Gene 221 (1998) 1–9 The Drosophila ebony gene is closely related to microbial peptide synthetases and shows specific cuticle and nervous system expression Bernhard T. Hovemann a,*, Rolf-Peter Ryseck b, Uwe Walldorf c, Klemens F. Sto ¨ rtkuhl d, Irmgard D. Dietzel d, Eliana Dessen a a Universidade de Sao Paulo, Instituto de Biociencias, 05508 Sao Paulo, Brazil b Universita ¨ t Hohenheim, Institut fu ¨r Genetik, 70593 Stuttgart, Germany c Bristol-Myers Squibb, Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA d Ruhr-Universita ¨t Bochum, Fakulta ¨t fu ¨r Chemie, AG Molekulare Zellbiochemie, 44780 Bochum, Germany Received 25 May 1998; accepted 13 August 1998; Received by A.E. Sippel Abstract The previously detected ebony (e) locus (Caizzi et al., 1987) consists of a complex gene structure that is divided into seven exons. An open reading frame encoding the putative Ebony protein of 98.5 kDa exhibits homology to a family of peptide synthetases (Stachelhaus and Marahiel, 1995), in good correlation with the proposed function as b-alanyl-dopamine synthetase. Multiple ebony transcripts are detected throughout development. P-factor mediated transformation of genomic DNA rescues the cuticle, electrophysiological and behavioural phenotypes. Fusion of the ebony reading frame with that of b-galactosidase of E. coli reveals expression in cuticle and nervous system. Strong staining in the first and, to a lesser extent, in the second optic neuropile may reflect the pronounced visual defect observed in ebony mutants. In addition, weak central brain and thoracic ganglion expression is detected in flies. Conservation of a multidomain protein structure known from peptide synthetases should have functional implications on the putative reaction mechanism of peptide bond formation. © 1998 Elsevier Science B.V. All rights reserved. Keywords: cDNA sequence; Gene structure; Peptide synthetases; Synaptic transmission; Tissue specific expression; Visual signal transduction 1. Introduction 1963). Moreover, b-alanine and dopamine have been found to accumulate in ebony pupae and young adults The ebony mutation (Bridges and Morgan, 1923) has ( Hodgetts, 1972; Hodgetts and Konopka, 1973), been known for a long time to give rise to pleiotropic whereas the amount of b-alanyl-dopamine is reduced at effects. Most obvious is the cuticle phenotype (Brehme, eclosion. These observations have led Wright and others 1941). Throughout larval development, spiracle sheaths ( Wright, 1987) to conclude that the affected genetic appear darkened; in ebony pupae, pigmentation is lighter locus encodes b-alanyl-dopamine synthetase. In addi- when compared to wild type, whereas in adults a darken- tion, electrophysiological measurements show an abnor- ing of the cuticle is observed that varies in strength mal electroretinogram (ERG) of ebony mutants with depending on the allele. The biochemical defect which missing on and off transients (Hotta and Benzer, 1969; underlies this phenotypical colour change results from Heisenberg, 1971). A number of behavioural defects the inability of the ebony mutant to incorporate have also been reported. Ebony flies suffer from a severe b-alanine into the pupal case (Jacobs and Brubaker, visual impairment. Phototaxis and optomotor response are drastically reduced (Heisenberg, 1972). They show * Corresponding author. Fax: +49 234 7094105; reduced mating success in light when compared to wild e-mail: bernhard.hovemann@ruhr-uni-bochum.de type. In contrast, relative mating success increases in darkness ( Kyriacou, 1981). This may partly be due to Abbreviations: cDNA, DNA complementary to mRNA; ERG, the visual deficits frequently resulting in mismounting electroretinogram; kb, kilobase(s); mRNA, messenger RNA; PBS, phosphate buffered saline; poly(A), polyadenine. by the male and subsequent abortion of the courtship 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0378-1119(98)00440-5