Uetz and Ito: Genomic two-hybrid

1. Uetz and Ito: Genomic two-hybrid A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae Nature 2000 A comprehensive two-hybrid anlaysis to explore the yeast protein interactome PNAS 2001

2. What’s the question? • How many protein-protein interactions are there? • These papers were done after the genomic sequence was available – no more partial digestions

3. Uetz • 2 approaches – one by one or pooled • Inserted 6000 ORFs into activation-domain plasmids • Gap-repair transformation • 16 384-well microtiter dishes • Made 192 DNA binding-domain clones

4. Alternative approach • Pooled approx. 6000 transformants • Same PCR products and recipient plasmids used to generate two collections of transformants – 64 barcoded 96-well plates • 5345 (of 6,144) ORFs were cloned into both AD and BD plasmids • Screened by pairwise mating

5. Method • Mating mixes were transferred to selective plates to identify (select) for diploids that contained both plasmids and activated both URA3 and lacZ • 87 of 192 showed positive reactions, i.e. interactions with another protein (281 pairs) • 68% were identified in indep. Interaction (41%), or multiple times in a single experiment (27%). • 32% were identified only once • What does this say about saturation or coverage of the experiment?

6. Two-hybrid mating arrays Select for transormants (leu) Select for interactions (trp, leu, his)

7. What are your conclusions from this table?

8. Results • 12 positive DNA-binding domain hybrids common to both screens gave 48 putative interactions in the array screen and 14 in the library screen. • What is your conclusion? What are other possible reasons for this difference?

9. Why the difference? • The His selection for 14 days in the mating/array assay was less stringent than the 2-day URA selection for the pooled strains. • Probably needed to select for the plasmids first and then for the interaction

10. How to start evaluating this data? • Have these interactions been shown in other ways? (109/957) • Why weren’t all previously shown interactions found? • They suggest it was the stringency of their assay, including using a Cen vector,using only full-length ORFS in both vectors, some may contain sequence errors or not have an insert.

11. Can we use this to infer function of unknown proteins? • YGR010W and YLR328W interact and interact with CAR2 (ornithine aminotransferase) – may function in Arg metabolism • Human gene can complement in yeast – so these new genes may provide insight into human disease

12. They did find interactions between genes in the same pathway • Two autophagy genes • Two genes in the cytoplasm-to-vacuole targeting pathway • Several mutations in these two pathways are allelic – what does that mean? • Present more interactions of interest – but how do you get a better handle on these? • More info: http://depts.washington.edu./sfields/.

13. Models for how this data might be used to relate or infer pathways

14. Ito - approach • Proteomics – could use a top-down approach (mass spec of complexes) or a bottom-up approach (genomics)

15. method

16. methods • Used PCR-generated ORFs • Cloned all of them and confirmed the clones • Used dual selection (ADE+ and HIS+) • Pooled each 96-well plate (62 pools, 95% of ORFs) • To get all possible matings, did 3,844 mating reactions by using a multisample filtration apparatus

17. What was the identification and analysis procedure? • PCR of inserts from positive colonies (lots of sequencing) • Database of interacting proteins: http://dip.doe-mbi.ucla.eduy) • Biomolecular Relations in Information Transmission and Expression database of KEGG: http://www.genome.ad.jpybritey) • All data is at: http://genome.c.kanazawa-u.ac.jpyY2H.

18. What was found and comparison of the datasets

19. Venn diagram comparison

20. Putative Networks

21. Caveats • Used two markers for selection • Used multicopy plasmids • Neither screen was saturated • Conclusions: more interactions to be determined, need additional confirmation and, probably, tools