maintain
normal cellular activities
Li et al., 2004
Why focus on the interactome?
● large amounts of data produced by genomic
projects
● fully sequenced genomes are loaded with novel
genes
● identification of interactions would contribute to
functional interpretation of genome
Why use two hybrid screening?
● limitations of other methods
● functional prediction
- functions of characterized proteins give insight into novel
proteins
● emphasis on complementary approaches
● yeast-two hybrid system was the only well-established
genomic approach for a genomewide scale analysis
Two Hybrid screening
● method to detect protein-protein interactions
● transcription factor split into two fragments:
bait
protein
prey
protein
binding domain (BD) & activation domain (AD)
● each fragment is fused to a different protein
BD: bait protein & AD: prey protein
● if the two proteins interact, the BD & AD bind
to allow for transcription of reporter gene
Pandey & Mann, 2000
Methods
● Open reading frames (ORFs) individually
cloned into bait expression vector & prey
expression vector
● each bait plasmid introduced into MATa
cell
● each prey plasmid introduced into MATα
cell
bait
vector
prey
vector
transform
MATa
MATα
Methods (cont.d)
● mate MATa & MATα haploid cells
● diploid cells will have active Ade,
His, & Ura reporter genes
● positive clones will survive on
media lacking adenine, histidine,
and uracil
● amplify inserts to obtain sequence
tags
Ade
His
Ura
His
- Ade
- His
- Ura
Pandey &
Mann, 2000
Outline of extensive two-hybrid analysis
● transformants divided
into bait & prey pools
● pools systematically
mated
● positive clones selected
● sequence tagging to
obtain interaction
sequence tags (ISTs)
Results (condensed)
-
ISTs: pair of tag sequences for bait & prey
Comparing results
-
comparing with an independent yeast two-hybrid project that used different
strategies
The Overlap in results
● They only share a small
overlap of interactions.
● The known interactions
from the YPD were
shared significantly
higher between data sets.
Genome Wide Two Hybrid Interaction Map
● Removal of redundancy
led to the core data.
● To confirm no background
noise they analyzed dataset
from YPD.
● Confirmed large cluster
of interactions.
Interaction Networks
The positives of the approach
● For genome wide interaction mapping this is the
most feasible approach.
● Hypothetical networks arise which become of
interest for studies.
● Better understanding of cellular processes can
be developed by testable hypothesis of these
networks.
The cons of the approach
● Dataset from different projects lack the
ability to provide large overlap.
● In the nature of large scale screenings,
interactions tend to escape detection.
● The two hybrid method itself has the concern
of reliability.
Topics of Discussion
● The project fails to restate ~90% of the two hybrid
interaction identified in conventional studies.
● These protein to protein interaction have been added the
the database creating large networks. The need for
bioinformatics to read these networks has become
necessary.
● To avoid complexes sharing protein components in silico
there needs be data collected on architecture and their
occurrence.
Further Readings
-
Ito, T., Tashiro, K., Muta, S., Ozawa, R., Chiba, T., Nishizawa, M.,
Yamamoto, K., Kuhara., Sakaki, Y. (2000). Toward a protein-protein
interaction map of the budding yeast: A comprehensive system to
examine two-hybrid interactions in all possible combinations between
the yeast proteins. PNAS, 97: 1143-1147.
-
Uetz et al. (2000). A comprehensive analysis of protein-protein
interactions in Saccharomyces cerevisiae. Nature, 403: 623-627.