Definition of Interactomics
= 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
Using forward genetics, where could the mutation be (while still having the same or a similar phenotype)?
in the same gene
in different genes involved in the same pathway
How can be tested if a mutation is in different genes, which are involved in the same pathway?
(Given that the mutations are recessive)
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
What are “modifier screens” and how does it work?
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
Which types of mutation can “modifier screens” uncover?
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
How come genes interact due to a mutation but not its protein products?
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)
Name the methods to detect protein-protein interactions
Affinity chromatography
Yeast 2-hybrid screen
What’s affinity chromatography?
= 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
Explain Yeast 2-hybrid screen.
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.
-> 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
How do Protein microarrays work and how do they differ to DNA microarrays?
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
what is a 2D-page?
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)
How does Mass spectrometry work?
Protein samples (e.g. “spots“ from 2D-page) are cut into 5-10 aa pieces and mass is determined
Determine whole aa sequence based on precise masses
Search Database to determine origin-protein
Repear 1-3 for all proteins to get the “proteome“
Last changeda year ago