Table II

Strains and plasmids used in this study

pRG61 (17), pDR8 (17), SKY191 (24), SKY54 (17), and pJG4–5 (3) have been described previously. Bacteriomatch II reporter strain and library were from Stratagene.

PlasmidsSelection inComment/description
YeastE. coli
Baits
    pGLS20aG418RKmRTEF1 promoter ensures expression of cI in yeast, while lpp/lacUV5promoter provides for expression in E. coli
    pGLS22/23a HIS5 CmRSimilar to pGLS20, see text for details
Reporters
    pRG61 URA3 KmRλcI operators direct transcription of the gusA gene; pRG61 is less sensitive and lower background reporter than pDR8.
    pDR8 URA3 KmRλcI operators direct transcription of the gusA gene; pRG61 is less sensitive and lower background reporter than pDR8
    pOR6 URA3 ApRλcl operators direct transcription of the lacZ gene
Activation Domain Fusions
    pJG4–5 TRP1 ApRGAL1 promoter provides efficient expression in yeast of a gene fused to a cassette consisting of nuclear localization sequence, transcriptional activation domain, and HA epitope tag
    pAC-AMP-αLPLaN/AApRTandem lpp/lacUV5 promoters provide efficient expression in E. coli of a gene fused to E. coli RNAP α subunit residues 1–248. Plasmids differ in replication origins (pACYC and ColE1, respectively)
pBR-UV5-αLPa
StrainsRelevant GenotypeComment/Description
S. cerevisiae SKY191MATα trp1, his3, ura3, cIop-LYS2Reporter strains in which the expression of the LYS2
S. cerevisiae PRT475aMATa trp1, his3, his5, ura3, cIop-LYS2    reporter gene is directed by a weak promoter bearing
S. cerevisiae PRT50aMATα trp1, his3, his5, ura3, cIop-LYS2    a λcI DNA binding site
E. coli KJ1567aΔhisB463, Δ (gpt-proAB-arg-lac)XIII zaj::Tn10 [FlacIq HIS3 aadA KanR]Reporter strain in which the expression of the HIS3 and aadA reporter genes is directed by a weak promoter bearing a λcI DNA binding site
BacterioMatch II reporter strain (Stratagene) Δ (mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 hisB supE44 thi-1 recA1 gyrA96 relA1 lac [FlaqIq HIS3 aadA Kanr]Reporter strain in which the expression of the HIS3 and aadA reporter genes is directed by a weak promoter bearing a λcI DNA binding site
E. coli AG58A(RP28) a ΔhisB463, Δ (gpt-proAB-arg-lac)XIII zaj::Tn10 [FlacIq lacZ KanR]Reporter strain in which the expression of the lacZ reporter gene is directed by a weak promoter bearing a λcI DNA binding site
LibrariesVectorComment/description
YTH Human HeLa Cell Library pJG4–5Hela S-3 cells, Primary size: 9.6 × 106 Primer: UdT, average insert size: 1.5 kb
BacterioMatch II Human HeLa Cell Library pTRG: see Stratagene Manual for detailsHela S-3 cells, Primary size: 4.5 × 106 Primer: UdT, average insert size: 1.3 kb
  • a Reagent produced in this study.