Journal of Neurochemisrry Raven Press, Ltd., New York 0 1990 International Society for Neurochemistry

Drosophila GABAergic Systems: Sequence and Expression of Glutamic Acid Decarboxylase F. Rob Jackson, Laurel M. Newby, and Shankar J. Kulkarni Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts, U.S.A

Abstract: A mammalian glutamic acid decarboxylase (GAD) cDNA probe has been utilized to isolate Drosophilu cDNA clones that represent a genomic locus in chromosome region 64A. Deletion analysis indicates that this chromosomal locus encodes an enzymatically active GAD protein. The in vitro translation of cRNA representing a Drosophila cDNA clone yields a 57-kDa protein that can be immunoprecipitated by an anti-GAD antiserum. A GAD-immunoreactive protein of the same size can also be detected in Drosophila head extracts. The nucleotide sequence derived from two overlapping Drosophilu cDNA clones predicts a 57,759-dalton protein composed of 5 10 residues that is 53% identical to mammalian GAD. Sequence comparisons of mammalian and Drosophila GAD identify two highly conserved regions

(270% identity), one of which encompasses a putative cofactor-binding domain. Transcriptional analyses show that expression of the Drosophila Gad gene commences early in embryonic development (4-8 h) and continues in all later developmental stages. A 3.1-kb class of mRNA is detected throughout embryogenesis, in all three larval stages, in pupae, and in adults. This transcript class has a widespread distribution in the adult CNS. A smaller 2.6-kb transcript is expressed in a developmentally regulated manner; it is detected only in embryos and pupae. Key Words: Insect-Neurotransmitters, inhibitory-Glutamic acid decarboxylaseGene-DOPA decarboxylase. Jackson F. R.et al. Drosophila GABAergic systems: Sequence and expression of glutamic acid decarboxylase. J. Neurochem. 54, 1068-1078 (1990).

The sophisticated genetics and well-characterized development of the fruitfly Drosophilu rnelunogaster have made this insect an attractive experimental model for studying the morphogenesis and physiology of the nervous system (Hall, 1982,1984; Rubin, 1988). These advantages have proven useful for molecular and genetic investigations of neurotransmitter and neuromodulator systems (reviewed in Restifo and White, 1989). Genetic analyses, for example, have suggested that acetylcholine and biogenic amines, in addition to functioning as neurotransmitters and/or neuromodulators within the mature nervous system, are required for proper neural development (Greenspan et al., 1980; Hall and Greenspan, 1980; Budnik et al., 1986; Chase and Kankel, 1988). These genetic studies have complemented investigations in other organisms that indicate that neurotransmitters, including y-aminobutyric acid (GABA), can regulate neural growth during embryogenesis (reviewed in Kater et al., 1988; also see Mattson et al., 1987; Lankford et al., 1988; Goldberg and Kater, 1989).

It would be of interest to utilize a genetic approach in Drosophilu to study the development and function of GABAergic inhibitory neurons. In vertebrates and invertebrates, GABA is a major inhibitory neurotransmitter of the mature CNS (reviewed in Gerschenfeld, 1973; Roberts et al., 1976; Callec, 1985). Developmental studies indicate that GABA may also have morphogenetic functions during the growth and differentiation of the nervous system (reviewed in Lauder and Krebs, 1986; Redburn and Schousboe, 1987; also see Mattson et al., 1987). The widespread distribution of GABA-containing neurons in both mammals (e.g., McLaughlin et al., 1974) and insects (e.g., Schafer and Bicker, 1986) reflects the importance of GABAergic neurotransmitter systems for a variety of physiological processes. Indeed, alterations of GABAergic transmission have been implicated in the pathogenesis of a number of human neurological and psychiatric disorders (e.g., Roberts, 1986). The synthesis of GABA in diverse species including Drosophila is controlled by the enzyme glutamic acid

Received June 9, 1989; revised manuscript received August 15, 1989; accepted August 15, 1989. Address correspondence and reprint requests to Dr. F. R. Jackson at Worcester Foundation for Experimental Biology, 222 Maple Ave., Shrewsbury, MA 01545, U.S.A.

Abbreviations used: DDC, DOPA decarboxylase: GABA, y-aminobutyric acid; GAD, glutamic acid decarboxylase; nt, nucleotide; PLP, pyridoxal phosphate; TDC, tryptophan decarboxylase.

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DROSOPHILA GLUTAMIC ACID DECARBOXYLASE decarboxylase (GAD; EC 4.1.1.15) (Chen and Widmer, 1968; Roberts et al., 1976; Chude et al., 1979). In many species, multiple forms of GAD appear to be involved in the regulation of GABA synthesis. Previous studies of mammalian and insect GAD have demonstrated the existence of different enzymatic forms, distinguishable on the basis of physical and/or kinetic properties (Frontali, 1964; Chen and Widmer, 1968; Langcake and Clements, 1974; Chude et al., 1979; Denner and Wu, 1985; Spink et al., 1985). The structural relationships among these isozymes and their functional significance remain to be determined. Although numerous studies indicate that GABA is an inhibitory neurotransmitter in the CNS and PNS of insects (reviewed in Gerschenfeld, 1973; Callec, 1985), little is known about the ontogeny, distribution, and function of GABAergic neurons in Drosophila. Several results, however, indicate that GABAergic systems exist in Drosophila. It is known, for example, that extracts of whole adults or larval brains possess considerable GAD activity (Chen and Widmer, 1968; Chude et al., 1979). The partially purified Drusophila enzyme has kinetic properties similar to those of mammalian GAD (Chude et al., 1979). In addition, Buchner et al. ( 1 988) have demonstrated that an extensive subset of Drosophila CNS neurons react with both anti-GABA and anti-mammalian GAD antisera. Importantly, physiological studies show that GABA has neurotransmitter activity in the Drosophila nervous system. For example, GABA activates an anion-selective channel present in Drosophila larval muscle (Delgado et al., 1989). In addition, GABA can mimic the endogenous inhibitory neurotransmitter of certain Drosophila neuromuscular synapses in culture (Ikeda, 1980). Molecular studies of GAD have been made possible by the recent isolation of mammalian GAD cDNA clones (Kaufman et al., 1986; Julien et al., 1987; Bond et al., 1988). These molecular probes will be extremely useful in studies of the development of GABAergic pathways and the regulation of GABA synthesis. Classical genetic approaches, however, cannot easily be applied to studies of mammalian GABAergic systems. To facilitate genetic studies of this neurotransmitter system, we have identified and characterized the expression of a Gad gene in D. melanogaster. These studies have also allowed us to deduce the amino acid sequence of an invertebrate GAD. The comparison of neural decarboxylases among invertebrate and vertebrate species has identified conserved regions that appear to correspond to important domains of function. EXPERIMENTAL PROCEDURES Drosophila stocks and culture methods Drosophila cultures were reared at 25°C and 60% relative humidity on a standard medium consisting of cornmeal, agar, yeast, sucrose, dextrose, and wheatgerm. The wild-type flies used in some experiments were collected from a CantonSpecial strain that has been maintained in our laboratory for 4 years.

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Library screens Plaque hybridizations were performed according to standard procedures (Benton and Davis, 1977),using DNA probes radiolabeled by nick translation. A Drosophila head cDNA library in Xgtll (Itoh et al., 1985) was screened under reducedstringency hybridization conditions [hybridization: 6X saline sodium citrate (SSC), 42% formamide, 37°C; washes: 2X SSC, 50°C] with a 2.4-kb feline GAD cDNA fragment (Kaufman et al., 1986). Four Drosophila cDNA clones with inserts of -2.4 kb were isolated. These cDNA clones hybridize to one another and have identical patterns of digestion with restrio tion endonucleases. Sequences that hybridize to the feline cDNA probe are limited to a 0.3-kb Sau3AI fragment present in each cDNA. One of the Drusophila cDNAs (XcDmGADl) was employed in high-stringency hybridizations to isolate 17 additional clones from the same library.

RNA analysis Total RNA was extracted according to Chomczynski and Sacchi (1987) and enriched for poly(A)+ transcripts using oligo(dT)-cellulose chromatography (Maniatis et al., 1982). Five-microgram samples of poly(A)+ RNA were denatured in formamide/formaldehyde buffer, fractionated through 1% agarosel2.2 M formaldehyde gels (Lehrach el al., 1977), and transferred to Genescreen” membranes (NEN). RNA markers (BRL) were used as size standards. Northern blots were hybridized with 32P-labeled single-stranded RNA probes. RNA probes were synthesized with T3 or T7 RNA polymerase from cDNA fragments subcloned into pBS or pBluescript SK- (Stratagene). Transcription reactions (20 p l ) contained 40 mM Tris-HC1 (pH 7 . 9 , 6 mM MgC12, 2 mM spermidine, 1 m M each of ATP, GTP, and CTP, 10 mM dithiothreitol, RNasin ribonuclease inhibitor (20 units; Promega Biotec), 50 pCi [32P]UTP(800 Ci/mmol), 2 pg linearized plasmid DNA, and 30-50 units RNA polymerase. Reactions were incubated at 37°C for 1 h and terminated by addition of 0.5 units RQ1 DNaseI (Promega Biotec). Labeled RNA was separated from unincorporated [32P]UTPby centrifugation through Sephadex G-50m. The direction of transcription for the 3. I-kb GAD mRNA was confirmed by hybridizing single-stranded RNA probes representing the two strands of a 1.5-kb XcDmGADl cDNA fragment to blots of adult poly(A)+ RNA. Only the RNA probe representing the putative antisense strand of XcDmGAD1 detected a transcript.

DNA sequence analysis DNA sequencing was performed according to standard methods (Sanger et al., 1977; Biggin et al., 1983).To sequence the XcDmGAD1 cDNA, two EcoRI fragments (1.5 and 0.9 kb) comprising the insert were gel-purified and subcloned into bacteriophage M13. The 1.5-kb fragment was also digested with Sau3AI or HaeIII, and the resulting fragments were randomly subcloned into the BamHI or SmaI sites of M13 mp19. The complete sequence of the 0.9-kb fragment was determined from the sequences of smaller overlapping restriction fragments. Contiguous sequences were assembled for the two EcoRI fragments using the Staden package ( 1 987) on a VAXIVMS 111750. Synthetic oligonucleotide primers were used to obtain the sequence of the opposite strand for all regions in which only one strand had been sequenced. On average, each nucleotide position was determined two and four times, respectively, for the 0.9- and 1.5-kb fragments. The sequence of the hcDmGAD2 cDNA fragment was determined from a set of overlapping deletions constructed in J. .Veeurochem., VoI. 54, No. 3, 1990

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F. R. JACKSON ET AL.

pBluescript SK- using the ExuIlI Erase-a-Base system (Promega Biotec). Single-stranded DNA for sequencing was rescued by superinfection with bacteriophage M 13K07. Amino acid sequence comparisons were performed using the software of Staden ( 1 982).

these experiments the increase in GAD activity was linear for incubations of up to 1 h, but not for longer incubation times (data not shown). Thus, 30- and 60-min incubations were used to assay the activity of deletion-bearing flies and their siblings.

In vitro translations and immunoprecipitations

Chromosome and tissue in situ hybridizations The protocols of Engels et al. ( 1986) were used to prepare

Sense RNA for translation experiments was synthesized from a 2.2-kb EcuRI fragment of hcDmGAD2 subcloned into pSP65pA. This vector is a modified form of pSP65 containing a 46-residue dA/dT tract inserted into the PstI site of the polylinker (Slavicek et al., 1988). Hence, it permits the synthesis of polyadenylated RNA. The DNA construct was linearized with Hind111 prior to RNA synthesis. Capped RNA was produced according to Sldvicek et al. (1988) by including 0.5 mM diguanosine triphosphate [G(S’)ppp(S’)C] in the transcription reactions. In vitro translations were performed with 2 pg of RNA and 50 pCi of [35S]methionine (- 1,200 Ci/mmol; NEN) using rabbit reticulocyte lysates (Slavicek et al., 1988).Translation reactions were stopped by adding either sodium dodecyl sulfate gel loading buffer or anti-GAD antiserum. The immunoprecipitation of translation products was performed as described (Slavicek et al., 1988) using 3 pl of anti-GAD antiserum 1440 (Oertel et al., 1981) in a total volume of 50 pl.

Protein gel electrophoresis and immunoblotting Labeled translation products and Drosophilu protein extracts were fractionated through 12%acrylamide gels (36 parts acrylamide/l part bisacrylamide) using a Mini-Protean II apparatus (Biorad). For the visualization of translation products, gels were fixed in 10% acetic acid, treated with Amplify (Amersham), and dried for autoradiography. Protein extracts were prepared from adult heads by homogenization in 20 mhf potassium phosphate, pH 7.4, in the presence of protease inhibitors ( 1 mMphenylmethylsulfonyl fluoride and 25 m M benzamidine). Homogenates were centrifuged in the cold (4°C) for 10 min at 14,000 g to remove cuticle and debris. The supernatant was retained as a crude protein extract. Total protein in these extracts was assayed by the method of Lowry et al. (1951). Gels containing Drosophilu extracts were blotted to nitrocellulose using the Mini-Protean blotting system. Duplicate blots were probed with sheep preimmune serum or anti-GAD antiserum 1440 at a final dilution of 1:1,000. Phosphatase-conjugated rabbit antibody to sheep IgC (Kirkegaard and Perry Labs) was resuspended at a concentration of 0.1 mg/ml in distilled water and used at a dilution of 1500 to detect anti-GAD antibodies. The application of antibodies and phosphatase staining were performed using the ProtoBlot System (Promega Biotec).

GAD enzyme assays GAD assays were performed using the rapid filtration method of Chude and Wu (1976). Enzyme activities were assayed in a 14,000 g supernatant of fly homogenates ( 100 flies/ml) by measuring the conversion of ~-[3,4-~H]glutamic acid (52 Ci/mmol; NEN) to [3H]GABA. Each assay utilized 250 pl of supernatant (equivalent to 25 flies). Background was determined for blanks (homogenization buffer or heatkilled extracts, containing labeled substrate solution) and subtracted from the total counts per minute before activities were calculated. Five assays per time point were performed for each extract. Total protein was determined by the method of Lowry et al. ( I 95 1 ) using bovine serum albumin as a standard. To define an optimal incubation time for GAD assays, we incubated wild-type extracts for 30, 60, or 120 min. In J. Neurochem.. Vol. 54, No. 3, 1990

and hybridize salivary gland polytene chromosomes. DNA fragments were labeled with biotin- 1 1-dUTP by nick translation. Tissue in situ hybridizations were performed according to Robinow and White ( 1 988), using 3H-labeled RNA probes. Sense and antisense probes were synthesized from a 1.5-kb EcoRI fragment of XcDmGADl cloned into the transcription vector pBS.

RESULTS Isolation and sequence of Drosophila cDNA clones We used a 2.4-kb feline GAD cDNA (Kaufman et al., 1986) as a probe to obtain cross-hybridizing Drosophila cDNA clones (see Experimental Procedures for details). Four clones, each possessing an insert of -2.4 kb, were isolated from a Drosophilu head cDNA library (Itoh et al., 1985). Sequences from one of the Drosophila cDNAs (XcDmGADl) were used to obtain additional cDNA clones from the same library. In all, 2 1 cDNA clones have been isolated and characterized. On the basis of size and restriction endonuclease digests, they fall into at least three classes. One class is represented by XcDmGAD 1 and by a larger clone (XcDmGAD2), which is equivalent in size (3.1 kb) to the predominant class of mRNA seen on Northern blots (see Figs. 4 and 5). The sequence of the XcDmGAD2 cDNA (see below) indicates that it contains the entire proteincoding domain of the corresponding mRNA. The second class of cDNA is represented by clone XcDmGAD3, which is also 3.1 kb in size but differs from XcDmGAD2 in 5’ and 3‘ sequences. A third class is identified by a 2.8-kb cDNA known as XcDmGAD4. Whereas class 1 and 2 cDNAs hybridize to feline GAD cDNA sequences, XcDmGAD4 does not hybridize to this heterologous probe. Genomic Southern analyses indicate that all four of these cDNA clones represent a single gene (data not shown). The entirety of the XcDmGADl cDNA and most of the XcDmGAD2 clone have been sequenced to deduce the primary structure of the Drosophilu translation product (Fig. 1). The two cDNAs are nearly identical in a 3’ 1.5-kb region that has been sequenced in both clones. The DNA sequence derived from these clones terminates with a poly(A) tract and contains consensus polyadenylation signals at nucleotide (nt) positions 2979 and 2983. The only lengthy open reading frame in this orientation begins at nt position 18 1 and terminates at position 17 16, leaving a l .3-kb untranslated region at the 3‘ end of the mature mRNA. The first ATG of the sequence is located at position 187, in frame with this open reading frame. The sequences flanking this ATG agree well with the consensus derived by Cavener

DROSOPHILA GLUTAMIC ACID DECARBOXYLASE

+ 2.4 kb

I

PI

E

I

259

1071

AlsThrAlaGlyrhrThrvslLe~GlqAl~Ph~AspAEpl~~A8~Th~l~~Al~A~pl~~ GCCACTGCCGGCACCACTGTGCTGCCAGCCTTCGATGATATCMCACGATTGCGGATATC

DmGADl

219

1020

T CysGlnLysTyrAsnCy6~rp~etHiElleAEpA~~A~~T~pGl~GlyGlyLeuL~u~ct

TGCCAGMCTACMCTGCTGCATGCACATCGATGCTGCWGGGGCGGTGGATTGTTCATG

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19

19 59

479 19

ProAsnPhePheArqserrlcIleScrStrrlarlavalrl~A~pva~A~pPh~

CCCARCTTCWCCCCTCGATCATCTCGTCGGCCCCGGTCMCGAGGC~ATGTGGACTTT 400 499

99 540

ATGCTGGACGAGATCCATCGCTGGGCGACGACTTGTMGGGGTTTCAGTCTAGTTTGGC TCGGCTCGTCTCGGTTCGGWCGGCTTCGATTCCTMTGGTA~TCTCACACMCCMGA TGTATCATTTAGGT~GCATATCAGGAGACCGGCTGMGAGCCCAGAGATCCGAGAGC

119

600

119 660 151

120

179 180 199 840 219 2 fV

900

1680

~etLcuAspClullcHisA~yLe"GlyA~pA~pLeU'.*

1740 1800 1860

CGGCGATTATGGGTATCCCGGTMTCATCAGAGTTCMTGTTGMTGTACATTTGTTTGT 1920 ATCTATMCGTGTTGCTAGATCWCCTTTACGATTTCTCATTCTGTTATCCGTTGACCGT 1980 T A T T C G T T M G C G T C G T T A T C C G T T C G T C C C G G A G T T G G ~ G T A C T A ~ T L T M2040 TCCAGGCGTTTTACTTTTACGTTCGTTGGCMCTATATCTAMTCMTGGACGGCGLGAG 2100 G A G M T C C A C M G T T A T A T T T G M T A T C T A T A T G T A T A C C G ~ T M C G A T A G M T T C 2160 nGAGCAT~GARCCCACCGCTCCACACACGCACCACC~CG~GACAGTCCCAGTCCC 2220 A C A C ~ G T A C T A T G A T W T A T M T G A T A T G T A T G T ~ T A T G A T A T A G ~ C T G T T G G M2280 CCCTATCTT~CATATCAGGAGMGATGMGAGCAGTTGAG~CCTTAGTTCMTTCGTT2140 GATTTCACTTTGTTTGCAMTAGAGMTGTTTTCIUTITTGCG 2400 T A C C T T T A T ~ C G A C A T C G A I U G I G I C C T G T G C G T T G ~ M T T G T A T A G A C ~ C C A2160 MGAGRGCGGATCTGTGCCGCAGMCCCAGTTAGTCCATCTATCGMCTGTGCTATATAT 2520 AGAGACGTGMTACGTATMTCGMTTAGTTGGAGTTTAGWTCMC~TGGCTGACA 2580 AGTATATATGTATTGCCCGGAGTCMTTMGCTCMTTACGMTGTTGGMGATTCAGAT 2640 TTTAGTTMGTTTTMTCAGTTAGTTGTTCCAG~GGTTGGTAGTG~TGTCCCTA T2100 A T G ~ G M C A C M T C A G A C A T A T T A C A T T C T A C C T A T G C A C ~ C T C G T T T G T C M T T 2760 C C G C ~ G T 1 U C G ~ T M T G G C A T ~ C T ~ ~ C M C M ~ A T A G T A C2020 T T T ~ G CGTGTTATCTMCTTTATCCAGCACCACGCTCATT~ATGTATGTAT~TTMTGTATG 2800 T A T A T G T A T T T C C A G A T C C ~ T C M T G C C ~ C M G M T T A T T C C M C A T C T A T A 2T9~4 0 T C C R l v \ T G M T A T A T I A T A T A T A T G M T A T A T G A G T ~ G T M T C R l v \ G C 3000

960

G

C

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FIG. 1. Restriction maps and sequences of the XcDmGADl and XcDmGAD2 cDNA clones. E, EcoRI; PI, Pvul; PII, Pvull; S, Scal. Hatched rectangles show the sequenced portions of each clone. The filled rectangle identifies a segment of the XcDmGAD2 insert that has not been sequenced. The only extensive open reading frame present in the derived cDNA sequence begins at nt 181 and terminates at an ochre stop codon at nt 1717. The conceptual translation of this open reading frame is shown above the nucleotide sequence. Consensus polyadenylation signals are underlined. Within a 1060-bp region of coding domain common to both clones (nt 651-1 716), XcDmGAD2 and hcDmGADl are identicalwith the exceptionof 14 polymorphic base substitutions.These are indicated for XcDmGADl below the sequence. The resulting amino acid substitutions (three of them) in the predicted XcDmGADl product are shown above the conceptual translation.

( 1 987) for Drosophila translation start sites. Within the

single long open reading frame, there are 14 base substitutions between XcDmGAD1 and XcDmGAD2, only three of which result in amino acid substitutions (see legend to Fig. 1). Two of these are conservative in nature, whereas the third one is a Thr-to-Lys substitution. Although these two cDNA clones differ in at least 14 positions, preliminary studies of genomic organization show that they represent the same gene (L. M. Newby and F. R. Jackson, unpublished results).

Predicted sequence of the Drosophila protein The conceptual translation of the open reading frame in XcDmGAD2 predicts a 510-residue protein of 57,759 daltons that is similar in sequence to mammalian GAD (see below). The algorithm of Kyte and Doolittle (1982) indicates that the predicted amino acid sequence has features characteristic of a soluble protein; specifically, it is largely hydrophilic and lacks hydrophobic segments capable of spanning a lipid bilayer (data not shown). The fly amino acid sequence can be aligned with

residues 81-585 of cat GAD (Fig. 2); i.e., the amino terminus of the Drosophila product is 80 residues shorter than that of cat GAD (Kobayashi et al., 1987). Overall, the proteins have a sequence identity of 53%. However, two discrete domains are more highly conserved. One domain is 140 residues in length and includes a putative cofactor [pyridoxal phosphate (PLP)]binding site (NPHK) (Bossa et al., 1977) that is conserved in the predicted fly protein (see box in Fig. 2). In this region (arrows), there is 74% identity between the proteins. Similarly, there is a 100-residue segment near the amino terminus of the Drosophila protein that is 70% identical to the corresponding region of feline GAD. These segmental identities between the cat and fly proteins are summarized diagramatically at the bottom of Fig. 2.

Recognition of cRNA translation product and endogenous Drosophila proteins by anti-GAD antibodies cRNA representing a Drosophila cDNA clone was translated in vitro to characterize the encoded product. J. Neurochem., Vol. 54. No. 3, 1990

F. R. JACKSON ET AL.

1072

cGAD

ASSTPSSSAT SSNAGADPNT TNLRPTTYDT WCCVAHGCTR KLCLXICCPL QRTNSLEEKS RLVSAIKERP SSKNLLSCLN

30 80

=GAD

SDRDGRlRRT ETDISNLFAR DLLPA-RNGL E Q T V P F L L N V D I L L N W R R TIDRSTIVLD

139

fGAD

PISLNPNGYKL SERTGKLTAY DLMPTTVTAG PETREILLKV IDVLLDFVKA TNDRNEKVLD

60

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FHHPHPLLEG IIEGlNLCLSD HPESLLQILV DCRDTLKYGV RTGHPRIINQ LSTGLDIICL

199

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FHHP---EDH KRLLDLDVPD PALPLQQLIE DCATTLKYPV KTGHPHIPNP LSNGLDLISM

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AGEWLTSTAN TNIITYEIAP WFVLMEQITL KKIIREIVGWS SKDGDGIFSP OGAISNIYSI

259

AGEWLTATAN TNPIFTYEIAP V F I L n L N W L IKIIREIIGWS G--GDSIWIP GGSISNLYAF

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LAARHKMrPN YKEHCSVCLP CTLVMLrSDQ CHYSIKSUVI VCGLGTDHCI WPSDEHGKM

235

IPADLEAKIL E A K Q K G W P L YYNATAGTTV Y W l O P I Q E I ADICEKYNLW L H V D M W C G G

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MG VLLQCSAILV KEKGIL-JGCN QMCAGYLFQP

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LLIISRTHRHP RFTGVEPADS V Z I I G ALLQCSTiHI KEDGLLISCN QMSAEYLFMT

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.....................................

197

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DKQYDISYDT GDKVIQCGRH NDIFKLWLQW PAKGTEGFEQ QQDRLIELVQ YQL-KRIREQ

414

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EFEIVFDGEP E H T N V C W Y I PQSLRGIPDS PERREKLHRV A P K I M L M n E SGTTnVGYQP

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SDRFHLILEP E C W S F W W PKRLRCVPHD AKKLVELGKI CPIIKGRMHQ KGTLIIVGYQP

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-76

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- 25-

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551 411

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Percent Identity 32%

70%

90

100

44%

74%

34%

70 140 I Resldues

110

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COOK 80

FIG. 2. Alignment of the predicted fly protein (fGAD) with cat GAD (cGAD).The cat GAD protein is numbered according to Kobayashi et al. (1987). Stars indicate identical residues. The two pairs of arrows delimit segments of the proteins that are >70% identical (domain 1, 70%; domain 2, 74%). The box encompasses a conserved pyridoxal-bindingsite. Pads (-) have been added to both sequences for optimal alignment. The percentage identity of correspondingsegments of the two proteins is shown diagramatically at the bottom of the figure. Filled rectangles represent highly conserved domains, whereas hatched rectangles show less conserved domains of the two proteins. The horizontal line at the N terminus representsthe 80-residue segment present only in the cat protein.

Sense RNA (cRNA) including the entire coding domain of the GAD2 cDNA was synthesized and then translated in a reticulocyte lysate (see Experimental Procedures). The translation of this cRNA yielded a 57-kDa protein (Fig. 3A, lane l), as predicted from the cDNA sequence. More importantly, this protein product could be immunoprecipitated by a specific polyvalent anti-GAD antiserum produced against purified rat GAD (Fig. 3A, lane 3 ) (Oertel et al., 1981). Translation reactions lacking GAD2 cRNA (Fig. 3A, lanes 2 and 4) did not produce detectable protein products. When used in immunoblotting experiments, the same anti-GAD antiserum cross-reacted with two size classes of protein (57 and 76 kDa) present in DrosophiZa head extracts (Fig. 3A, lane 6). The 57-kDa species is identical in size to the in vitro-translated product of the GAD2 cDNA clone. Control preimmune serum did not cross-react with endogenous proteins (Fig. 3A, lane 5 ) . These immunological results, considered with functional data presented below, confirm that the Drosuphila cDNA clones identify a Gad gene. J. Neurorhem.. Vol. 54, No. 3, 1990

c

1.51

+/+

w+w+

FIG. 3. In vitro and in vivo expression of Drosophila GAD. A: AntiGAD immunoreactivity of cDNA-encoded and endogenous Drosophila proteins. cRNA corresponding to the sense strand of the GAD2 cDNA clone was used for in vitro translations. RNA synthesis, in vitro translations, gel electrophoresis, immunoprecipitations, and immunoblottingwere performedas described in Experimental Procedures. Lanes 1-4 show [35S]methionine-labeledproducts of in vitro translation reactions performed with (1 and 3) or without (2 and 4) the addition of cRNA. The translation products loaded into lanes 3 and 4 were immunoprecipitatedwith anti-GAD antiserum before electrophoresis..Lanes 5 and 6 show immunoblots of Drosophila head protein (20 pg each; lane 5, preimmune serum; lane 6, anti-GAD antiserum). 8: In situ hybridizationof the 1.5-kb cDNA fragment of hcDmGAD1 to polytene chromosomes. This probe hybridizes to a single site in region 64A3-5 of the third chromosome (arrowhead). A 0.9-kb EcoRl fragment from XcDmGADl, which encompasses the remainder of the XcDmGADl cDNA, hybridizes to the same chromosomal location (not shown). C: GAD enzyme activities (mean k SEM, nmol/min/mgprotein)in wild-type flies (+/ +) and in siblings from the cross of Df(3L)HR277//n(3LR~Tnn68to the wild type. Df(3L)HR277 is a third-chromosome deletion that removes region 63B12-64812. in(3LR)TMGB is a dominantly marked third chromosome that bears a normal copy of the Gad locus. The activities of wild-type, TM68/+, and Df/+ [i.e.. Df(3L)HR277/+] extracts were 0.92 f 0.07,1.15 f 0.09, and 0.63 k 0.04 nmol/min/mg protein, respectively (mean f SEM, n = 5). The data shown in this figure were calculated from 60-min incubations of extracts. Comparable results were obtained using a 30min incubationof extracts (data not shown).

DROSOPHILA GLUTAMIC ACID DECARBOXYLASE Reduction of GAD enzyme activity by deletion of chromosome region 64A We wished to demonstrate that the gene identified by our cDNA clones regulated GAD activity in vivo. To address this question, the chromosomal location of the gene was mapped by hybridizing cDNA sequences to salivary gland polytene chromosomes. Sequences from XcDmGAD 1 hybridized to a single site in region 64A3-5 of chromosome 3L (Fig. 3B). To determine if this locus exerted dose-dependent effects on GAD activity, we compared enzyme activities in adult flies bearing one or two copies of the 64A chromosomal interval. Such flies were produced using the chromosomal deletion Dj‘3LIHR277, which removes sequences between regions 63B and 64B (Lindsley and Zimm, 1986). In theory, a deletion-bearing fly that carries only one dose of the 64A region should produce 50%less enzyme than a normal fly carrying two doses of the same region (Stewart and Merriam, 1974; Hall and Kankel, 1976; Greenspan, 1980; and references therein). GAD activities were assayed in wild-type (CantonSpecial) flies and in two types of sibling progeny produced by crossing the wild type (+/+) to Df(3L)HR277/ In(3LR)TM6B flies. [In(3LR)TM6B is a third chromosome “balancer” that carries a normal copy of the 64A region (Craymer, 1984).] As shown in Fig. 3C, Df(3L)HR277/+ extracts displayed a 45 or 32% decrease in activity relative to TM6B/+ or wild-type extracts, respectively (both reductions were statistically

-

7.5

-

4.4

3.1 Kb-

- 2.4 1.4

FIG. 4. Transcriptionalexpression of the Drosophila Gad locus. A blot containing poly(A)+RNA from 0- to 24-h embryos, first-instar larvae, adults, adult heads, and adult bodies was hybridized to a labeled RNA probe (spec. act. = 1O9 cpm/bg RNA) representing the antisense strand of a 1.5-kb cDNA fragment of the XcDmGADl clone (nt 651-2,160). Autoradiography was carried out for 5 h at -70’C. On a longer exposure (11 h), a second minor transcript of 2.6 kb could be visualized in embryo RNA. Subsequent hybridization of the blot with a Drosophila 5C actin cDNA probe (Fyrberg et al., 1983) demonstrated that all lanes contained equivalent amounts of RNA (not shown). Densitometric scanning of autoradiograms, using a Kontes fiber optical scanner, showed that the 3.1-kb transcript was -8- to 10-fold more abundant in head than in body RNA.

I073

1 2 3 4 5 6 7 8 9

-

3.1 2.6-

FIG. 5. Developmental expressionof DrosophilaGad transcripts. The antisense cRNA probe described in Fig. 4 was hybridized to blots containing poly(A)+RNAs from the following stages: lane 1, 0- to 4-h embryos; lane 2, 4- to 8-h embryos; lane 3, 8- to 12-h embryos; lane 4, 12- to 16-h embryos; lane 5,first-instar larvae (-14 h posthatching);lane 6, second-instar larvae (-43 h posthatching); lane 7, third-instar larvae (-67 h posthatching); lane 8, early pupae (1-3 days post pupariation); lane 9,adults. All lanes except 4 are from the same Northern blot.

significant;p < 0.05). In other words, when compared to siblings (TM6B/+), in which genetic background differences are minimized, deletion-bearing flies have approximately half the normal amount of enzyme activity. These functional data strongly suggest that a gene in region 64A encodes an enzymatically active GAD protein. Expression of Drosophila GAD transcripts Drosophila cDNA sequences have been used in RNA blot analyses to examine the expression of GAD transcripts. An RNA probe representing the antisense strand of the XcDmGAD1 clone hybridizes to a 3.1kb poly(A)+ RNA present in embryos, larvae, and adults (Fig. 4). RNA probes corresponding to the sense strand of XcDmGADl did not hybridize to adult poly(A)+ RNA (not shown). The 3. I-kb mRNA is also detected by three different DNA probes (nt 65 1-2 160, nt 216 1-3010, and nt 2030-28 1 1) representing various portions of the XcDmGADl cDNA clone (data not shown). Densitometric measurements of the autoradiogram represented in Fig. 4 demonstrate that this transcript is 8- to 10-fold more abundant in head than in body RNA. Since heads contain a far greater proportion of neural tissue than do bodies, it is likely that the transcript is preferentially expressed in the nervous system. The complete developmental profile of GAD transcript expression is shown in Fig. 5. The 3.1-kb class of transcripts can be detected in all developmental stages, with expression commencing between 4 and 8 h of embryogenesis. In addition, a smaller 2.6-kb transcript is also detected by cDNA sequences. This transcript displays an interesting pattern of developmental regulation; it is found only in particular embryonic stages (4-8 and 8- 12 h) and in pupae. Spatial distribution of GAD transcripts In situ hybridization of cDNA sequences to sections of adult heads indicates that the expression of transcripts is limited to neural tissue. Figure 6 illustrates hybridization of a labeled antisense probe to a horiJ. Neurochem., Vol. 54, No. 3. 1990

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FIG. 6. Spatial expression of Gad transcripts in the adult CNS. A XcDmGADl antisense RNA probe was hybridized to 6-cm horizontal sections of adult head tissue. Top: A bright-fieldphotomicrographof a section from a relatively ventral level. Anterior is at the top. The antennal nerves (an) can be seen entering the ventralsurface of the antennal lobes. The eyes and optic lobes are apparent on either side of the central brain. r, retina; la, lamina; m. medulla; lo, lobula. Bottom: A dark-field photomicrograph of the same section. Prominent signal can be seen over cell bodies of the antennal lobes as well as over cortical regions of the optic ganglia (arrows). The clusters of grains at the anterior and posterior margins of the section are due to nonspecific sticking of the probe to the cuticle surrounding the CNS. A control sense probe also stuck to the cuticle, but did not hybridize to brain tissue (not shown).

zontal section at the level of entry of the antennal nerves. This probe hybridizes to cell bodies that are widely but not uniformly distributed throughout the adult CNS. Significant hybridization signal can be seen over clusters of cell bodies in the ventral cortex of the antennal lobes. Prominent clusters of putative GABAergic neurons are also present in cortical regions of the optic ganglia. Additional hybridization studies indicate that transcripts are also expressed in a subset of cell bodies of the Drusophila larval CNS, but not in salivary glands or in imaginal disc tissues (data not shown). Hence, expression appears to be primarily restricted to the nervous system as expected for a gene encoding GAD. Similarity between GAD and DOPA decarboxylase A search of the NBRF-PIR protein sequence database (release 17.0; July 1988) identified a single protein with significant similarity to Drusophila GAD. This protein, Drosuphilu DOPA decarboxylase (DDC) (aromatic amino acid decarboxylase), is an enzyme that participates in the synthesis of the neurotransmitters dopamine and serotonin. DDC, like GAD, utilizes the cofactor PLP (Bossa et al., 1977). For both enzymes, PLP is postulated to bind to a lysine residue (K) of the tetrapeptide sequence NPHK (Bossa et al., 1977; Eveleth et al., 1986; Morgan et al., 1986; Kobayashi et al., 1987). We have demonstrated that Drosophilu GAD cDNA sequences do not hybridize to a cloned DroJ. Neurochem., Yol. 54, No. 3, 1990

sophilu Ddc gene, even under reduced-stringency hybridization conditions (data not shown). However, an extensive segment of the Drusophila GAD protein has significant sequence similarity (3 1% identity) to a region of Drosophila DDC (residues 2 17-445) that encompasses the PLP-binding domain (Fig. 7). As previously noted by Kobayashi et al. (1 987), cat GAD is also similar to the same domain of Drusophila DDC (Fig. 7). Indeed, fly GAD and cat GAD are equivalently similar (30-3 1 % identical) to this region of DDC. DISCUSSION Drosophila GAD We have isolated multiple Drosophila cDNA clones that hybridize to a cat GAD cDNA probe. Several lines of evidence indicate that these clones identify a Drusophifu Gad locus: (a) Most importantly, the sequence derived from two overlapping cDNAs (XcDmGAD1 and XcDmGAD2) predicts a 5 10-residue protein of 57,759 daltons with extensive similarity to cat GAD (53% identity for 504 residues). The predicted Drosophilu protein is comparable in size to mammalian GAD proteins (59-67 kDa; Martin, 1987) and contains a putative binding site for the cofactor PLP. (b) A cRNA representing a Drosophilu cDNA clone can be translated in vitro to produce a 57-kDa protein that is recognized by an anti-GAD antiserum. The same antiserum cross-reacts with a 57-kDa protein present in

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70%identity) are apparent from the comparison of fly and cat GAD proteins. One of these conserved domains (domain 2) includes the putative cofactor-binding site; the other one (domain 1) is in a more aminoterminal region (see Fig. 2). These highly conserved domains are separated by a less conserved region. This bipartite structure is reminiscent of hydroxylases, for which two discrete domains of function have been postulated (Ledley et al., 1985). In hydroxylases, the substrate specificity domain is thought to reside near the amino terminus, whereas the general “hydroxylase” domain is located in a more carboxy-terminal region. By analogy to hydroxylases, domain 2 of GAD proteins may correspond to the “decarboxylase” domain. We propose that domain 1 includes residues that contribute to the substrate specificity of GADS. This proposition can be tested by performing a site-directed mutagenesis of the GAD protein.

Expression of the Drosophila Gad locus Transcription from the Drosophilu Gad locus commences early in development (4-8 h) and prior to the formation of morphologically recognizable synapses. In RNA blot analyses, two size classes of GAD mRNA

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(3.1 and 2.6 kb), with distinct patterns of developmental regulation, can be detected. Our preliminary analysis of cDNA clones suggests that multiple 3.1-kb mRNAs arise from the Drosophilu Gad locus. The hcDmGAD1 and XcDmGAD2 cDNA clones represent one of these mRNAs. A different 3.1-kb transcript may be represented by another full-length cDNA clone (XcDmGAD3) we are presently characterizing. This second full-length clone was also isolated from a head cDNA library, and therefore could encode the larger GADimmunoreactive protein that is seen in head protein extracts (Fig. 3; Buchner et al., 1988). Whereas the 3.1-kb mRNA class is expressed throughout development, the 2.6-kb transcript class has been seen only in 4- to 12-h embryos and pupae. Thus far, we have not isolated cDNA clones that represent this smaller transcript. However, its homology to the 3. I-kb mRNA suggests that it could represent a splicing variant from the Gudlocus. It is an intriguing possibility that each GAD transcript encodes a distinct form of enzyme that is expressed at a specified stage of development and/or in a defined subset of neurons. The Drosophilu Ddc locus, for example, expresses two developmentally regulated transcripts that are capable of encoding distinct protein isoforms (Eveleth et al., 1986; Morgan et al., 1986). A similar regulated expression of GAD protein might be one mechanism by which GABA synthesis is spatially and/or developmentally modulated. In this regard, it is of interest that certain Munducu neurons transiently express GABA immunoreactivity during pupal metamorphosis (Homberg and Hildebrand, 1988). We have begun to study the functional organization of Drosophila GABAergic systems by defining the spatial expression of GAD transcripts in the adult CNS. Transcripts corresponding to a 3.1-kb size class of mRNA are localized in clusters of cells widely distributed throughout the CNS. These anatomical regions include the subesophageal ganglion, lateral protocerebrum, antennal lobes, and optic lobes. In general, the observed pattern of mRNA localization is consistent with the known distribution of GABAergic neurons in Drosophilu and other insects. For example, local interneurons and projection neurons of the Manduca and honeybee antennal lobes are immunoreactive for GABA (Hoskins et al., 1986; Schafer and Bicker, 1986). Many of the Manduca antennal lobe neurons have physiological properties characteristic of GABAergic neurons (Waldrop et al., 1987). It has also been demonstrated that a large number of perikarya of the Drosophila and honeybee visual systems cross-react with anti-GAD and/or anti-GABA antisera (Schafer and Bicker, 1986; Buchner et al., 1988). Our in situ hybridization results suggest that GAD transcripts are more abundant in cells of the medulla and lobula than in cells of the optic lamina. These results are in agreement with studies of Buchner et al. (1988), which demonstrated prominent GAD and GABA immunostaining in the medulla and lobula, but very little staining in the lamina. J. Neurochem.. Vol. 54, No. 3, 1990

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The XcDmGAD1 probe used for transcript analysis may hybridize to several different 3.1-kb mRNAs that are expressed from the Drosuphila Gad locus. Thus, the observed spatial distribution and developmental profiles of transcription probably represent the aggregate expression of a population of related mRNAs. Studies with transcript-specific probes may yield greater insights into the mechanisms governing the cellular and developmental regulation of enzymatic activity. Evolution of decarboxylases GADs have been characterized primarily in vertebrate species (reviewed in Martin, 1987). In mammals and birds (quail), immunoblotting experiments have identified two distinct GAD subunit sizes (of 59 and 62 kDa) in brain tissue [Legay et al. (1986); also see Denner et al. (1987) for evidence that 40- and 80-kDa subunits exist in the mammalian brain]. It is of interest that Drusuphila brain tissue also contains two GADimmunoreactive species (Fig. 3; Buchner et al., 1988), whereas in certain “lower” vertebrates (trout and frog), only a single GAD species is seen (Legay et al., 1986). In mammals and Drosophila, transcriptional analyses have suggested that single Gad genes express multiple mRNAs that could encode distinct protein isoforms (Benson et al., 1988; present work). In addition, preliminary results suggest that a tandemly duplicated pair of Gad genes may exist in the Drusuphila genome (L. M. Newby and F. R. Jackson, unpublished results). Thus, the phyletic array of GAD subunit sizes might reflect differences in genomic organization (i.e., one versus two genes) as well as differences in RNA splicing patterns or the posttranslational processing of GAD proteins. Kobayashi et al. (1987) previously reported a similarity between GAD and DDC based on an interspecies comparison of cat GAD and Drusuphila DDC. They noted a similarity within 90-residue segments that encompass the putative cofactor-binding domain of each protein. We have shown that GAD and DDC from the same species (i.e., D. melanugaster) are 31% identical within an -230-residue domain. This extensive similarity suggests that these distinct decarboxylases are evolutionarily related. Interestingly, the Drusophila GAD and feline GAD sequences have approximately the same degree of similarity (30-31%) to this 230residue domain of DDC. These equivalent similarities suggest that Gad and Ddc genes arose from a duplication event that occurred prior to the divergence of chordates and invertebrates. It has been postulated that the pyridoxal-dependent decarboxylases of phylogenetically distant organisms evolved from a common ancestor (Dunathan and Voet, 1974; Bossa et al., 1977). Thus, it is of related interest that the primary structure of a plant tryptophan decarboxylase (TDC, EC 4.1.1.28) has recently been delineated (De Luca et al., 1989). In the plant Catharanthus roseus (commonly known as periwinkle), TDC catalyzes the production of tryptamine for the biosynthesis of alkaloids (De Luca et al., 1989). Remarkably, J.

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there is extensive amino acid sequence similarity among the TDC, DDC, and GAD proteins (De Luca et al., 1989). Moreover, we have found that Drosophila GAD and mammalian GAD are equivalently similar to comparable 140-residue segments of DDC (35% identity) and TDC (34% identity) that contain the presumed cofactor-binding domain (unpublished results). That is to say, GAD proteins as a class are no more similar to this domain of animal DDC than they are to the comparable region of plant TDC. The simplest interpretation of these painvise similarities is that GADlike and DDC-like activities evolved before the divergence of the plant and animal kingdoms. The presence of functionally similar (and possibly structurally related) GADs in animals, plants (Satyanarayan and Nair, 1985), and bacteria (Cozzani et al., 1970; Strausbauch and Fischer, 1970a,b) is consistent with this interpretation. Note added in proof: During a search of the most recent NBRF protein database (version 20.0), we discovered that Drosophila GAD and other PLP-dependent decarboxylases have significant similarity (25% identity for Drosuphila GAD) to bacterial histidine decarboxylase (Vaaleret al., 1986). This finding lends direct support to the idea that procaryotic and eucaryotic PLP-dependent decarboxylases evolved from a common ancestor.

Acknowledgment: We are indebted to A. Tobin and D. Kaufman for providing a feline GAD cDNA clone. Antiserum to GAD was provided by the Laboratory of Clinical Science, NIMH, where it was developed under the supervision of Dr. I. Kopin with Drs. W. Oertel, D. Schmechel, and M. Tappaz. K. Elliott, B. Walker, and S. Quinn provided technical assistance for various portions of this study. We thank L. Restifo for help with tissue in situ hybridizations and for critical readings of the manuscript, J. Richter for help with in vitro translations and immunoprecipitations, P. Salvaterra for a Drosuphila head cDNA library, J. Hirsh for Drosuphila Ddc sequences, J. Bonner, K. Matthews, and the Indiana University Stock Center for Qf(3L)HR277.J. Goodchild for synthetic oligonucleotides, and R. OConnell and D. Yang for assistance with computer analyses. We also thank S. DiBartolomeis for comments on the manuscript. This research was supported by NIH grant NS25914, the McKnight Foundation, and institutional funds from the Worcester Foundation.

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