Mol Gen Genet (1991) 230:97-103 © Springer-Verlag 1991 Genetic and biochemical characterization of little isoxanthopterin (lix), a gene controlling dihydropterin oxidase activity in Drosophila melanogaster Francisco J. Silva, Baltasar Escriche, Eugenio Ordofio, and Juan Ferr6 Department de Gen~tica, Universitat de Valencia, 46100 Burjassot, Valencia, Spain ReceivedJanuary 14, 1991 ~ June 24, 1991 Summary. Dihydropterm oxidase catalyses the oxidation of 7,8-dihydropteridines into their fully oxidized prod- ucts, and is involved in the biosynthesis of isoxanthop- terin. Fifteen Drosophila melanogaster mutants, selected for their low pterin and isoxanthopterin content, were assayed for dihydropterin oxidase activity. The activity was around 100% in most mutants tested, slightly re- duced in red, g and dke, and undetectable in lix. In flies carrying various doses of the lix + allele, a correlation was found between enzyme activity and the number of lix + copies in the genome. The results suggest that lix is the structural gene for the dihydropterin oxidase en- zyme. Isoxanthopterin was quantitated in strains carry- ing deficiencies for the region in which lix has been mapped by recombination. This allowed us to assign the lix locus to the 7D10-7F1-2 segment of the X chro- mosome. Key words: Drosophila melanogaster - lix - little isoxan- thopterin - Dihydropterin oxidase - Pterin biosynthesis Introduction The biosynthesis of pteridines in Drosophila involves a series of reactions and intermediates that are not yet fully elucidated. The picture is somewhat complicated by the fact that from a common precursor, H4-pyru- voylpterin, the pathway ramifies into several branches that lead to several end-products with different physio- logical functions (Fig. 1). Drosopterins, a set of five or more structurally related red-orange pigments (Rokos and Pfleiderer 1975; Theobald and Pfleiderer 1978), function as screening pigments in the eye (Phillips and Forrest 1980). H4-biopterin has recently been proposed, as in mammals, to act as cofactor for the aromatic amino acid hydroxylases (Guillamdn and Ferr6 1988; Guilla- mdn et al. 1989; Bel and Ferr6 1989). Isoxanthopterin Offprint requests to: F.J. Silva accumulates mainly in the testis sheath, and the onset of its synthesis is much earlier than for the eye pteridines (Fan et al. 1976); in spite of much effort (Forrest and Smith 1975), its physiological role still remains obscure. Finally, biopterin can be presently regarded as an end- product of unknown function, which is found in many tissues but accumulates mainly in the eye. Much work has been done on the biochemistry and genetics of the eye pigments of Drosophila since the early studies by Ephrussi and Beadle (Ephrussi 1942). How- ever, the genes that control their synthesis are known only for some steps (Fig. 1). Thus, rosy (ry, 3-52.0) is the structural gene for xanthine dehydrogenase (Yen and Glassman 1965; Keith et al. 1987), and other genes are known that modulate the activity of this enzyme: low xanthine dehydrogenase (lxd, 3-34.5), cinnamon (cin, 1- 0.0), and maroon-like (mal, 1-64.8) (Warner and Fin- nerty 1981 ; Schott et al. 1986). Punch (Pu, 2-97) is the structural gene for guanosine triphosphate (GTP) cyclo- hydrolase I (Mackay and O'Donnell 1983; Weisberg and O'Donnell 1986), and the activity is also strongly re- duced by prune (pn, 1-0.8), some alleles of raspberry (ras, 1-32.8), and to a lesser extent by clot (cl, 2-16.5), red Malpighian tubules (red, 3-53.6) and rose (rs, 3-35.0) (Mackay and O'Donnell 1983). Based on gene dosage experiments, purple (pr 2-54.5) has been proposed to be the structural gene for Hg-pyruvoylpterin synthase (Yim et al. 1977; Krivi and Brown 1979). sepia (se, 3- 26.0) seems to be a good candidate for the structural gene for Hz-acetylhomopterin synthase and clot (cl, 2- 16.5) a regulatory gene for this activity (Wiederrecht and Brown 1984; Wiederrecht et al. 1984). Finally, Hen- na (Hn, 3-23.0) has been shown to be involved in the synthesis of H4 pterin (Ferr6 et al. 1986; Guillam6n and Ferr6 1988; Guillam6n et al. 1989). In an effort to characterize other genes controlling the biosynthesis of pteridines in Drosophila melano- gaster, we have developed a sensitive assay for dihydrop- terin oxidase activity, which we have applied to mutants affected in pteridine biosynthesis. There were several rea- sons for the choice of this enzyme. On the one hand,