Bi190 Advanced Genetics 2011 Lecture 11/ho10 Genome to Function

 

Sternberg 2011

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

Bi190 Advanced Genetics 2011 Lecture 11/ho10 Genome to Function

 

Sternberg 2011

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

Bi190 Advanced Genetics 2011 Lecture 11/ho10 Genome to Function

 

Sternberg 2011

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.

DBD

ACT

Bi-partite transcriptional activators

DBDReporter gene

ACT

Reporter gene

Reporter gene

+

2-Hybrid Assay in yeast

DBD

ACT

Bait Prey

Reporter gene

+

DBD-Bait Prey-ACT

Bi190 Advanced Genetics 2011 Lecture 11/ho10 Genome to Function

 

Sternberg 2011

Some proteins are “sticky” and interact promiscuously. Proteins can interact in the yeast two-hybrid 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).

Bi190 Advanced Genetics 2011 Lecture 11/ho10 Genome to Function

 

Sternberg 2011