Download Bi190 Advanced Genetics 2011 Lecture 11/ho10 Genome to Function

Bi190 Advanced Genetics
2011 Lecture 11/ho10
Genome to Function
A genome sequence enables systematic (global) analysis of gene function.
0. Experiments that establish relationships between genes, proteins, cells and functions.
As we have seen, there are a numbe of experiments that connect genes and their gene
products.
1. Genome Content Mapping
The protein content of a genome can help define genetic modules, that is genes that always
(or almost always) act together. The MAP kinase cascade is an example of a module.
Inferring pathways from occurrence of genes.
3. Gene Knockouts
Sternberg 2011
Bi190 Advanced Genetics
2011 Lecture 11/ho10
Genome to Function
Systematic analysis of gene function reveals that under standard laboratory conditions only a
fraction (20%-40%) of genes are essential or have discernable phenotypes. One can analyze
phenotypes more carefully, or look at genetic interactions, or run selection experiments, e.g.
with bar-coded yeast deletion strains.
Targeted knockouts are best made by gene replacement, using the Rothstein method (which
was used by Cappechi and colleagues for mouse knock-outs)
4. Phenotypic profiling
Sternberg 2011
Bi190 Advanced Genetics
2011 Lecture 11/ho10
Genome to Function
Bar-coded yeast knockouts
5. Protein-protein interactions.
There are several high throughput methods for examining protein-protein interactions. Tandem
affinity tagged pulldown of a protein complex, and its analysis by liquid chromatography and
mass spectrometry (LC-MS-MS) has proven to be powerful and reasonably accurate.
The yeast two-hybrid assay (Stan Fields) was based on Brent & Ptashne’s discovery of the
bipartite nature of transcriptional activators. This method is not expensive as it relies more on
biology than machines.
Bi-partite transcriptional activators
DBD
ACT
+
Reporter gene
DBD
Reporter gene
ACT
Reporter gene
2-Hybrid Assay in yeast
DBD-Bait
Prey-ACT
ACT
Bait
DBD
Prey
+
Reporter gene
Sternberg 2011
Bi190 Advanced Genetics
2011 Lecture 11/ho10
Genome to Function
Some proteins are “sticky” and interact promiscuously. Proteins can interact in the yeast twohybrid assay, but never see each other in vivo! This can be sorted out by gene expression
analysis or subcellular localization studies.
6. Multiple support networks.
Empirically, each high-throughput screen or assay has particular biases and caveats. Thus, we
are more confident if a connection is support by multiple lines of evidence. For example,
synthetic lethality and high copy suppression; genetic interaction plus physical interaction.
7. Localization screen.
The location of each gene product in (or outside) a cell is a crucial piece of information.
Molecules that are in the same complex should colocalize, and co-localization would support the
possibility of direct physical interactions.
Double labeling of proteins in yeast.
RFP for each cell component.
GFP for each open reading frame (ORF).
Sternberg 2011
Bi190 Advanced Genetics
2011 Lecture 11/ho10
Genome to Function
Sternberg 2011