TABLE 2.
Results of the liquid β-galactosidase assay with ONPG as substrate to demonstrate NifL-NifA interactions
| Plasmid to which GAL4 BD was translationally fused | Plasmid to which GAL4 AD was translationally fused | β-Galactosidase activity (Miller units)a | Growth on SD plates lacking Leu, Trp, and His/3ATb | Interacting peptides |
|---|---|---|---|---|
| pBG502 (GAL4 BD:NifA) | pBG505 (GAL4 AD:NifL) | 5.60 ± 0.22 | ++ | NifA and NifL |
| pBG503 (GAL4 BD:NifL) | pBG504 (GAL4 AD:NifA) | 5.71 ± 0.37 | ++ | NifA and NifL |
| pAS2-1 (GAL4 BD) | pBG504 (GAL4 AD:NifA) | 0.63 | −− | None |
| pAS2-1 (GAL4 BD) | pBG505 (GAL4 AD:NifL) | 0.53 | −− | None |
| pBG502 (GAL4 BD:NifA) | pGAD424 (GAL4 AD) | 0.50 | −− | None |
| pBG503 (GAL4 BD:NifL) | pGAD424 (GAL4 AD) | 0.71 | −− | None |
| pVA3-1 (GAL4 BD:p53) | pTD1-1(GAL4 AD:SV40 large T antigen) | 35.9 ± 2.61 | ++ | p53 and large T antigen |
| pAS2-1 (GAL4 BD) | pABCT2 (GAL4 AD) | 1.13 | −− | None |
The β-galactosidase activity assay was performed as described in the MATCHMAKER two-hybrid system (20). Briefly, cells from 1.5-ml samples of exponential culture were collected by centrifugation and resuspended in 300 μl of Z-buffer (20). A 100-μl aliquot of the resuspended cells was lysed by quick freeze-thaw (treatment with liquid nitrogen followed by thawing at 37°C). To measure the β-galactosidase activity in the cell lysate, a 0.7-ml sample of the Z-buffer–β-mercaptoethanol solution was added to each tube followed by 0.16 ml of Z-buffer–ONPG (4 mg of ONPG per 1 ml of Z-buffer). The time of ONPG addition was recorded, and the tubes were incubated at 30°C with shaking. When yellow color was visible, 400 μl of 1 M NaCO3 was added to each tube to terminate the reaction, and the time was recorded. The tubes were then centrifuged for 10 min at 10,000 × g to remove cellular debris, and the optical density at 420 nm was recorded. β-Galactosidase units were defined as the amount of enzyme which hydrolyzes 1 μmol of ONPG to o-nitrophenol and d-galactose per min.
S. cerevisiae CG1945 was transformed with plasmid DNA by utilizing the YEASTMAKER yeast transformation system (22) (CLONTECH Laboratories, Inc.). Transformants were selected on appropriate nutrition-deficient SD medium selection plates by incubating at 30°C for 2 to 3 days. Single colonies from each transformation experiment were purified and were used to determine growth characteristics on SD media lacking His.