Interactomics and Proteomics

= is concerned with interactions between genes or proteins

types of interactions:

• genetic interactions

  -> two genes are involved in the same functional pathway

• physical interaction

  -> direct physical contact between two proteins or protein and DNA

• in the same gene

• in different genes involved in the same pathway

with a complementation test:

• Cross two homozygous mutants and observe heterozygous offspring phenotypes

  • Mutations in the same gene will not complement

    -> offspring have mutant phenotype

  • Mutations in different genes will complement

    -> offspring have wild-type phenotype

• Do pairwise crosses for all mutants to identify complementation groups

• Typically each complementation group represents a different gene

• If many mutations are recovered in the same genes, this implies saturation

Modifier screens

= way to find new genes involved in the pathway leading to a mutant phenotype

How does it work?

Identify new mutants, which alter the phenotype of the original mutant by performing forward saturation mutagenesis in the background of a particular mutant phenotype

• Back mutations 

• -> a second mutation in the same gene that corrects the previous mutation and restores phenotype to wild-type

• Enhancers 

  -> mutations that make the original mutant phenotype more extreme 

• Suppressors 

  -> mutations that make the original mutant phenotype less extreme

• Epistasis

  -> A mutation in one gene allows a mutation in a second gene to be revealed

mutation might affect:

• different steps in same enzymatic pathway

• steps in different enzymatic pathways, the products of each required for phenotype

• cis or trans acting factors influencing gene expression

• different components of a signal transduction pathway

• different peptides that physically interact with each other (protein-protein interactions)

• many more possibilities

-> most progress on detection of e)

• Affinity chromatography

• Yeast 2-hybrid screen

= purify a target protein by passing total proteins through a chromatography column to see what other proteins come out with the target.

-> detects direct or indirect interactions

Methods:

• Co-immunoprecipitation

  • Link specific antibodies of the target protein to column

    -> causes the target protein (and all proteins bound to it) to be retained in the column

• Biotin-affinity chromatography

• GST-fusion chromatography

• Direct purification

cDNA libraries used to make fusions of “bait“ and “prey” proteins. The recombinant proteins are expressed in yeast. If there is a bait-prey interaction transcription of a reporter gene is induced.

-> detects direct or indirect interactions

-> protein interaction maps

False positives: bait activates transcription alone

False negatives: 2 proteins normally change structure and stop interacting when fused to bait or prey

Can be applied to yeast and drosophila

limitations:

• autoactivation: some baits can activate transcription themselves

• some baits or prays may be lethal when expressed in yeast

• interaction in yeast does not guarantee interaction in other organisms

• protein fusion may alter protein structure/function

proteins can be purified and immobilized on a plate

many proteins - 1 molecule for hybridization to determine interaction with the target.

problems:

• hard to produce large amounts of purified proteins

• proteins are less stable than DNA

gel electrophoresis in which proteins are first seperated by charge (IEP) then by size.

results in multiple “spots“ on the gel

-> good for comparison (treatment-control / human-chimpanse)

Quantitative change: size of the spot (amount of protein)

Qualitative change: location of the spot (size or charge of protein)

1. Protein samples (e.g. “spots“ from 2D-page) are cut into 5-10 aa pieces and mass is determined

2. Determine whole aa sequence based on precise masses

3. Search Database to determine origin-protein

4. Repear 1-3 for all proteins to get the “proteome“