Two-hybrid interaction between Czf1p and Efg1p

DNA-binding domain fusionGa14p-activation domain fusionβ-Galactosidase activitya
CZF1-lexAbGAL4AD (unfused)1.9 ± 1.4
lexA (unfused)GAL4AD-EFG1c4.4 ± 0.9
CZF1-lexAGAL4AD-EFG159.4 ± 10.4
CZF1-lexAGAL4AD-SLK19d0.35 ± 0.12
lexA-FosdGAL4AD-EFG112.8 ± 1.3
lexA-FosGAL4AD (unfused)10.5 ± 0.9
  • a Strain EGY40 carrying the lacZ reporter plasmid pSH18-34 (Golemis et al. 1996) was transformed with the lexA plasmids (pNLexA, pBOM1a, and plexA-Fos) and GAL4AD plasmids (pGAD-C2, pKLEF4, and pC1.1.2) using standard methods (Ausubel et al. 1989). For quantitative analyses, cells were grown overnight in synthetic complete medium lacking uracil, histidine, and leucine. Cells were diluted into fresh medium and grown to exponential phase for 3–5 hr to an OD600 of 0.4—0.9. Cells were harvested and β-galactosidase activity was determined using o-nitrophenyl β-d-galactopyranoside as a substrate as described in the Clontech Yeast Protocols Handbook. Analyses were done in quadruplicate.

  • b CZF1-lexA was constructed by PCR amplification of the CZF1 gene with primers 5′-GGTGAATTCCATTCAAACGAAAACTATCTGGT-3′ and 5′-GGTGGATCCTACTTCTGTATTCAACAATACCT-3′ and cloning of the PCR product into vector pNLexA (Golemis et al. 1996).

  • c GAL4AD-EFG1 was constructed by PCR amplification of EFG1 with primers 5 ′-CGGAATTCTCAACGTATTCTATACCCTATTAC-3′ and 5′-CGGGATCCGATGTACACGAATGATATTTATTA C-3′ and cloning of the PCR product into vector pGAD-C2 (James et al. 1996).

  • d Control fusion proteins that are not expected to interact with either Efg1p or Czf1p. Plasmids plexA-Fos and pC1.1.2 encoding GAL4AD-SLK19 were generously provided by R. Brent (Massachusetts General Hospital, Boston) and R. Kamieniecki (Tufts University, Boston).