Fig. 1. Molecular cloning, expression and functional analyses of Drosophila MBF1. (A) Deduced amino acid sequences of fly, silkmoth, human and yeast MBF1. Residues that are identical to those of fly MBF1 are shaded. Thick bar represents the well-structured domain; 1-4 denote four amphipathic helices. (B) Drosophila mbf1 partially rescues sensitivity of yeast mbf1 disruptant to aminotriazole. Indicated Saccharomyces cerevisiae strains were tested for aminotriazole sensitivity. Yeast MBF1 (trp1-1 ura3-52 leu2-P1) and mbf1 (trp1-1 ura3-52 leu2-P1 mbf1::LEU2), and a yeast genomic MBF1 construct pMBF1 have been described previously (Takemaru et al., 1998). pDmMBF1 carries Drosophila mbf1⁺ cDNA in place of the yeast MBF1 coding region in pMBF1. As a control, these four yeast strains showed essentially the same growth in the absence of aminotirazole (data not shown). (C) Expression of MBF1 as revealed by immunostaining with anti-MBF1 antibody. (1) Syncytial blastoderm stage embryo. (2) Stage 16 embryo. Staining of CNS along the midline and tracheal staining on the margin of the embryo. (3-5) Dissected tissues from a 3rd instar larva: (3) strong staining in both the somatic and germ cells of the developing testis; (4) nuclear staining in the polyploidal salivary gland; and (5) CNS. (D) Expression of a FTZ-F1-dependent reporter gene in mbf1⁺ and mbf1^- homozygous embryos. Expression of a transgene fPE-lacZ carrying the ftz proximal enhancer and the hsp70 minimal promoter fused to lacZ (upper panels) or its mutant derivatives (middle and bottom panels) was analyzed by X-gal staining. Mutation in FTZ-F1-binding sites but not in the mbf1 locus affected the reporter gene expression. The fPE-lacZ is silent in ftz-f1 mutant background (Yussa et al., 2001).