The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila (bric A brac/tramtrack/trnscrlptlonal regulation/amlno-terminal domain/enbryogenesls)

Abstract

The Drosophila bric A brac protein and the transcriptional regulators encoded by tramt and Broad-Complex contain a highly conserved domain of =115 amino acids, which we have cafled the BTB domain. We have iden-tifed six additonal Drosophila genes that encode this domain. Five of these genes are developmentally regulated, and one of them appears to be functionally related to bric a brac. The BTB domain defines a gene family with an estimated 40 members in Drosophila. This domain is found primarily at the N terminus of zinc finger proteins and is evolutionarily conserved from Drosophia to mammals. Many conserved protein domains that mediate specific molecular functions have been identified. In addition to clarifying the mechanisms underlying a variety of biological processes , the characterization of such domains has led to the classification ofgenes into families, helping to assign function to other genes and facilitating the identification of interesting new genes. We have recently identified the Drosophila gene bric A brac (bab), which is required for pattern formation along the proximal-distal axis of the leg and antenna (1). Mutations in bab cause homeotic transformation and fusion of tarsal segments. The graded expression pattern in tarsal primordia, together with analysis of bab mutants, suggest that the concentration of the nuclear bab protein determines tarsal segment specification. In addition, bab is required during morphogenesis of the ovary (D.G., unpublished data). bab contains a motif of-115 amino acids also found in the zinc finger proteins encoded by the Drosophila genes tramtrack [ttk (2)1 and Broad-Complex [BR-C (3)]. The ttk protein binds transcriptional regulatory elements of the seg-mentation genes fushi tarazu and even-skipped. It is required during embryogenesis, presumably to repress inappropriate transcription ofthese genes, and possibly other segmentation genes, including odd-skipped, hairy, and runt (4-7). ttk is also required during cell fate determination in the eye (8). BR-C is an early response locus in the ecdysone-induced regulatory cascade that initiates metamorphosis, and its products are thought to activate downstream regulatory genes (3). The transcripts of both ttk and BR-C are alternatively spliced, producing proteins in which the motif they have in common with bab is at the N terminus, fused to different sets of zinc fingers near the C terminus (3, 7). We have called this motif the BTB domain, for BR-C, Itk, and kab (1). In this study, we used PCR to search for additional BTB domain-encoding genes in Drosophila. We have identified and characterized six additional members of this family.ll Five of them are transcribed in spatially and temporally regulated patterns, suggesting that these genes have specific and distinct functions during development. We estimate that the BTB domain family contains as many as 40 members in Drosophila. MATERIALS AND METHODS Equal amounts of HindIll-or EcoRI-digested Drosophila genomic DNA were loaded onto multiple lanes of an agarose gel and transferred to nylon. The filter was then cut into strips and hybridized with a 32P-labeled probe encoding the bab BTB domain in 0.25 M NaH2PO4/0.25 M Na2HPO4, pH 7.2/7% SDS/1 mM EDTA/1% bovine serum albumin, at 55TC. Each strip was washed at a different stringency. The final wash conditions were as follows: 0.1% SDS/0.1x standard saline/citrate (SSC) at