Table 1. Interaction trap-compatible two-hybrid system plasmids and strains
| Plasmid name/source | Selection | Number of operators | Comment/description | |
|---|---|---|---|---|
| in yeast | in E. coli | |||
| LexA fusion plasmids: | ||||
| pEG202 (pMW101, 103) | HIS3 | ApR | ADH1 promoter expresses LexA followed by polylinker; basic plasmids to clone bait as LexA fusion. n.b.: E. coli marker for pMW101 is CmR, for pMW103, KmR. | |
| pJK202 | HIS3 | ApR | pEG202 derivative, incorporating nuclear localization sequences between LexA and polylinker (enhanced ability to translocate bait to nucleus) | |
| pNLexA | HIS3 | ApR | Polylinker is upstream of LexA; allows fusion of LexA to carboxyl terminus of bait, leaving amino-terminal residues of bait unblocked. | |
| pGilda | HIS3 | ApR | GAL1 promoter expresses LexA followed by polylinker, for use with baits whose continuous presence is toxic to yeast. | |
| pEG202I | HIS3 | ApR | pEG202 derivative, which can be integrated into yeast HIS3 gene after digestion with KpnI; ensures lower levels of bait expression | |
| Reporter plasmids: | ||||
| pMW111 | URA3 | KmR | 1 lexA | lexA operators direct transcription of the lacZ gene: Sensitivity to transcriptional activation is a function of operator number. |
| pMW109 | URA3 | KmR | 2 lexA | |
| pMW112 | URA3 | KmR | 8 lexA | |
| Activation domain fusion plasmids | ||||
| pJG4-5, pYesTrp2 | TRP1 | ApR | Library or prey expression plasmids; GAL1 promoter provides efficient expression of a gene fused to a cassette consisting of nuclear localization sequence, transcriptional activation domain, and HA or V5 epitope tags | |
| Genotype | ||||
| LEU2/LYS2 selection strains: | ||||
| SKY48 (MATα) | trp1, his3, ura3, lexAop-LEU2, cIop-LYS2 | 6 lexA 3 cI |
Stringent selection for interaction partners of cI-fused baits; most sensitive lexA-responsive LEU2 reporter | |
| SKY191 (MATα) | 2 lexA 3 cI |
Most stringent lexA-responsive LEU2 reporter; and more sensitive cI-responsive LYS2 reporter versus SKY48 | ||
| SKY473 (MATa) | 4 lexA 3 cI |
To be used as mating partner for SKY48 and SKY191 strains | ||
| Selection | ||||
| in yeast | in E.coli | |||
| Control set of plasmids: Testing specificity | ||||
| pEG202-Ras | HIS3 | ApR | Expresses LexA-Ras fusion protein; use as negative control for activation assay, positive control in repression assay, and as test bait in interaction assay | |
| pEG202-hsRPB7 | HIS3 | ApR | Expresses LexA-hsRPB7 fusion protein; use as weak positive control for activation assay | |
| pGKS3 | HIS3 | ApR | Does not express any LexA-fusion protein; use as negative control for repression assay | |
| pSH17-4 | HIS3 | ApR | Expresses LexA-GAL4 fusion protein; use as strong positive control for activation assay | |
| pJG4-5-Raf1 | TRP1 | ApR | Raf interacts with Ras; control in interaction assay. | |
| We note that SKY48 is described rather than EGY48 as host strain; this strain possesses additional features allowing the potential of adapting a two-hybrid screen to a two-bait two-hybrid screen, as described in Serebriiskii et al. (1999). Additional information on interaction trap-compatible reagents is provided online at http://www.fccc.edu/research/labs/golemis/InteractionTrapInWork.html. | ||||
