Summary of New Dual Bait-Compatible Reagents
| cI Fusion plasmids | |||
| Plasmid name (frames) | Selection in yeast/in E. coli | Comment/Description | |
| pGKS3# AB pGKS4* AB | HIS3 | ApR KmR | ADH1 promoter expresses cI followed by polylinker |
| pGKS6# ABC | ADH1 promoter expresses cI followed by polylinker | ||
| pGKS7* AB | Modified ADH1 promoter expresses ∼5× higher level of expression of cI bait | ||
| pGKS8# AB | ZeoR | Dual purpose vector. ADH1 promoter expresses cI followed by polylinker, whereas cI-responsive gusA reporter cassette (with 3cI ops) is integrated into the same plasmid backbone | |
| pGBS9* AB pGBS10* AB | G418R | KmR | ADH1 promoter expresses cI followed by polylinker Modified ADH1 promoter ensures higher level of expression of cI |
| pGMS11* A | ZeoR | GAL1 promoter expresses cI followed by polylinker; for | |
| pGMS12* B | G418R | KmR | use with baits whose continuous presence is toxic to yeast |
| Reporter Plasmids | ||||
| Plasmid name | Selection in yeast/in E. coli | No. of operators | ||
| pRG64* pRG62* | 4 cI 2 cI | cI operators direct transcription of the gusA gene: sensitivity to transcriptional activation is a function of operator number | ||
| pRG61* | URA3 | KmR | 1 cI | |
| pDR8* | 8 lexA 3 cI | LexA-responsivelacZ reporter is comparable with pMW112, whereas cI-responsive gusA reporter has sensitivity comparable to pRG62 | ||
| LEU2/LYS2Selection Strains | |||
| Strain name | Genotype | No. of operators | |
| SKY48 | MATαtrp1, his3, ura3, lexAop-LEU2, cIop-LYS2 | 6 lexA 3 cI | Stringent selection for interaction partners of cI-fused baits; most sensitive LexA-responsive LEU2reporter |
| SKY191 | 2 lexA 3 cI | Most stringent LexA-responsive LEU2 reporter; and more sensitive cI-responsive LYS2 reporter versus SKY48 | |
| SKY473* | MATa his3, leu2, trp1, ura3, lexAop-LEU2 clop-LYS2 | 4 lexA 3 cI | Sensitivity of LEU2reporter is intermediate between sensitivity of LEU2 in SKY48 and SKY191. Sensitivity of LYS2 reporter is the same as sensitivity of LYS2 in SKY191. Can be used as mating partner for SKY48 and SKY19 strains. |
[i] Reagents newly constructed (*) or modified (#) (change of reading frame and/or sequence of polylinker) in this work are indicated. SKY48 and SKY191 have been described previously (Serebriiskii et al. 1999), but are noted here to provide context for SKY473. A complete listing of dual bait-compatible reagents is provided athttp://www.fccc.edu/researchy/labs/golemis/InteractionTrapInWork.html, as are links to detailed protocols for system use. The newly described cI plasmids provide options to regulate expression levels of baits using either constitutive or galactose inducible promoters (useful for toxic baits), and to use HIS3, ZeoR or G418R as selectable markers in yeast, and either ApR or KmR as selectable markers in E. coli, to maximize compatibility with other yeast two-hybrid systems. A newly developed DR8 dual reporter contains bothlacZ and gusA genes, simplifying transformations, whereas the pRG reporter series allows variation of sensitivity levels for gauging cI-responsive transcription. Finally, the SKY473 reporter strain is an extremely robust MATa reporter strain that is optimal as an interaction mating partner with pre-existing Dual Bait or other two hybrid strains.