blumberg-lab.bio.uci.edu

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: 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 bait protein prey protein 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 transform prey vector

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

MATa MATα

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.