Fig. 1

The yeast one-hybrid system was used to identify Foxd3 bound to the myf5 -82/-62 cassette. (A) The yeast one-hybrid assay of clones transfected with the plasmids indicated the following: pHISi-6× (-82/-62), which contains six repeats of -82/-62; pGADT7-zfoxd3, which contains foxd3 with the GAL4 activation domain; pHISi-m4m5, which contains the mutated -82/-62 cassette; p53HIS/pGAD53m, the positive control; p53HIS/pGAD424, the negative control. Yeasts that harbored plasmids containing wild-type -82/-62 grew under growth-inhibiting conditions (histidine and leucine were absent, 60 mM of 3-amino-1,2,4-triazole was present) when Foxd3 fused to the activation domain was expressed. Yeasts containing plasmids with mutated -82/-62 did not grow. (B) The colony-lift filter method was used to perform β-galactosidase assays. Yeasts were transformed, as indicated, with each of the following plasmids: pLacZi-6× (-82/-62), the bait plasmid that contains six repeats of -82/-62 and carries the lacZ reporter gene; pLacZi-m4m5, which contains four repeats of the mutated -82/-62 sequence; p53BLUE/pGAD53m, the positive control; p53BLUE/pGAD424, the negative control. Positive β-galactosidase activity, shown in blue, was detected only when the foxd3 activation domain fusion was expressed in yeasts containing wild-type -82/-62. No activity was detected in yeasts with mutated -82/-62.