Biochim&a et Biophysica Acta, 1216 (1993) 339-341 339 Elsevier Science Publishers B.V. BBAEXP 90576 Short Sequence-Paper DR-78, a novel Drosophila melanogaster genomic DNA fragment highly homologous to the DNA-binding domain of thyroid hormone-retinoic acid-vitamin D receptor subfamily Enrique Martln-Blanco * and Thomas B. Kornberg Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-0554 (USA) (Received 25 May 1993) Key words: Cloning; Ecdysone; Genomic library; Nuclear receptor; PCR; (Drosophila) Degenerate oligodeoxyribonucleotides were designed for both ends of the DNA-binding domain of members of the nuclear receptor superfamily. PCR amplified Drosophila melanogaster DNA was purified and cloned (DR plasmids). Genomic ADASH clones were identified at high stringency with an amplified DR-78 plasmid DNA and isolated. The partial sequence shows a very probable open reading frame which would encode a peptide highly homologous to members of the thyroid hormone-retinoic acid-vitamin D receptor subfamily. The fragment corresponds to a single copy gene and was mapped at position 78D of chromosome three by in situ hybridization. Different members of the nuclear receptor super- family have been described in Drosophila melanogaster, see, e.g., Refs. 1-4. Ecdysone receptor, Ultraspiracle (Usp), a gene coding for an homologous of vertebrate RXR genes, and E75 protein seem to be involved in ecdysone response during metamorphosis. FTZ-F1, Knirps and Knirps-related are probably participating in segmentation processes. To search for new members of the nuclear receptor superfamily, we designed two degenerate primers cor- responding to the two zinc fingers of the DNA binding domain of vertebrate receptors [5] (Fig. 1). After PCR amplification of genomic Drosophila DNA and poste- rior cloning, we isolated and sequenced several clones with partial homology to different receptors. For one * Corresponding author. Present address: Instituto de Investiga- ciones Biom6dicas, CSIC, Departamento de Bioqulmica, Facultad de Medicina, Universidad Aut6noma de Madrid, Madrid 28029, Spain. Fax: + 34 1 5854587. The DR-78 sequence data reported in this paper have been submit- ted to the EMBL/GenBank Data Libraries under the accession number X73045. Abbreviations: PCR, polymerase chain reaction; hRAR, human retinoic acid receptor; RXR, retinoid X receptor; v-erbA, viral onco- gene erbA; rGR, rat glucocorticoid receptor; hGR, human gluco- corticoid receptor; cER, chicken estrogen receptor; hER, human estrogen receptor; hPR, human progesterone receptor. of them, DR-78, we screened a ADASH genomic Drosophila library (kindly provided by J.N. Jan Labora- tory, UCSF) and isolated several A clones. Southern blot of restriction enzyme-digested ge- nomic DNA from Drosophila melanogaster was probed with the DR-78 clone. Hybridization under high-strin- gency conditions gave only a single band indicating that the DR-78 fragment derives from a single copy se- quence (data not shown). Physical maps of genomic clones were aligned and the PCR probe was localized by hybridization to a 3.6 kb EcoRI fragment. DR-78 nucleotide sequence and its conceptual translation product bear strong resem- blance to the nuclear receptor zinc fingers domain (C region) [6], with the characteristic structure C-X2-C- X13-C-X2-C and C-Xs-C-X12-C-X4-C (Fig. 2). In each of the genes of the nuclear receptor superfamily that have been examined, an intron had been found in between the two zinc fingers. This intron occurs either one codon after [7] or within the tenth codon after [8] the final cysteine residue of the first finger. DR-78 encodes a possible intron, of 57 bp, one codon after the cysteine, which presents good putative donor and acceptor splicing sites (Fig. 2A). DR-78 is highly homologous to members of the thyroid hormone-retinoic acid-vitamin D receptor sub- family [9] and would code for Glu, Gly and Gly at the discriminatory positions at the base of the first finger