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Tissue Specific Effects of Ommochrome Pathway Mutations in Drosophila Melanogaster

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Tissue Specific Effects of Ommochrome Pathway Mutations in Drosophila Melanogaster Genet. Res., Camb. (1991), 57, pp. 257-266 With 3 text-figures Printed in Great Britain 257 Tissue specific effects of ommochrome pathway mutations in Drosophila melanogaster RICK TEARLE Department of Genetics and Human Variation, La Trobe University, Bundoora, Victoria 3083, Australia (Received 16 August 1990 and in revised form 26 October 1990) Summary The tissue-specific effects of 17 mutations affecting the synthesis of brown eye pigment (xanthommatin) have been investigated by combining them with chocolate and red cells, two mutations causing ectopic pigmentation of the Malpighian tubules and larval fat body (which normally only synthesize pigment precursors). The majority of mutations block the pigmentation of four organs: the normally pigmented eyes and ocelli, and ectopically pigmented tubules and fat body. They represent genes that would appear to be required for the normal operation of the pathway per se and are likely to encode structural proteins. Mutations at 5 loci affect pigmentation of a subset of organs: cd and po affect only the eyes and ocelli; kar affects the eyes, ocelli and fat body; car causes excretion of pigment from tubules; and z affects pigmentation of the eyes alone. Of these loci, only z has been shown to encode a regulatory protein and the role of the remaining four gene products is not clear. Two mutations affecting the red eye pigments (drosopterins), bw and mal, do not substantially perturb brown pigment synthesis in any of the four organs. 1978) the conversion of tryptophan to kynurenine 1. Introduction is usually treated as a single step.] In addition to v The compound eye of Drosophila melanogaster con- and en, lesions in about 25 other genes have a major tains two types of light-screening pigments, the brown effect on the pathway, as judged by altered eye or ommochrome (xanthommatin) and a series of red ocellus pigmentation (Lindsley & Grell, 1968; pteridines (drosopterins). Xanthommatin is derived Phillips & Forrest, 1980). Most of these mutations from tryptophan via the ommochrome biosynthetic affect the formation of both xanthommatin and the pathway, which consists of four steps and has been drosopterins. well characterized biochemically (Fig. 1). Enzymes The pathway operates in four organs and at two catalysing each step of the pathway have been partially stages in the organism's life history (Fig. 2). During purified and characterised (Sullivan, Kitos and larval life dietary tryptophan is taken up by the Sullivan, 1976; Sullivan and Kitos 1976; Moore and Malpighian tubules and the larval fat body. The Sullivan 1978; Wiley and Forrest 1981) with the genes tubules convert excess tryptophan (over and above encoding tryptophan oxygenase (vermilion) and that required for protein synthesis) to 3-hydroxy- kynurenine-3-hydroxylase (cinnabar) identified and kynurenine and the fat body converts tryptophan to cloned (Searles and Voelker, 1986; Walker et al. 1986; kynurenine (Beadle, 1937a, b; Nissani, 1975). These W. Warren and A. J. Howells pers. comm.). The other intermediates are stored in the tissues which synthesize two steps in the pathway are catalysed by more than them. one form of the enzyme (Moore & Sullivan, 1978; Around the time of pupariation the larval stores of Wiley & Forrest, 1981) and have not been uncovered pigment precursors are released into the haemolymph, mutationally. [Because kynurenine formamidase is and along with tryptophan from the degradation of found in the same tissues as tryptophan oxygenase larval proteins, are taken up by the developing eyes and in vast excess to that enzyme (Moore & Sullivan, (Beadle & Law, 1938). Pigment deposition begins about 48 hours after pupariation. The capacity of the * Present address: Department of Biochemistry, University of developing eyes to synthesize pigment from tryp- Adelaide, G.P.O. Box 498, Adelaide, South Australia 5001, Australia. tophan alone (Nissani, 1975) indicates that all the 17-2 Downloaded from https://www.cambridge.org/core. IP address: 170.106.40.139, on 28 Sep 2021 at 20:51:57, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0016672300029402 R. Tearle 258 Tryptophan enzymes of the pathway are present in the eyes. The ocelli, which appear to become pigmented somewhat Tryptophan oxygenase later than the eyes, are dependent on exogenous I (v) sources of kynurenine (Beadle, 19376; Nissani, 1975) W-formal kynurenine and only the steps in the pathway from kynurenine to xanthommatin definitely occur in the ocelli. I Kynurenine fomiamidase As shown in Fig. 2, not all steps in the pathway operate in all organs, implying tissue-specific regu- lation of genes encoding structural pathway com- Kynurenine ponents. Because of the organ autonomous nature of most of the pathway mutants (Beadle & Ephrussi, Kynurenine 3-hydroxylase 1936), the requirement for a particular gene product (cn) in the eyes and ocelli can be gauged from the effect a I mutation has on pigmentation of these organs. 3-Hydroxykynurenine Analysis of the effect of mutations on the pathway in the tubules and fat body, however, presents a major difficulty as they do not normally deposit pigment. I Phenoxazinione synthase While it is possible to use biochemical assays to determine the levels of kynurenine and 3-hydroxy- Xanthommatin kynurenine, isolation of the tissues is difficult and Fig. 1. The ommochrome biosynthetic pathway. The tedious. This problem can be overcome by utilizing genes encoding enzymes which control two steps in the pathway have been identified: vermilion (v) which encodes mutants that cause these tissues to deposit pigment. tryptophan oxygenase and cinnabar (cn) which encodes Lesions in two genes, chocolate (cho) and red kynurenine-3-hydroxylase. Malpighian tubules {red) cause the ectopic synthesis of ommochrome in the Malpighian tubules from early in 1. Larval storage phase larval life (Lindsley & Grell, 1968). They also alter eye Tubules Fat body colour to a dark brown and darken the colour of the ocelli. Mutations at two other loci, red cells (re) and • TRP TRP - lysine (lys), can cause the ectopic deposition of 4 KYN ommochrome in the remnants of the disintegrating KYN larval fat body at 48 h after pupariation - the same 3HK time as it is occurring in the eyes (Jones & Lewis, 1957; Grell, 1961). The eyes and ocelli are normally pigmented. 2. Pigment synthetic phase The aim of the work presented here is to begin to Tubules Fat body investigate the tissue-specific regulation of pathway genes by analysing the organ-specific effects of mutant genes affecting ommochrome synthesis. This paper KYN- — KYN details the effects of 17 such eye colour mutations on the tubule phenotype of cho, and fat body phenotype 3HK- of re2, and combines these data with eye and ocellus phenotypes to delineate the organ-specific effects of Eyes Ocelli the genes. The possible modes of action of several genes are also discussed. TRP - KYN - • KYN 2. Materials and methods 4 4 Mutant stocks of D. melanogaster were obtained from 3HK - 3HK 4 4 a number of different sources, the majority from the XAN XAN Bowling Green and Caltech stock centres. The alleles used and their nomenclature are given in Table 1. Fig. 2. Temporal and tissue specific expression of the Material to be examined was maintained at 25 °C ommochrome biosynthetic pathway in D. melanogaster. except for stocks involving re2, which were maintained Pigment precursors are stored in the Malpighian tubules at 18 °C to enhance pigmentation of the larval fat and larval fat body during the larval phase. During the body. pupal phase the stored precursors and tryptophan are taken up by the eyes and ocelli. The substrates and To ensure that the eye, ocellus, tubule and fat body products of these steps in the pathway are tryptophan phenotypes could all be attributed to the ommochrome (TRP), kynurenine (KYN), 3-hydroxykynurenine (3HK) mutation, and were not due to an unrelated lesion on and xanthommatin (XAN). the same chromosome, two measures were taken. Downloaded from https://www.cambridge.org/core. IP address: 170.106.40.139, on 28 Sep 2021 at 20:51:57, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0016672300029402 Eye colour mutations 259 Table 1. Alleles of eye colour loci tested in combination with cho and re2. Alleles marked with an asterisk were backcrossed to the wild-type strain Canton S for 25-30 generations to standardize the genetic background Allelic combinations tested Locus with cho with re2 brown (bw) *bw' *bw> cardinal (cd) *cd', cdKP\ *cet"°, *cd'/cdKP2 *cd' carmine (cm) *cm' *cm' carnation (car) *car' *car' cinnabar (en) *cn', cn3Sk, *cn'/cn35k *cn' claret (ca) *ca', ca3, *ca'/ca3 *ca' garnet (g) *g',*gS3d,*g'l*gS3d *g53d karmoison (kar) *kar',Dj(3R)kar-SZJl/DJ(3R)kar-SZ12 *kar' light (It) It' It' lightoid (ltd) *ltd', *ltd'/DJ(2R)CyLbwVDc") *ltd> maroonlike (mal) mafz mat" orange (or) *or>, or48", *or'/or48a *or' pale ocelli (po) po' po' v nb > pink (p) *p , *p' , *p"/*p"" *PP ruby (rb) *rb', rb" *rb> scarlet (st) *st', st'/sf3c *st> vermilion (v) V, v36f, *v'/v36f V white (w) *w', w'"8, *w'/w"ls *w> zeste (z) V, z" *z< Firstly 17 mutant stocks were backcrossed to cho affects both ommochrome and drosopterin Canton-S for 25-30 generations to remove ex- pigment formation. The alteration to ommochromes traneous mutations and to standardize the genetic is complex, probably involving the synthesis of forms backgrounds. Where more than one allele was other than xanthommatin (Linzen, 1974; Ferre et al. available for a locus or a deficiency existed, trans- 1986) while the levels of drosopterins are severely heterozygotes were tested with cho. For crosses to re2, reduced. This complexity precludes simple biochemi- only outcrossed mutant stocks were used.
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