Yeast 2-hybrid assay for an entire genome Uetz et al. Nature(2000)403, 623-627 Two strategies 1.array approach: 6,000 activation domain hybrid transformants mated to 192 dNA binding domain fusion transformants only 20% of interactions (281 reproducible(many auto-activate) 3. 3 positives per interaction-competent protein 2.high-throughput screen"approach: 5, 345 ORFs cloned separately into DNA-binding and activation domain plasmids(2 reporter genes ) DBd fusions pooled and mated to ad fusions 12 clones per pool sequenced, gave 692 unique interactions (472 seen more than once 1. 8 positives per interaction-competent protein Ito et al.PNAS(2001)98,45694574 For both dBd and ad, make 62 pools of a96 proteins. Mate all pools against all Gave 4 549 interactions; 841 observed >3 times core data) The potential number of interactions is huge, and the number of real interactions is probably very large( 10,000); these studies only characterize a tiny fraction (low coverage)Yeast 2-hybrid assay for an entire genome Uetz et al. Nature (2000) 403, 623-627 Two strategies: 1. “array” approach: ~6,000 activation domain hybrid transformants mated to 192 DNA binding domain fusion transformants only 20% of interactions (281) reproducible (many auto-activate) 3.3 positives per interaction-competent protein 2. “high-throughput screen” approach: 5,345 ORFs cloned separately into DNA-binding and activation domain plasmids (2 reporter genes); DBD fusions pooled and mated to AD fusions; 12 clones per pool sequenced, gave 692 unique interactions (472 seen more than once) 1.8 positives per interaction-competent protein Ito et al. PNAS (2001) 98, 4569-4574 For both DBD and AD, make 62 pools of ~96 proteins. Mate all pools against all. Gave 4,549 interactions; 841 observed ≥ 3 times (= core data). The potential number of interactions is huge, and the number of real interactions is probably very large (>10,000); these studies only characterize a tiny fraction (low coverage)