Why do you think we needed another way to look at protein-protein interactions?

You should find this very familiar because of GAL4. The ORF for one protein is cloned into the DNA binding domain and the ORF for another protein is cloned into the transcriptional activation domain.

Draw yourself a diagram of how this might work from transcription to translation to protein interaction and gene activation.

Look up SNF1 at SGD and Proteome. What do you know about it's function, related sequences, and gene expression? What can you find out about protein interactions?

What is the phenotype from a snf1 deletion that the Yeast Consortium found? (it's at SGD)

How does the interaction lead to the activation of ß-galactosidase? What is the gene construct that allows this?

What is the question that generated Table 2 and what does the result tell you about the two hybrid assay? What domains were present in the 881 GAL4 construct that are missing in the later constructs?

Chien/Fields paper

What is the development in this paper that moves this technology to genome scale?

What's the deal about transcription factors and homodimers? Why do you think a homodimer is difficult to detect biochemically?

What are the potential problems of looking at segments of proteins in this assay?

How are they keeping all the plasmids in the yeast and looking for interactions?

Do you see any limitations to this assay?

How do they make the activation domain library?

Why would the activation domain library have some plasmids that would induce ß-galactosidase?

Ito and Uetz:

How do their methods of screening differ?

What is the difference in their results?

How did each group make this assay high throughput?

What are the reproducibility issues in each of these papers?