The Drosophila homeobox segmentation gene fushi tarazu (ftz) is expressed in a seven-stripe pattern during early embryogenesis. This characteristic pattern is largely specified by the zebra element located immediately upstream of the ftz transcriptional start site. The FTZ-F1 protein, one of multiple DNA binding factors that interacts with the zebra element, is implicated in the activation of ftz transcription, especially in stripes 1, 2, 3, and 6. An FTZ-F1 complementary DNA has been cloned by recognition site screening of a Drosophila expression library. The identity of the FTZ-F1 complementary DNA clone was confirmed by immunological cross-reaction with antibodies to FTZ-F1 and by sequence analysis of peptides from purified FTZ-F1 protein. The predicted amino acid sequence of FTZ-F1 revealed that the protein is a member of the nuclear hormone receptor superfamily. This finding raises the possibility that a hormonal ligand affects the expression of a homeobox segmentation gene early in embryonic development.

The Drosophila segmentation gene fushi tarazu (ftz) is expressed at the cellular blastoderm stage in a pattern of seven transverse stripes; the stripes lie out of register with the segmental primordia, spanning alternate segmental boundaries. The zebra element, a 740-bp DNA sequence upstream of the ftz translational start, directs striped expression of lacZ when introduced into the fly genome. We have purified to homogeneity a sequence-specific DNA-binding factor, FTZ-F1, that binds to two sites located within the zebra element and to two sites within the ftz protein-coding sequence. FTZ-F1 DNA-binding activity is first detected in extracts of 1.5-to 4-hr embryos, coincident with the time of ftz expression in stripes; the activity then diminishes before reappearing during late embryo, larval, and adult stages. When one of the FTZ-Fl-binding sequences in the zebra element is mutated by 2-or 4-base substitutions, the binding to FTZ-F1 is disrupted in vitro, and the intensity of lacZ expression is reduced in transformed embryos, especially in stripes 1, 2, 3, and 6. The results suggest that FTZ-F1 is a transcriptional activator necessary for the proper expression of the ftz gene.

Drosophila heat shock activator protein, a rare transacting factor which is induced upon heat shock to bind specifically to the heat shock regulatory sequence in vivo, has been purified from shocked cells to more than 95 percent homogeneity by sequence-specific duplex oligonucleotide affinity chromatography. The purified protein has a relative molecular mass of 110 kilodaltons, binds to the regulatory sequence with great affinity and specificity, and strongly stimulates transcription of the Drosophila hsp70 gene. Studies with this regulatory protein should lead to an understanding of the biochemical pathway underlying the heat shock phenomenon.

Fruit fly FTZ-Fl, silkworm BmFTZ-Fl, and mouse embryonal long terminal repeat-binding protein are members of the nuclear hormone receptor superfamily, which recognizes the same sequence, 5'-PyCAAGG PyCPu-3'. Among these proteins, a 30-amino-acid basic region abutting the C-terminal end of the zinc finger motif, designated the FTZ-Fl box, is conserved. Gel mobility shift competition by various mutant peptides of the DNA-binding region revealed that the FTZ-Fl box as well as the zinc finger motif is involved in the high-affinity binding of FTZ-Fl to its target site. Using a gel mobility shift matrix competition assay, we demonstrated that the FTZ-Fl box governs the recognition of the first three bases, while the zinc finger region recognizes the remaining part of the binding sequence. We also showed that the DNA-binding region of FTZ-Fl recognizes and binds to DNA as a monomer. Occurrence of the FTZ-Fl box sequence in other members of the nuclear hormone receptor superfamily raises the possibility that these receptors constitute a unique subfamily which binds to DNA as a monomer.The FTZ-F1 protein is a positive regulator of the fushi tarazu gene in blastoderm-stage embryos of Drosophila melanogaster (22). The BmFTZ-F1 protein, a factor present in the silkworm, Bombyx mon, is biochemically similar to . The embryonal long terminal repeat-binding protein (ELP), a mouse factor that is present in undifferentiated murine embryonal carcinoma cells but not in differentiated cells, represses transcription from the promoter of the long terminal repeat of Moloney leukemia virus (17). Molecular cloning of cDNAs for these three proteins revealed that they are members of the nuclear hormone receptor superfamily with Cys2-Cys2-type zinc fingers as DNA-binding domains (4,13,18). FTZ-F1, BmFTZ-F1, and ELP recognize the 9-bp sequence 5'-PyCAAGGPyCPu-3', which apparently does not have a direct or inverted repeat (16)(17)(18)(20)(21)(22). In contrast, other members of the nuclear hormone receptor superfamily usually bind to repeated sequences (1,6,24). Nevertheless, the FTZ-F1, BmFTZ-F1, and ELP proteins have high affinities to the binding-site DNA (23). These results indicate that the mechanism of binding of these proteins to DNA is somewhat different from that of other members of the nuclear hormone receptor superfamily.Using various mutant peptides in the DNA-binding domain of FTZ-F1, we demonstrated that in addition to the zinc finger motif, the basic region abutting the C-terminal end of the zinc finger motif is involved in sequence-specific DNA binding and that the 9-bp binding site is recognized by a single polypeptide. MATERIALS AND METHODSPlasmid construction. To express peptides carrying various lengths of the DNA-binding region of FIZ-F1 in Escherichia coli, three DNA fragments coding for amino acids 507 to 622, 507 to 581, and 507 to 593 preceded by methionine as the translational start site were made by using * Corresponding author.polymerase chain reactions (8) with FTZ-F1 cDNA as the template. Amplification with the N-...