A single locus encodes both phenylalanine hydroxylase and tryptophan hydroxylase activities in Drosophila

Vol. 267, No. 6 , Issue of February 25, pp. 4199-4206, 1992 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY id 1992 by The American Society for Biochemistry and MolecularBiology, Inc

A Single Locus Encodes Both Phenylalanine Hydroxylaseand Tryptophan Hydroxylase Activities inDrosophila* (Received for publication, April 19, 1991)

Wendi S. Neckameyer$ and Kalpana White From the Department of Biology, Brandeis University, Walthum. Massachusetts02254

We have used a full-length clone encoding rabbit tryptophan hydroxylase (TRH) to isolate the Drosophila homologue (DTPH).Southern analysis of Drosophila genomic DNA reveals a pattern indicative ofa single gene.The single transcript is expressed in adult head and body mRNA but is also detected in mRNA from early embryos. The embryonic transcript is ubiquitously expressed and appears to concentrate in yolk granules. In situ hybridization of TRH-homologousantisense RNA probeto sectioned tissue from third instar larvae demonstrated the presence of this transcript in fat body and cuticular tissue. Developmental immunoblot analysis usingantibodies raised against a /3-galactosidase-Drosophila fusion protein revealed a 46-kDa embryonic protein also detected in female abdomens and a SO-kDaprotein found in larval and adult stages. Immunocytochemical analysis of the Drosophila protein in the larval central nervous system showed that it appeared to be present in both serotonin- and catecholamine-containing neurons. A nonfusion protein generated in Escherichia coli hydroxylates both tryptophan and phenylalanine. We propose that there are only two aromatic amino acid hydroxylase genes in Drosophila: one encoding tyrosine hydroxylase, DTH, and DTPH, a gene encoding both tryptophan and phenylalanine hydroxylase activities.

Numerousstudiesinbothvertebratesandinvertebrates have implicated the biogenic amines in synaptic modulation and behavioralplasticity. The genes and theencoded proteins for tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2, TH’) and tryptophan hydroxylase (tryptophan 5monooxygenase, EC 1.14.16.4, TRH), the rate-limiting enzymes in the synthesis of dopamine and serotonin, respectively, have thus far been characterized primarily in mammalian species (for example,rabbit TRH, Grenettet al., 1987; rat TRH, Darmon et al., 1988; rat TH, Grima et al., 1985). These two enzymes belong to a gene family of which phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC

1.14.16.2, PAH) is the thirdmember. Current molecular data from amino acid sequence comparisons suggest that TH diverged from the ancestral gene before TRH and PAH diverged from each other (Grenett et al., 1987). These enzymes hydroxylate their respective amino acid substrates by similar mechanisms (Kaufman and Fisher, 1974), and characterization of the genes encoding these enzymes suggests that they arose froma singleancestral gene. Comparison of the deduced amino acid sequences of the hydroxylases suggests that the amino-terminal third of the protein contains the regulatory and substrate specificity domain, and the restof the protein is responsible forthe hydroxylase activity (Ledley et al., 1985). We have initiateda molecular genetic analysis of this gene family in Drosophila. The Drosophila central nervous system contains catecholamine and serotonin;however, the function of these neuroactive molecules is not understood. The Drosophila gene encoding T H has been cloned using a molecular probe for the vertebrategene ( D T H , Neckameyer and Quimn, 1989). The high degree of conservation between Drosophila TH and its vertebrate counterparts suggests that studies to characterize theregulation of T H in Drosophila may be useful in understanding vertebrateT H as well. In this paper we have used a full-length clone encoding rabbit TRH (Grenettet al., 1987) to isolateDrosophila cDNA clones by reduced stringency hybridization. We present evidence to suggest that Drosophila contains only two aromatic amino acidhydroxylase genes: D T H , and a gene encoding both tryptophan and phenylalanine hydroxylaseactivities. This gene will be referred to asDTPH. MATERIALSANDMETHODS

Southern Hybridization and Screeningof Drosophila cDNA Libraries-4pgof wild-type Canton S Drosophila genomic DNA was digested with various restriction enzymes (New England Biolabs), electrophoresed through a 0.7% agarose gel, and transferred to nitrocellulose filter paper (Southern, 1975). Low stringency conditions (6 X SSPE, 50 “C)were as described in Neckameyer and Quimn (1989). The probe (pTRH779) was a 2,441-base pair cDNA containing the complete coding region for rabbit tryptophan hydroxylase, the gift of Savio Woo of Baylor College of Medicine. (Ledley et al., 1987). Low stringency conditions for screening the Xgtll recombinant head* This work was supported by National Institutes of Health Grant specific Drosophila cDNA library (Itoh et al., 1985), similar to those NS23510. The GCG computer analysis was made feasible by National used for the Southern analysis, were as described in Neckameyer and Institutes of Health Shared Instrumentation Grant RRO 4671. The Quimn (1989). Higher stringency hybridization of the rabbit TRHcosts of publication of this article were defrayed in part by the homologous Drosophila cDNA clones was done at 42 “C in 50% payment of page charges. This article must therefore be hereby formamide, 3 X SSPE, 50 mM Tris-HC1 (pH 7.6), 1 X Denbardt’s, 1 marked “advertisement” in accordance with 18 U.S.C. Section 1734 mM EDTA, and 20 pg/ml salmon sperm DNA. The filters were then washed at 65 “C in 2 X SSPE, 0.2% SDS. solely to indicate this fact. A cDNA clone isolated from this library, XcDTPH.1, was used to T h e nucleotide sequence(s)reported in thispaperhas been submitted to the GenBankTM/EMBL DataBankwith accession number(s) probe a cDNA library containing sequences from0-3-h embryos M81833. (Poole et al., 1985), using higher stringency hybridization conditions $ T o whom reprint requests should be addressed. Tel.: 617-736- of 50% formamide, 5 X SSPE, 20 pg/ml salmon sperm DNA, 0.5% 3176; Fax: 617-736-3107. SDS, 20 mM Tris-HC1 (pH 7.6), 1 mM EDTA at 42 “C followed by The abbreviations used are: TH, tyrosine hydroxylase; TRH, washes in 0.2 X SSPE, 0.2% SDS at the same temperature. tryptophan hydroxylase; PAH, phenylalanine hydroxylase; SDS, soD N A Sequencing-DNA fragments fromcDNA clones isolated dium dodecyl sulfate; kb, kilobase(s). from the Xgtll library were subcloned into the polylinker regions of

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M13mp18 and M13mp19 (Messing, 1983; Yanisch-Perron et al., 1985) acrylamide gels and transferred to nitrocellulose using standard proand their sequence determined by the method of Sanger et al. (1977) cedures (Harlow and Lane, 1988).The affinity-purified antibody was using the Sequenase enzyme (U. S. Biochemical Corp.). The sequence used at a dilution of 1:500 and was visualized using an alkaline of hDTPH.l was multiply confirmed using M13 subclones of both phosphatase-conjugated antibody to rabbit IgG (Bio-Rad). polarities (with the exception of the last 5% of the sequence, containImmunocytochemistry-Central nervous systems from larval ining the 3"untranslated region, which was multiply sequenced on one stars were hand-dissected in Ikeda-Ringer solution, and the tissues strand). Double-stranded deletions of a representative embryonic were incubated for 2 h in 4% paraformaldehyde in phosphate buffer cDNA clone wereprepared as described by Henikoff (1987) and were (pH 7.2) followed by a 20- min incubation in methanol. The samples sequenced as described above. Computer analysis was done using the were washed 2 X 10 min, 4 X 30 min in PBT (phosphate-buffered Wisconsin GCG program. saline, 0.1% bovine serum albumin, 0.1% Triton X-100) and incuRNA Isolation, Blotting, and Hybridization-Polyadenylated RNA bated overnight in a 1:50 dilution of DTPH antibody in PBT. The was isolated from 0-6-h embryos, and adult heads and bodies of wild- samples were washed in PBT, incubated for 4-6 h in a 1:40 dilution type Canton S flies using the phenol-chloroform method. 2 pg of each of arabbit IgG conjugated to fluorescein (Cappel), washed again with sample was separated on formaldehyde, 1% agarose gels, blotted, PBT, andrinsed for 10 min in 5 mM sodium carbonate (pH 9.5). The hybridized, and washed using the high stringency conditions described samples were mounted in 5% n-propyl gallate in 20 mM sodium in Neckameyer and Quimn (1989). carbonate (pH 9.5), 80% glycerol and viewed under a fluorescence In Situ Hybridization to Polytene Chromosomes-Salivary gland microscope. All incubations were done at room temperature. Embryos chromosomes from third instar Canton S larvae were dissected and were dechorionated and devitellinized as described in Luo et al. (1990) fixed according to the procedure of Engels et al. (1986). Biotinylation and treated as described above except that all antibody incubations of probes, hybridization, and washing conditions were identical to were done in 1.5% normal goat serum. those described. Enzyme Assays-The 1.45-kb cDNA insert from XDTPH.l was RNA in Situ Hybridization to Tissues-For hybridization to whole subcloned into the T7 promoter vector pT7-7 (Tabor and Richardson, embryos, antisense digoxygenin-labeled RNA probes from DTPH 1985) and transformed into the Escherichia coli strain JM109/DE3 (cloned into SK+, Stratagene) and DTH (cloned into pGEM1, Pro- (Promega). Eighteen additional in-frame amino acids were inserted mega) were made exactly according to the manufacturer's protocols amino-terminal to the DTPHputative initiator methionine. Produc(Boehringer Manneheim). In situ hybridization was according to tion of the DTPH protein was induced by the addition of isopropyl Tautz and Pfeifle (1989). The embryos were dechorinated, fixed, and 1-thio-0-D-galactopyranoside to 2 mM at an AW of 0.7 followed by devitellinized as described in Luo et al. (1990). The probes were first incubation a t 37 "C for an additional 3 h. A single induced band of tested on Southern dot blots according to the manufacturer's sugges- 50 kDa was detected on Coomassie-stained 10% SDS-acrylamide gels; tion to confirm they were capable of detecting greater than (or equal this band specifically bound the DTPH antibody on immunoblots. to) 200 pg of DTPH or DTH cDNA. The cells were harvested and resuspended in 50 mM Tris-HC1 (pH Antisense tritium-labeled RNA probes from DTPH and DTH were 7.0), 1 mM dithiothreitol, 0.2 pg/ml pepstatin, 0.4 pg/ml leupeptin, made as described in Martin-Morrisand White (1990) and hybridized and 40 p~ phenylmethylsulfonyl fluoride and lysed by the addition to 6-pm sections of fixed Drosophila melanogaster third instarCanton of lysozyme to 0.5 mg/ml. The cells were then incubated a t room S larvae, also according to Martin-Morris and White (1990). Washing temperature for 10 min with DNase I to a final concentration of 2.5 and developing of hybridized tissues were exactly as described. Under pg/ml and centrifuged at 500 X g for 5 min. The pellet containing the the hybridization conditions used, the probes did not cross-react with DTPH protein was resuspended in the above buffer, and aliquots each other. were stored at -70 'C. Approximately 75% of the recombinant DTPH Preparation of Antibody against the Drosophila Tryptophan Hy- protein pelleted with the insoluble material; this value was estimated droxylase Protein-The 1.45-kb cDNA insert from XDTPH.1was from the relative intensities of the 50-kDa protein recognized by the subcloned into the P-galactosidase fusion vector pWR590-1 (gift of DTPH antibody from equivalent amounts of the soluble and insoluble Li-He Guo and Ray Wu; Guo and Wu, 1984).Isolation of an in-frame fractions on immunoblots. The assay for phenylalanine hydroxylase fusion protein was confirmed by DNA sequencing of the P-galactosid- activity was a modification of that described by Geltosky and Mitchell ase-DTPH junction as well as by induction of the fusion protein by (1980). The incubation mixture included 50 mM Tris-HC1 (pH 7.0), incubation of the cells in 1 mM isopropyl 1-thio-0-D-galactopyrano-2 mM dithiothreitol, 0.1 mg/ml catalase (Sigma), 0.4 mM L-phenylside (Sigma), blotting the cell extract onto nitrocellulose, and visu- alanine, crude protein, and 0.3 mM 2-amino-6-methyl-tetrahydrobializing the -110-kDa fusion product with an antibody to P-galacto- opterin (Sigma), a synthetic pterin cofactor, in a volume of 0.2 ml. sidase (Promega). Isolation and purification of the fusion protein The reaction was allowed to proceed for 30 min at room temperature in open air and stopped by the addition of an equal volume of 4 "C were according to Rio et at. (1986). The protein solution was dialyzed at room temperature with several 15% trichloroacetic acid. The denaturedprotein wasremovedby changes against 50 mM Tris-HC1 (pH 8.0), 10% glycerol, 0.5 M NaCl centrifugation, and the supernatantwas added to 0.8 ml of nitrosonand spun at 8,000 rpm for 10 min at 4 "C in an HB4 rotor (Sorvall). aphthol reagant, incubated at 55 'C for 30 min. After allowing the Protein concentration was determined using a modified Bradford tubes to cool to room temperature, 10 ml of 25% ethanol was added, assay (Bradford, 1976; Bio-Rad). The supernatant was electropho- and fluorescence emission was measured at 565 nm with an activation resed onto 1.5-mm-thick 7.5% acrylamide-SDS preparative gels, and at 470 nm in a Perkin-Elmer MPF-4spectrofluorometer. To assay tryptophan hydroxylase activity, the crude protein was the fusion protein product was cut out and stored a t -70 "C. The gel the above reaction mixture with 4 mM Lslice was pulverized by several passages through a 20-gauge needle, incubated in 0.5mlof substituted for L-phenylalanine. The reaction was and 50-100 pg of protein in asolution containing phosphate-buffered tryptophan saline and 50% Freund's complete adjuvant (and subsequently with stopped by the addition of 0.1ml of 70% perchloric acid, the denatured Freund's incomplete adjuvant) was injected at 4-5-week intervals proteins were removed by centrifugation, and the supernatant was into three female New Zealand White rabbits. The IgG fraction of added to 3 ml of 4 N HCl (based on the assays done by Kuhn et al., the rabbit serum was concentrated by ammonium sulfate precipita- 1980). The fluorescence was measured at 540 nm with an activation at 295 nm using a 31 filter. tion, and one-tenth of the total bleed (-2 ml) was loaded onto an The assays were performed with either tyrosine or 5-hydroxytrypaffinity column, prepared as follows. 10-30 mg of the fusion protein (prepared as described above, through the centrifugation through tophan included as standards. Controls included incubations without urea-sucrose) was dissolved in 5-10 ml of 6 M guanidine HCl and cofactor, without substrate, and with crude protein from E. coli allowed to bind overnight at 4 "C to cyanogen bromide-activated transformed with the pT7-7 plasmid alone. Substrate competition Sepharose (Pharmacia LKB Biotechnology Inc.). The antibody was assays were done with 1 mM trytptophan and 0.4 mM phenylalanine. eluted with 0.1 M glycine HC1 (pH 4.0). No DTPH- or P-galactosidaseimmunoreactive material was found in the column flow-through. RESULTS Immunoblot Analysis-Crude protein was extracted from embryonic, larval, pupal, and adult stages by grinding the samples with a Isolation and Characterization of Drosophila cDNA ClonesTeflon pestle in a 1.5-ml microcentrifuge tube containing phosphate- To determine if TRH-homologous sequences are present in buffered saline with 0.2 pg/ml pepstatin, 0.4 pg/ml leupeptin, and 40 the Drosophila genome, wild-type Canton S genomic DNA JGMphenylmethylsulfonyl fluoride. A brief centrifugation (2 min) at was digested with various restriction enzymes and hybridized 5 X the relative centrifugal force (4"C) followed to pellet cuticular debris. Protein concentrations were determined using the Bio-Rad at reduced stringency to a full-length cDNA encoding rabbit assay. Protein samples were electrophoresed through 10% SDS-poly- TRH (pRBTRH479, a gift of Dr. Savio Woo, Grenett et al.,

and Tryptophan Hydroxylase

Drosophila Phenylalanine

1987). Apattern suggestive of a single gene was revealed (Fig. 1B). This same probe was used to screen (again, at reduced stringency) a cDNA library made from adult head mRNA. Thelargest cloneisolated inthisscreen, XcDTPH.1, was hybridized under more stringent conditions to the same genomic Southern blot. XcDTPH.1 recognized the samegenomic bands as those recognized by rabbit TRH, confirming that this cDNA arosefromthesame locus (Fig. L4). Other (smaller) rabbit TRH-homologous Drosophila cDNA clones wereisolated in this screen, and all recognized this same pattern of restriction fragments. The relative simplicity of the pattern revealed by hybridization of the TRH-homologous probes to thegenomic Southern blot suggests the majority of the TRH-homologoussequences reside in a single restriction fragment (the 4.5-kb EcoRI, the 5-kb PstI, and the -14-kb PvuII fragments). BamHI and Sac1 cut Drosophila genomic DNA less frequently,andtheresultingTRH-homologous fragment is larger (greater than 15 kb). At thereduced stringency conditions for Southernhybridization (Fig. 1B),a weakly hybridizing pattern of fragmentscan bedetected. These correspond to DTH-homologous sequences. No other bands are seen. The insert from XcDTPH.1 was sequenced in its entirety and was found tobe 1,449 nucleotide base pairs in length, not including a13-nucleotide poly(A)tail (Fig. 2). Thereis a single large open reading frame from nucleotides 1 to 1,367, but the ATG at nucleotides 10-12 most likely codes for the initiator methionine. The immediate upstream sequence, G A A A (AUG) agrees well with the Drosophila initiation site consensus C/A A A A/C (AUG) (Cavener, 1987). Assuming that this ATG does code for the initiator methionine, the deduced molecular mass of the predicted 453-residue protein is 51,539, which is very similar to thoseof the TRH and PAH proteins deduced from thesequences of the mammaliangenes. A poly(A)tail is found81 base pairsfrom the TAG stopcodon a t nucleotides 1,368-1,370, and the polyadenylation signal AATAAA is found 16 nucleotidesupstream of the poly(A) tail (underlined in Fig. 2). Thelargestindependently isolated clonesrecovered in the screening of the 0-3-h embryonic cDNA library, XcDTPH.4 and XcDTPH.5, contain identical inserts of 1.5 kb. Both begin a t nucleotide 84 of the XcDTPH.1 sequence and contain very long poly(A) tails. The sequences of the embryonic and adult head cDNAs are colinear, with 13 scattered single base substitutions, only two of which result -

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FIG. 2. Nucleotide sequence of the Drosophila. cDNA clone XcDTPH.1 and deduced amino acid sequence of the TPH protein. The nucleic acid sequence of DTPH is shown in A . Each amino acid is represented by a capital letter shown underneath the first nucleotide of the triplet codon. The stop codon is denoted by an asterisk, and the polyadenylation signal is underlined. The serine residue postulated to be phosphorylated by the Ca2+/calmodulin-dependent protein kinase type I1 is denoted by the arrow. The sequencing strategy is shown in B. The coding sequence of DTPH is represented by the heavy line, and the 3"untranslated region is denoted by the lighter line. Only the HpaIIrestriction enzyme sites are shown, for simplicity, although fragments from several different restriction enzyme digests were sequenced. Direction and extent of sequence determined are shown by the arrows.

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FIG. 1. Restriction enzyme analysis of wild-type Canton S Drosophila. Genomic DNA (4 pg) was digested with the indicated enzymes, electrophoresed through a 0.7% agarose gel, blotted onto nitrocellulose, and probed at moderate stringencywith the Drosophila cDNA clone XcDTPH.1 ( A ) or at reduced stringency with the fulllength clone encoding rabbit T P H ( B ) .

in an amino acidchange. Both of theseare conservative changes. The embryonic cDNA library was constructed from the Oregon R strain of D. melanogaster and the adult head cDNA library from Canton S. We presume that thenucleotide substitutions arise from differences between the two strains and that thecDNAs represent the same transcript. In vitro transcription followed by in vitro translation of the DTPH head cDNA with a rabbit reticulocyte system shows that the DTPHcDNA is capable of directing the synthesis of

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DTPH FIG.3. Expression of the Drosophila TPH gene. 2 pg of poly(A) RNAfrom adultheads, bodies, and 0-6-hembryos from Canton S flies was electrophoresed through formaldehyde denaturing gels,blotted onto nitrocellulose,and hybridizedwithXcDTPH.1 ( D T P H )or XcDTH.1 ( D T H ) .Equivalent exposures are shown. The -1.7-kb transcript seen with DTH is not the result of cross-hybridization with DTPH but specifically hybridizes to the 3“untranslated region of the DTH transcript.

(notshown) shows thatthereisgreateridentity between DTPH and rat PAH and rat TRH than between DTPH and Drosophila T H at both the nucleotide and amino acid levels, implying, as has been suggested by Grenett et al. (1987), that T H diverged from the ancestralgene early in theevolution of this family. There is50% identity between the deduced amino acid sequences of DTPH and rabbit TRH and 58.7% identity between DTPH and rat PAH. In comparison, there is a 46% identity on the amino acid level between DTH and DTPH. Although there is somewhat more identity between DTPH and rat PAH thanbetween DTPH and rabbit TRH, there do exist residues in common between the Drosophila protein and tryptophan hydroxylase which are not foundin phenylalanine hydroxylase. These residues are found throughout the proteins, including both theregulatory and hydroxylase domains. RNA in Situ Hybridizations-RNA in situ hybridization experiments were undertaken to characterize the distribution of the DTPH and DTH transcripts further. DTPH and DTH cDNAs were cloned into transcriptionvectors, linearized, and antisense RNA probes were made using digoxygenin-labeled nucleotide. As expected, there was no detectable expression of DTH in0-6-h embryos, consistent with the Northern data (Fig. 3). However, DTPH transcripts were strongly detected in 0-6-h embryos (and less strongly, but stillclearly present, in 0-40-min embryos; data not shown). The expression was ubiquitous throughout the embryo; however, the signal appeared to concentrate ina yolk granules. Third instar larvae were fixed, embedded in paraffin, sectioned,and hybridized to tritium-labeled DTPH or DTH antisense RNA probes (Fig. 4). Not unexpectedly, since dopamine is an intermediate in the formation of melanin, the majority of DTH expression was detected in cuticulartissue. Note the intense stainingof mouthparts by the DTH probe in Fig. 40. The majority of DTPH expression was localized to fatbody (the Drosophila equivalent of liver tissue), aswell as to mouthparts, although the intensityof cuticular expression was significantly less for DTPH than DTH. Developmental Immunoblot Analysis-Antibodies against the DTPHprotein were generated to aid in further characterization of this locus. A 0-galactosidase-DTPH fusion protein wasexpressed in E. coli, and the denatured antigen from acrylamide gels was injected into New Zealand White rabbits. After two boosts of antigen within a 2-month period, serum from all three injected rabbits recognized a single 50-kDa species on immunoblots in Drosophila head extracts as well as the fusion protein band used as antigen. These proteins

a50-kDa protein (data not shown), the size expected for translation of the cDNA. The size of this proteinis essentially identical to those of the vertebrate TRH proteins. Since the embryonic cDNA clones lack the initiator AUG, they cannot successfully be translated in vitro. Phosphorylation of the hydroxylase enzymes by several kinases has been implicated in their regulation in vivo. A serine residue inthemammalian enzymes believed to be phosphorylated by the Ca“jca1modulin-dependent protein kinase type I1 (Ehret etal., 1989) is conserved in DTPH (the residue is denoted inFig. 2 by the arrow). Chromosomal Localization-In situhybridization of the DTPH cDNA to third instar larval salivary gland polytene chromosomes was performed to confirm the presence of a single site for this gene, as well as to initiate a screen for possible mutants. The gene was localized to 66A on the left arm of the third chromosome, proximal to the DTHlocus a t 65B (data not shown). No other sites were detected. T o date, no previously isolated mutant strains map in region this which would qualify as potential TRH mutations. Northern Analysis-The insert from XcDTPH.1 was hybridized to poly(A)+ RNA blots and revealed a single transcript of about 1.75 kb which was enriched in adult heads and present in adultbodies, as expected for a gene encodingTRH. However, this transcript was alsodetected(although a t a lower level) in polyadenylated RNA isolated from 0-6-h embryos, a time pointa t which no serotonin-containing neurons are present (Fig. 3). Hybridization of this same Northern filter to a Drosophila T H probe showed that DTH transcripts arenot expressed in 0-6-h embryos. The -300-base pair difference in length between the transcript and the cDNAs is probablycomposed of 5”untranslated sequences,which is comparable to the size of the 5”untranslated region of DTH.‘ Unlike the Drosophila T H transcript, the DTPH transcript FIG.4. Expression of DTPH and DTH in the third larval does not appear to contain a long 3’-untranslated region. The instar. Sagittal sections of the anterior region of third instar larvae importance of that sequence (if any) is notclear. are shown. A and C show the bright-field photomicrographs for DTPH Homology with Other Hydroxylases-Alignment of the de- and D T H , respectively. In the dark-field exposures for DTPH and duced DTPH, Drosophila TH, rat PAH, and rat TRH proteins DTH ( B and D, respectively), grains are observed in mouthparts and in fat body exclusively for DTPH. fb, fat body; m, mouthparts; cu, cuticle. Anterior is left. Bar, 100 pm. W. S. Neckameyer, unpublished results.

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lateral neurons were in locations and in a pattern similar to the previously defined serotonin neurons (Vall6s and White, 1988). Simultaneous labeling experiments with serotonin antibodies confirmed that the DTPH immunoreactivity localized to serotonin-immunoreactive neurons (data not shown). DTPH immunofluorescencewas alsodetected in the ring gland (Fig. 6C) and inprocesses in the ventral ganglion (Fig. 6B), which appeared identical to those seen with antibodies against serotonin(Vall6s and White,1986). No staining above background was seen in gut, malpighian tubules, cuticle, or imaginal discs (data not shown);however, background staining with preimmune serum is quite high in these tissues and may obscure a real signal. The DTPH-immunoreactive dorsolateral neurons and the unpaired medial neurons were in the same location as the neuronal sets detected by glyoxylic acid-induced histofluorescence (Budnik and White, 1988), suggesting that the catecholamine-containing neurons are also DTPH-immunoreactive. Immunocytochemical studiesdonewithan antibody raised in rabbits against mammalian T H (Pel-Freez) andwith the DTPH antibody revealed that the TH-immunoreactive patternis a subset of thepatternseenwiththeDTPH antibody. This is consistent withlocalization the of the DTPH transcript to dopamine-containingtissues. Cells corresponding in position to the thoracic dorsolateral catecholamineneurons were not detected. However, these cells were only observed in some samples with glyoxylic acidinduced histofluorescence and were not observed with theless sensitive dopamine or T H immunocytochemistry (Budnik et al., 1986; Budnik and White, 1988). The DTPH antibodyalso recognizes yolk granules in 0-6hembryos (datanotshown).Thisisconsistent with its localization in immunoblots (Fig. 5) to embryonic stages and to female but not male abdomens. Enzyme Assays-To demonstrate that DTPHdirected the hydroxylation of bothphenylalanineandtryptophan,the adult head DTPHcDNA, which can direct the synthesisof a 50-kDa protein species in vitro, was placed under the control of a n inducible T 7 promoter and introduced intoE. coli. The 50-kDa speciesis presumably a singlesubunit of a multimeric FIG.5. Developmental immunoblot analysis of DTPH. 100 enzyme; however, the recombinant human PAH subunit profig of crude protein extracted from 0-6-, 6-12-, 12-18-, and 18-24-h embryos, first, second, and third larval instars, early and late pupa, duced in E. coli is capable of hydroxylating phenylalanine to and heads, thoraxes, and (male or female) abdomens from wild-type tyrosine in thepresence of exogenous cofactor (Ledley et al., Canton S flies were loaded onto 10% SDS-PAGE gels, electroblotted 1987). E. coli protein extracts containing theinduced DTPH onto nitrocellulose, and incubated with a 1:500 dilution of affinity- protein were assayed for the ability hydroxylate to both phenpurified antibody to DTPH. The proteins were visualized by a color ylalanine and tryptophan (Fig. 7). Similarly induced E. coli reaction using alkaline phosphatase-conjugated secondary antibody (Bio-Rad). The molecular masses of the marker proteinsare indicated containing thepT7-7 plasmid alonewas incapable of hydroxylating either substrate, consistent with the fact that these in kDa to theside. bacteria do not contain iron-dependent aromatic amino acid hydroxylases. Therefore, all activity assayed must result from the induced D T P H 50-kDa species. Although it is frequently difficult to generate a recombinantbacterialprotein with enzymatic activity (see, for example, Katarova et al., 1990), the crude extract containing theinduced protein was clearly able to hydroxylate both tryptophan and phenylalanine in assays similar to those usedfor the mammalian enzymes. Incubation of the reaction mixtures in tubesopen to the air proved essential foractivity. This is consistent with the dependence on molecular oxygen for hydroxylation by the vertebrate enzymes. A B C Phenylalanine hydroxylase activity of the DTPH protein FIG.6. Immunocytochemicalanalysis of a first larvalinstar was decreased by the inclusion of tryptophan in the reaction with aDTPH. The central nervous system was hand-dissected from mixtures (Fig. 7A). As would be expected, tryptophan hydroxthe larvae and incubated with affinity-purified antibody to DTPH and fluoroescein isothiocyanate-conjugatedanti-rabbit IgG as sec- ylase activity was also decreased by the inclusion of phenylondary. Three different focal planes are shown to highlight the alanine (Fig. 7B), suggesting that thetwo substrates compete for bindingto the DTPH protein. pattern of staining. A, medial; B, ventral lateral; C, dorsolateral.

were not recognized with any preimmune serum (data not shown). Developmental immunoblot analysis showed a single protein comigrating a t 50 kDa in extracts from all larval and pupal stages and adult heads(Fig. 5). Surprisingly, a 45-kDa specieswas recognized in embryonic extracts. The 50-kDa band first appears in12-18-h embryos, just as the level of the 45-kDa protein begins to decrease. The level of the 50-kDa species is maintained through the adult stage and increases somewhat in third instar larvae, late pupae, and adult head. The 45-kDaspecies is also detected infemale but not inmale abdomens. Immunocytochemistry-Antiserum from one of the three rabbits proved useful for immunocytochemistry. The central nervous system was dissected from all larval stages and incubated with a 1:50 dilution of affinity-purified antibody. At all of these developmental stages, distinct neurons were detected in the ventral ganglion and the brain lobes (Fig. 6). Preimmune serum from this rabbit did not show any specific staining. The patternof DTPH-immunoreactive neuronswas more complicated than expected on the basis of known serotonin neurons. The pattern in the ventral ganglion was analyzable because of itssegmentalnature.Threesegmental subpatterns were readily identified 1)pairs of ventral lateral neurons (Fig. 6B, also detected in the focal plane of 6A); 2) dorsolateral neurons in the abdominal neuromeres(Fig. 6C); and 3) medial unpaired neurons(Fig. 6A). The pairsof ventral

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had fully expected to find a complex Southern pattern under low stringency conditions with the rabbit TRH probe. However, we observed a simplepattern indicativeof a single gene. This implied that either theDrosophila PAH and TRHgenes had diverged further from each other relative to the vertebrate enzymes, and the rabbit TRH probe recognized only one of the two genes, or that therewas only one gene encoding both enzymes. The pattern of restriction fragments in Drosophila genomic DNA recognized by thefull-lengthrabbit TRH cDNA is the same patternrecognized by several rabbit TRHhomologous clones isolated from Drosophila cDNA libraries. Amore weakly hybridizing pattern of fragments could be protein detected after hybridization of the probes at reduced strin0.4mM L-phe c .4mM L-trp c-". 1mM L-trp, 0.4 mM c .1mM L-trp. 0.4 mM gency. These bands correspond to those recognized by both L-phe L-phe mammalian and Drosophila T H probes. This suggests that FIG. 7. Enzymatic assays of induced DTPH protein in E. onlytwo aromaticamino acidhydroxylasegenes existin coli. The cDNA insert from k D T P H . l was placed under the control Drosophila: one corresponding to TH, and the other correof an inducible T7 promoter and transformed into E. coli. Increasing Drosophila amounts of crude extract showed increasing tryptophan hydroxylase sponding to TRH and PAH. While screening the cDNA libraries for rabbit TRH-homologous sequences, dupliactivity, which could be competed out by the inclusion of L-phenylalanine. B , the same extract also directed the production of tyrosine cate filters were hybridized under more stringent conditions from phenylalanine, which could be competed out by the presence of with DTH. Those clones with strong homology to the verteL-tryptophan inthe reaction mixture ( A ) .Relative fluorescence from brate TRHgene hybridized, although more weakly, to DTH, nonsubstrate, noncofactor, and plasmid controls wasused as base indicating that thehybridization conditions were sufficiently line. This figure shows the results of two independent assays from a reduced to detect cross-hybridization atoless similar species. single extract. A single locus was detected after in situ hybridization of the D T P H probe t o salivary gland chromosomes, and a single Reactions incubated without either pterin cofactor or with- transcript was detected in both early embryos and in adults out either L-phenylalanine or L-tryptophan were also inca- on Northern blots. Nucleic acid and amino acid comparison pable of producing the expected product, demonstrating that indicated that DTPHis highly homologous to both mammathe hydroxylation was specific to the exogenous substrate lianPAHandTRHand less so toTH.Thesedataare and, similar to the mammalian aromatic aminoacid hydrox- consistent only with either a single locus encoding both PAH ylases, was absolutely dependent on the pterin cofactor for and TRH or two separate but contiguous genes. The simple activity. As mentioned above, reactions incubated with no genomic Southern pattern refutes the latter possibility. AdDTPH protein showed no activity, demonstrating that both ditionally, there is only a single class of genomic clones with phenylalanine and tryptophan hydroxylase activities extrap- homology to tryptophan hydroxylase.3 Since Drosophila must olate to zero. The protein, which pelleted with the insoluble require TRH and PAH activities, we propose that this single material, was clearlyactive. The soluble fraction was too locus provides both these functions. dilute t o yield significant activity. The unexpected transcript detected in poly(A)+ RNA from In some preparations, increasing activitywas seen for both 0-6-h embryos could be specific for PAH function andwould hydroxylation reactions with increasing amounts of previ- explain the presence of this transcript at a stage in developously saturating substrate (not shown) because of the varia- ment prior to the presence of defined serotonergic neurons. bility of induction. Additionally, the protein pelleted with The 0-6-h transcript most likely encodes the 45-kDa embryinsoluble material, making uniform resuspension more diffi- onic protein that is sequestered yolk in granules and detected cult. Under the most optimal conditions, a band could be seen in female but not in male abdomens. Using an antibody to on a Coomassie-stained gel which was specifically recognized dopa decarboxylase, Konrad and Marsh (1987) detected no by the DTPH antibody. We estimate this level of induction dopa decarboxylase expression in Drosophila ovaries or in 0to yield no more than 5% DTPH protein. Under less optimal 14-h embryos.Valles and White (1988) did not detect the conditions, no Coomassie-stained gel band could be detected presence of serotonin either in ovaries or 0-16-h embryos. It although a 50-kDa species could be detected on immunoblots is therefore highly likely the RNA and protein expression with the DTPH antibody. We estimate the specific activity detected in earlyembryogenesis reflects aPAH component of of the crude DTPH protein induced under the conditions this locus. The cDNAsisolated from a 0-3-h embryonic described in Fig. I to be 29 nmol of 5-hydroxytryptophan library are essentially identical to those isolated from head formed per mg of protein/30 min at 25 "C and 119 nmol of tissue, suggesting that the 45-kDAspeciesdoes not arise tyrosine formed per mg of protein/30 min at 25 "C for tryp- through alternative splicing but more likely through posttophan and phenylalanine hydroxylase activities, respectively. translational modification(s). Assuming that the45-kDa emHowever, all induced E. coli cultures demonstrated both tryp- bryonic species largely (or exclusively) functions as a phentophan and phenylalaninehydroxyase activities. ylalanine hydroxylase, one could speculate that the substrate preferences of the 45- and 50-kDa DTPH species might be DISCUSSION determined by changes in the amino-terminalregulatory doBased on theevidence presented in this paper and discussed main or by differential phosphorylation of potential sites. It below, we propose that in Drosophila a single locus encodes should also be noted that the hydroxylase enzymes in verteboth TRH and PAH activities. This is in contrast to vertebrates are multimeric and that the differences between the brates, inwhich independent loci encode PAH and TRH. The 45- and 50-kDa species could form a conformational change vertebratePAHandTRH genes share extensive identity in theholoenzyme resulting in altered substratespecificities. (rabbit trytophan hydroxylase shares 230 of 434 amino acid The presence of the DTPH transcript in dopamine-conresidues with human phenylalanine hydroxylase; Grenett et G. Grasso and K. White, unpublished observations. al., 1987) and are more similar to each other than to TH. We

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ytryptophan. In dopaminergic neurons, tyrosine is hydroxyltaining tissues such as mouthparts, which do not contain serotonin, suggests that this transcript is present in these ated by TH, andremoval of the product may help stimulate the phenylalaninehydroxylase reaction. It is also conceivable tissues to encode a protein that can hydroxylate phenylalaspecific kinases, proteolytic ennine to tyrosine.The DTPH transcript is detected in fatbody, that other factors such as zymes, and/or phosphatases may help regulate the hydroxylthe Drosophila equivalent of mammalian liver (a tissue in which PAH is highly expressed). DTH, under identical hy- ation reactions. The cloning of a Drosophila PAH homologue has recently bridizationconditions, does not cross-hybridizewith the DTPH transcript in fat body (Fig. 4). This transcript must been reported (Moraleset al., 1990). Weagree on much of the a substantially also encode a protein capableof hydroxylating tryptophan to nucleotidesequence data,buttheyreport 5-hydroxytryptophanasthefirststepinthesynthesis of different (andless similar with either PAHor TRH) deduced serotonin. (Drosophila synthesizesserotoninthrough a 5- amino-terminal protein sequence (within the first 120 residues). Thedifferences cannot be accounted for by alternative hydroxytryptophanintermediate;LivingstoneandTempel, splicing. Additionally, little characterization of the gene or 1983.) The level of expression of this transcript in the nervous system is expected t o be significantly reduced relative to that gene product(s) was reported, and their deduced amino acid sequence was compared with only vertebrate TH and PAH found in other tissues since tryptophan hydroxylase is the rate-limiting enzyme in the synthesis of serotonin, which is and not with vertebrateT R H or with Drosophila TH. We suggest thattheancestral hydroxylase gene most specifically localized to a small population of cells within the nervous system (Valles and White, 1988). DTH expression in strongly resembles PAH in structure and function and that the nervous system is similarly reduced relative to that found the other hydroxylases evolved and were refined for use in the nervous system (and, for bothDTPH and DTH, cuticular in cuticular tissue. Additional evidence that D T P H does encode T R H activity tissue). The estimated evolutionary distance between PAH comes from the immunocytochemical data. Previously iden- and TRH is-600 million years (Grenettet al., 1987), approxtified serotonin-immunoreactive cells are also DTPH-immu- imately the same as that between vertebrates and invertebrates. We propose that in Drosophila, and presumably in noreactive, but the pattern of immunoreactive neurons includes both the dopamine- and serotonin-containing neurons.other invertebratespecies, the ancestralhydroxylase gene has diverged to form T H a n d a gene encoding both TRH and It isnot likely that the DTPH immunoreactivityinthe PAH functions. In all mammalianspecies examined thus far, dopamine-containingneuronsiscaused by cross-reactivity againstTH,assuchcross-reactivity was not observed on separate enzymes exist for the hydroxylation of tyrosine, in tryptophan, and phenylalanine. Comparative enzymatic studimmunoblots,norcantheantibodyimmunoprecipitate uitro translated DTH. The DTPH transcript is expressed in ies of the Drosophila aromatic amino acid hydroxylases with will enableustodetermine dopamine-containing tissue,so one could expect tosee DTPH theirvertebratecounterparts which regions of the proteins are involved in which activities protein in these tissues, as well. If 5-hydroxytrptophan (and therefore serotonin) is generated in Drosophila by an evolu- (substrate recognition, hydroxylation), and will aid in charhydroxylationreactionarily diverged hydroxylase, it isunlikely that we would be acterizingthosefactorsregulatingthe tions. Given the neuronal localization of TRH and the nonable to detect serotonin-containing neurons with the DTPH neuronal distribution of PAH in vertebrates, understanding antibody. In vertebrates,TPH and PAH can hydroxylate each other’s the regulation of the Drosophila counterpart(s) may increase substrates (Ichiyama et al., 1976; Renson et al., 1962). That our understanding of the evolution of the biogenic amines in the 50-kDa protein is capable of PAH activity, at least in the nervous system. uitro, is suggested by the PAH activity profile presented by Ackmwkdgrnents-Wegratefully acknowledge the critical comGeltosky and Mitchell(1980). They assayed PAH activity in ments of Guissepina Grasso, Michael Lisbin, Linda Martin-Morris, Drosophila and found activity in larval instars with a peak in and Christian Brandes. We also appreciate thegift of the rabbitT P H pupal stages. At these stages of the Drosophila life cycle, the cDNA probe (pTRH779) from Dr. Savio Woo. We would especially like to thank Dr. Colin Steel of the Chemistry Department at Branfly would requireboththesynthesis of serotonininthe nervous sytem and tyrosine as partof the cuticular metabo- deis University for his generosity for the use of the spectrofluorometer. lism. The DTPH proteininduced in E. coli is capable of hydroxREFERENCES ylating both tryptophan and phenylalanine. As mentioned Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 earlier, the mammalian PAH and T P H enzymes are able to Budnik, V., and White, K. (1988) J. Comp. Neurol. 268,400-413 hydroxylate both substrates, as well. However, in vertebrates Budnik, V., Martin-Morris, L., and White, K. (1986) J. Neurosci. 6, 3682-3691 there are clearly two distinct enzymes with nonoverlapping tissue distribution. InDrosophila, there is a single gene prod- Cavener, D. (1987) Nucleic Acids Res. 15, 1353-1361 M., Guilbert, B., Leveil, V., Ehret, M., Maitre, M., and uct which must be regulated to prevent the hydroxylationof Darmon, Mallet, J. (1988) J. Neurochern. 51, 312-316 an inappropriate substrate in the different tissues. Ehret, M., Cash, C. D., and Hamon, M. H., and Maitre, M. (1989) J. These considerations suggest that the activityof the same Neurochern. 52, 1886-1891 protein must be regulated differentially in serotonergic and Engels, W. R., Preston, C. R., Thompson, P., and Eggleston, W. B. dopaminergic neurons.Given the similarityof the amino acids (1986) BRA Focus 8, 6-8 Geltosky, J. E., and Mitchell, H. K. (1980) Biochern. Genet. 18, 781substrates, the fact that these enzymes share the same pterin 791 cofactor, and that both serotonergic and dopaminergic neuGrenett, H. E., Ledley, F. D., Reed, L. L., and Woo, S. L. C. (1987) rons contain theenzyme dopa decarboxylase, what regulatory Proc. Natl. Acad. Sci. U. S. A. 82, 617-621 controls prevent the production of serotonin in dopaminergic Grima, B., Lamouroux, A., Blanot, F., Biguet, N. F., and Mallet, J. (1985) Proc. Natl. Acad. Sci. U. S. A . 82, 617-621 neurons? One such regulatory control may involve end prodGuo, L. H., Stepien, P. P., Tso, J. Y., Brousseau, R., Narang, S., uct inhibition by tyrosine. In serotonergic neurons, there is Thomas, D. Y., and Wu, R. (1984) Gene (Arnst.) 29, 251-254 no TH activity to hydroxylate tyrosine toL-dopa, and there Harlow, E.,and Lane, D. (1988) Antibodies: A Laboratory Manual, may be negative feedback from the build-up of tyrosine to Cold Spring Harbor Laboratory,pp. 471-510, Cold Spring Harbor, help force the reaction kinetics to the production of 5-hydroxNY

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