A Rhodopsin Gene Expressed in Photoreceptor Cell R7 of the Drosophila Eye: Homologies with Other Signal-Transducing Molecules

The Journal of Neuroscience, May 1987, 7(5): 1550-I 557

Charles S. Zuker, Craig Montell, Kevin Jones, Todd Laverty, and Gerald M. Rubin Department of Biochemistry, University of California, Berkeley, California 94720

We have isolated an opsin gene from D. melanogaster that example, Pak, 1979; Hardie, 1983; Rubin, 1985). The com- is expressed in the ultraviolet-sensitive photoreceptor cell pound eye of Drosophila contains 3 distinct classesof photo- R7 of the Drosophila compound eye. This opsin gene con- receptor cells, Rl-6, R7, and R8, distinguishableby their mor- tains no introns and encodes a 383 amino acid polypeptide phological arrangement and the spectral behavior of their that is approximately 35% homologous to the blue absorbing correspondingvisual pigments (reviewed by Hardie, 1983). In ninaE and Rh2 opsins, which are expressed in photoreceptor each of the approximately 800 ommatidia that make up the eye cells RI-6 and R8, respectively. Amino acid homologies be- there are 6 outer (Rl-R6) and 2 central (1 R7 and 1 R8) pho- tween these different opsins and other signal-transducing toreceptor cells (Fig. 1). The photopigments found in the Rl- molecules suggest an important role for the conserved do- R6 cells, the R7 cell, and the R8 cell differ in their absorption mains of rhodopsin in the transduction of extracellular sig- spectra (Harris et al., 1976) most likely becausedifferent opsin nals. genesare expressedin these distinct classesof photoreceptor cells. The 6 peripheral cells (RI-6) contain the major visual Phototransduction, the neuronal excitation processtriggered by pigment, a rhodopsin that absorbsmaximally at 480 nm (Ostroy light, provides an ideal model system for the study of sensory et al., 1974). The gene encoding this visual pigment has been transduction in the nervous system.Rhodopsin, the major pho- isolated by virtue of its homology to the bovine opsin geneand toreceptor of both vertebrate and invertebrate eyes, consistsof has been shownto correspondto the genetically defined ninaE an apoprotein, opsin, covalently bound to a chromophore, gen- locus (O’Tousa et al., 1985; Zuker et al., 1985). Of the 2 central erally 1 I-c&retinal (reviewed in Fein and Szuts, 1982; Har- photoreceptor cells, R7 contains a UV-sensitive pigment and grave, 1982). Light activation of rhodopsin is the first step in R8 a blue nonadapting pigment (Harris et al., 1976; Hillman et the visual response,a biochemical cascadethat converts the al., 1983). We have previously reported the isolation and anal- energyof an absorbedphoton into a receptorpotential (reviewed ysis of a Drosophila opsin gene that is transcribed specifically by Stryer, 1983, 1985; Kiihn, 1984; Stieve, 1986). The chro- in the R8 photoreceptor cell (Cowman et al., 1986). We report mophore is isomerized by light from the 1 I-cis to the all-truns here the isolation and analysis of a novel opsin gene that is configuration, which in turn leadsto a conformational change homologousto the opsins expressedin the Rl-6 and R8 pho- in the opsin moiety. Thesephotoactivated rhodopsin molecules then trigger the cascade of events that results in a transient change of the cation conductances of the photoreceptor cell membrane (reviewed by Fain and Lisman, 1981). Several ver- CO tebrate opsins have been sequenced(Ovchinnikov et al., 1982; Psc Hargrave et al., 1983;Pappin and Findlay, 1984)and the genes E for bovine and human opsinshave been analyzed (Nathans and Hogness, 1983, 1984; Nathans et al., 1986). These vertebrate R7 opsinsare highly homologousin amino acid sequenceand struc- Rl-6 ture. Drosophila is an attractive experimental organismin which R8 to study signal transduction in the visual system using a com- bined molecular,.genetic, and physiological approach (see,for PC

Received Sept. 26, 1986; accepted Nov. 25, 1986. This work was supported by grants from the NIH to G.M.R. C.S.Z. is a fellow ofThe Jane Coffin Childs Memorial Fund for Medical Research. C.M. is supported by a NIH postdoctoral fellowship, and K.J. by an NSF predoctoral fellowship. Figure 1. Adult ommatidial unit. The rhabdomeres (microvilli con- We thank S. Mount, D. Kalderon, K. Moses, and J. Nathans for their comments. taining the visual pigments) of R 1 -R6 form an asymmetrical trapezoidal We also thank A. Tomlinson for his help with Figure 1, and K. Fryxell and E. Meyerowitz for communicating their results. We particularly want to thank Mark shape around the central rhabdomeres of the R7 and R8 cells. The R8 Stoneking and Alan Wilson for their help and advice in the construction of the cell is located below the R7 cell and extends through the proximal half phylogenetic trees. of the retina. Inset shows cross sections through the distal (upper) and Correspondence should be addressed to Charles S. Zuker, Department of Bi- proximal (her) regions of the retina. CO, cornea1 lens; psC, pseudo- ology, B-022, University of California at San Diego, La Jolla, CA 92093. cone; PC, pigment cells; CC, conecells; RI-6, R7, and R8, photoreceptor Copyright 0 1987 Society for Neuroscience 0270-6474/87/051550-08$02.00/O cells. Adapted from Tomlinson (1985). The Journal of Neuroscience, May 1987, 7(5) 1551

H = 1.00 kb 15 mM sodium citrate), 0.5% SDS at 65°C. The cDNA library (a gift from B. Yedvobnick and S. Artavanis-Tsakonas) was made from poly(A) RNA isolated from the heads of adult flies (O-24 hr after eclosion) of the Oregon R (P2) strain. DNA probes. Nick-translation of DNA was carried out as described by Maniatis et al. (1982). All probes for in situ hybridization to head sections were made as described previously (Hafen et al., 1983). Oligonucleotide probes. Two oligonucleotides were used as probes. The first [TATGTGCCIGAGGGTAA(C/T)CTGAC(C/T)] was 24 residues long, degenerate at positions 18 and 24, contained inosine at position 9, and encoded the peptide YVPEGNLT. The second [CAGGCCAAGAAGATGAATGTCAA(A/G)TCCCTI] was a 30-residue oligonucleotide, degenerate at position 24, contained inosine at position 30, and encoded the peptide QAKKMNVKSL. Synthetic oligonucleotides were purified by thin-layer chromatography on silica gel plates (Silica Gel 60 F-254; EM Reagents) in n-propanol : NH,OH : H,O H =O.l5kb (55:35:10), and were end-labeled with -+2P-ATP, as described by Man- Figure 2. Restriction map of XDmRh3 and structure of the RNA it iatis et al. (1982). Hybridizations with end-labeled oligonucleotide probes encodes. Shown is a map of XDmRh3 indicating the restriction sites for were carried out at 42°C in 7 x SSC, 0.1% bovine serum albumin, 0.1% Hind III, Bum HI, and Sal I (top map). Also shown, in expanded view, PVP-40,O. 1% Ficoll. Filters were washed in 7 x SSC, 0.5% SDS at 42°C. is the region of the 2.4 kbase Hind III fragment encoding the RNA Four genome equivalents of a total genomic library (Maniatis et al., (lower map). The restriction sites for Eco RV, Pst I, Bgl II, and Hind 1978) were screened. II endonucleases are indicated. The diagram below the map shows the In situ hybridization to tissue sections. Preparation of 8 PM frozen structure of the Rh3 RNA as deduced by comparison of the nucleotide sections of adult heads and hybridization of )H-labeled probes were as sequences of a cDNA clone and the genomic clone, and Sl nuclease described by Hafen et al. (1983) except that the acid and pronase mapping and primer extension data (see Materials and Methods); note treatments were omitted in the pretreatment of the slides. the lack of introns in Rh3. DNA sequence analysis. DNA sequencing was carried out on ran- domly sheared fragments according to the chain termination procedure of Sanger et al. (1977). Ml3 mp18 and mp19 were used as sequencing toreceptor cells. We show that this opsin geneis transcribed in vectors and TGl (the gift of Toby Gibson, MRC Laboratory of Mo- lecular Biology, Cambridge, England) as the host strain. Reactions were the UV-sensitive photoreceptor cell, R7, of the compound eye. carried out as described by Bankier and Barrel1 (1983) with olJ5S-dATP In the accompanying paper (Monte11et al., 1987), we describe as the radioactive nucleotide. The genomic sequence of the Rh3 opsin the isolation and characterization of an additional opsin gene gene was determined over both strands. The complementary DNA that isexpressed in a nonoverlapping subsetof R7 photoreceptor (cDNA) sequence was determined on 1 strand. Primer extension and SI nuclease analysis. Primer extensions were cells. carried out by hybridizing 5 ng of a synthetic 20 base oligonucleotide (complementary to positions + 18 to +37) in separate 20 ~1 reactions Materials and Methods to either an Ml3 clone containing the 0.75 kbase Hind III-Bgl II frag- Isolation of poly(A)+ RNA. RNA was extracted from the heads of the ment of XDmRh3 (see Fig. 2), 20 pg of head poly(A)+ RNA, or 20 pg appropriate mutants, as described by O’Hare et al. (1983). Heads of of body poly(A)+ RNA. Reverse transcription was then carried out as adult flies (O-24 hr after eclosion) for Oregon R (P2), sev (sevenless; described for cDNA synthesis by Maniatis et al. (1982). Sl nuclease Harris et al., 1976) ora (outer rhabdomeres absent), and sev oru mutants protection experiments were carried out as described by Maniatis et al. (Harris et al., 1976) were separated from bodies as described by Oliver (1982). Single-stranded DNA probes used in the S 1 endonuclease pro- and Phillips (1970). Poly(A)’ RNA was isolated by affinity chromatog- tection experiments were prepared by synthesizing a radiolabeled second raphy on oligo(dT) cellulose columns (Blumberg and Lodish, 1980). strand on M 13 templates. The newly synthesized material was separated Blotting and hybridization of DNA and RNA. Fractionation of the from the template after restriction enzyme cleavage by boiling in 30% RNAs on formaldehyde gels, transfer onto nitrocellulose paper, and dimethyl sulfoxide (DMSO) and gel purified on a 1% agarose gel. prehybridization were carried out exactly as described by Chung et al. In situ hybridization topolytene chromosomes. Polytene chromosome (198 1). Hybridizations with nick-translated DNA probes were carried squashes (Canton S strain) were prepared as previously described (Zuker out at 65°C in 750 mM NaCl, 100 mM NaH,PO, (pH 6.8), 75 mM sodium et al., 1985). Hybridization with biotinylated DNA probes was carried citrate, 0.04% bovine serum albumin, 0.04% PVP-40, 0.04% Ficoll, out according to Langer-Sofer et al. (1982) with the following modifi- 0.5% SDS. Filters were washed in 0.2 x SSC (1 x SSC is 150 mM NaCl, cations: DNA was nick-translated using Bio- 16-dUTP (Enzo Biochem)

Ftgure 3. In situ hybridization to salivary gland chromosome squashes. XDmRh3 was biotinylated as described in Materials and Methods and used as a hybridization probe to determine its chromosomal location. Shown is the 92 region of chromosome III of Dro- sophila melanogaster (Canton S). The arrow indicates the site of hybridization at 92D 1; no other sites of hybridization were observed. and hybrids weredetected using a Detek-I-HRP detectionkit (Enzo Biochem). Results and Discussion Isolation of hDmRh3, a DNA segmentencoding a novel Drosophila opsin The different spectralsensitivities of the various photoreceptors of the Drosophila eye indicate the presenceof multiple rhodopsins (reviewed in Hardie, 1983). The gene encoding the opsin expressedin the 6 peripheral photoreceptor cells (Rl-6) has been isolated by virtue of its sequencehomology to the bovine rhodopsin gene (O’Tousa et al., 1985; Zuker et al., 1985). This gene correspondsto the genetically identified ninaE locus (Sca- varda et al., 1983). We have usedthe ninaE gene to searchfor cross-homologoussequences in the Drosophila genome and thereby isolated the gene encoding the opsin expressedin the central, blue-absorbingR8 photoreceptor cells(Rh2 opsin; Cow- man et al., 1986); neither the ninaE nor the Rh2 opsin genes appear to be expressed in the central R7 photoreceptor cell (Zuker et al., 1985; Cowman et al., 1986). Therefore, at least one additional rhodopsin gene, the one encoding the opsin expressedin the R7 photoreceptor cell, must exist in the Dro- sophila genome. Low-stringency hybridizations of Drosophila genomic and cDNA libraries with the cloned ninaE and Rh2 probes did not reveal any additional homologous sequences. Assumingthat functionally significant regionsof rhodopsin might be conserved among the different opsin genes,we designedoli- gonucleotideprobes (see Materials and Methods) corresponding to 2 of the most highly conserved regions between ninaE and Rh2 opsins in order to screena genomic library with greater sensitivity. One of these regions encodesan 8 amino acid sequencethat is conserved between the 2 Drosophila and bovine rhodopsins(Nathans and Hogness,1984; O’Tousa et al., 1985; Zuker et al, 1985; Cowman et al., 1986). The other encodesa 10 amino acid sequencethat is unique to Drosophila opsins (Zuker et al., 1985; Cowman et al., 1986); vertebrate opsinsl&k this 10 amino acid region. Positive clones were isolated and counterscreenedwith radiolabeled ninaE and Rh2 gene-specific probesto eliminatethe ninaE and Rh2 cognatesequences. Three of the genomic clones that hybridized strongly with both oligonucleotide probes representedoverlapping sequences,here- after referred to as Rh3. Homology to the oligonucleotideswas confined to a 2.4 kbase Hind III fragment (Fig. 2). The ninaE and Rh2 geneshave been cytogenetically mapped to chromosomal positions 92B8- 11 and 9 1D l-2, respectively (Zuker et al., 1985; Cowman et al., 1986). The Rh3 cloneswere mappedby in situ hybridization to salivary gland chromosomes to chromosomalposition 92D (Fig. 3). This cytogenetic location correspondsto that of X512, a genomic clone isolated by Levy et al. (1982) on the basisof its encoding a head-specifictran- script. No mutations affecting vision or visual input-mediated behavior have been isolatedat or near this cytogenetic location.

Rh3 encodesan opsin We usedthe 2.4 kbase Hind III fragment of XDmRh3 (seeFig. Figure 4. Nucleotidesequence and deducedamino acid sequenceof 2) to screen a Drosophila cDNA library and isolated several DrosophilaRh3 opsin.The sequenceshown was determined on both strandsof the genomicclone (2.4 kbaseHind III fragment;see Fig. 1). The regionfrom nucleotide400 to 1270was also sequenced in a cDNA c clone.The underlinedregion at - 28 to - 34 showsthe putative TATA box. The star at nucleotide+ 1 indicatesthe positionof the start of 1589-1599(note the presenceof 2 overlappingAATAAA consensus transcription,as determined by primerextensions and S 1 nucleasemap- sites).The Rh3 opsinprotein sequenceis shownaligned under the pingdata. The putative poly(A) additionsignal is underlined at position nucleotidesequence. The Journal of Neuroscience, May 1987, 7(5) 1553

Cytoplasmic

.338

Figure 5. Proposed structure of Dro- sophila Rh3 opsin. Drawing is a mod- ified version of the model of Hargrave (1982) and Ovchinnikov (1982). Pu- tative transmembrane domains were determined by the algorithm of Kyte N and Doolittle (1982). Amino acid residues are indicated by their single letter codes. Black solid circles indicate identities among the Drosophila ninaE, Rh2, Extracellular and Rh3 opsins.

cDNA clones. Using Ml 3 dideoxynucleoside triphosphate se- contains more than 1 potential N-linked glycosylation site (con- quencing, we have determined the DNA sequence of one of sensus,Asn-X-Ser, Asn-X-Thr). Figure 5 showsthe proposed those cDNA clones and of the 2.4 kbase Hind III genomic structure of the Rh3 opsin, basedon the algorithm of Kyte and fragment of XDmRh3 (see Fig. 2). The genomic sequence we Doolittle (1982) and the models of Ovchinnikov (1982) and characterized included the same coding region as that present Hargrave (1982). It is worth noting that amino acid residues in X512 (C. Monte11 and G. M. Rubin, unpublished observa- 198-205 and 258-267, correspondingto oligonucleotide probes tions; K. Fryxell and E. Meyerowitz, personal communication). 1 and 2, are 71% (5/7) and 90% (g/10) conserved in Rh3 (see Figure 4 shows the nucleotide sequence and the deduced amino Materials and Methods). At the nucleotide level, oligonucleotide acid sequence of the Rh3 gene. The structure of the RNA (Fig. 1 shows 83% identity (20/24, including degenerateresidues) 2) and the position of the start of transcription were determined with the correspondingregion of the Rh3 gene,and the oligonu- by primer extension and Sl nuclease mapping analyses (data cleotide 2 sequenceis 86% conserved (26/30, including degen- not shown). erate residues). Rh3 encodes a 383 amino acid polypeptide that shares 130 and 125 amino acid identities with the ninaE and Rh2 opsins, Rh3 is expressedin the UV-sensitiveR7 photoreceptor cells respectively; 116 of these residues are conserved between all 3 Previous comparision of the amino acid sequencesof the ninaE polypeptides. In addition, the protein encoded by Rh3 contains opsin with the Rh2 opsin revealed a high degreeof homology all of the structural features expected of a visual pigment protein: (67%; Cowman et al., 1986). Both of theseopsins have absorp- 7 hydrophobic domains separated by hydrophilic sequences, a tion maxima in the blue region of the spectra (reviewed by presumed retinal-binding site in the seventh transmembrane Hardie, 1983). In contrast, Rh3 is only approximately 35% ho- domain (Lys 328) a series of potential phosphorylation sites mologous with the ninaE and Rh2 proteins (Fig. 5). In order (Ser and Thr residues) in the C-terminal region of the polypep- to determine in which photoreceptor cell type the Rh3 opsin is tide chain, and a glycosylation site(s) in the extracytoplasmic expressed,and thus what spectral behavior it mediates,we ex- face (Asn 5 and Asn 13). This is the only Drosophila opsin that amined the levels of mRNAs homologousto this genein mutant 1554 Zuker et al. l DrosophilaOpsin Genes

Fimre 6. Exvression of Rh3 tran- s&ipts. Poly(Aj’ RNAs were extracted .5 at different stages ofdevelopment, from adult heads of wild-type, sev, and sev oru flies, and from wild-type adult bodies. The RNAs (2.0 Ils/&ne) were gel fractionated. blotted. and hvbridized to a DNA prode deriveh fromnucleotides 1070-20 16 of Rh3, as described in Ma- terials and Methods. Lane E, embryos; lanes1, 2, 4, 6, and 8, days of development; A, newly eclosed adults. X-Hind III and OX 174 Hae III fragments were used as size markers. strains that lack specific photoreceptor cells and have deter- (outer rhabdomeresabsent) flies, which lack the 6 peripheral mined the sites of accumulation of these mRNAs by in situ outer photoreceptor cells (Rl-R6), and from the double mutant hybridization to tissue sectionsof the wild-type eye. sev oru, which has only the central R8 photoreceptor cells (lack- We isolated total poly(A)’ RNA from wild-type Oregon R ing Rl-6 and R7). The RNAs were fractionated on agarose- flies at different times during development, and from the heads formaldehyde gels and hybridized to a radiolabeled DNA frag- and bodiesof wild-type adult flies. We also isolated RNA from ment consistingof the 3’ region of Rh3 (nucleotides1070-20 16; headsof flies homozygousfor the sevenless(sev) mutation, which Fig. 4). This sequencedoes not hybridize to any of the other lack the central R7 photoreceptor cell, from the headsof oru Drosophila opsin genes.Rh3 hybridizes to a 1.5 kbase RNA

Figure 7. Spatialdistribution of Rh3 transcriptsin tissuesections of adult heads.Shown is the hybridization of an F&3-specificprobe to adult retinas (exposure, 30 d). Longitudinal (a) and tangential (b) sections of adult heads are shown. The R7 cell is located above the R8 cell and extends through the distal half of the retina (see Fig. 1). Re, retina; la, lamina ganglionaris; co, cornea1 lens; ib, interommatidial bristles. The Journal of Neuroscience, May 1987, 7(5) 1555

t MAQQWSLQRLAGRHPQDSYEDSMSSIFTYTNSNSTRGPFEGPNYHIAPRWV- 52 MAQQWSLQRLAGRHPQDSYEDSMSSIFTYTNSNSTRGPFEGPNYHIAPRWV- 52 MRKMSEEEFYLFKNISSVGPWDGPQYHIAPVWA- 33 MNGTEGPNFWPFSNATGWRSPFEYPQYYIAEPWQ- 36 MGPPGNDSDFLLTTNGSHVPDHDVTEERDFAWVV 34 MF.SFAVAAAQLGPHFAPLS-NGSWDKVTPDMAHLISPYWNQFPAt4DPIW-- 49 MF.RSHLPETPFDIAHSGPRFQAQSSGNGSVLDNVLPDMAHLNVPYWSRFAPMDPMM-- 56 MESGNVSSSLFGNVSTALRPEARLSAETRLLG--WNVPPEELRHIP-EHWLTYPEPPESM 57 MEPLCNASEPPLRPEARSSGNGDMFLGWNVPPDQIQYIP-EHWLTQLEPPASM 53

~ l **I l ***** l , l l hG YHLTSVWMIFWIASVFTNGLVLAA TMKFKKLR--HPLN WILVNLAVADLAETVIASTISVVN QVYGYFVLGHPMCVLEG 130 hR YHLTSVWMIFWTASVFTNGLVLAA****l TMKFKKLR--HPLNl WILVNIAVADLAETVIASTISIVN QVSGYFVLGHPMCVLEG 130 hB FYLQAAFMGTVFLIGFPLNAMVLVA TLRYKKLR--QPLN YILVNVSFGGFLLCIFSVFPVFVA SCNGYFVFGRHVCALEG 111 hRh FSMLAAYMFLLIVLGFPINFLTLYV TVQHKKLR--TPLN YILLNIAVADLFMVLGGFTSTLYT SLHGYFVFGPTGCNLEG 114 AR GMAILMSVIVLAIVFGNVLVITA --IAKFERLQTVTN YFITSLACADLVMGLAVVPFGASH ILMKMWNFGNFWCEFWT 110 Rhl AKILTAYMIMIGMISWCGNGWIYI FATTKSLR--TPAN LLVINLAISDFGIMITNTPMMGMGIN LYFETWVLGPMMCDIYA 127 Rh2 SKILGLFTLAIMIISCCGNGWVYI FGGTKSLR--TPAN LLVLNLAFSDFCMMASQSPVMIIN FYYETWVLGPLWCDIYA 134 Rh3 NYLLGTLYIFFTLMSMLGNGLVIWV FSAAKSLR--TPSN ILVINLAFCDFM-VKTPIFIYN SFHQGYALGHLGCQIFG 134 Rh4 HYMLGVFYIFLFCASTVGNGMVIWI FSTSKSLR--TPSN MFVLNLAVFDLIMC-LKAPIFIYN SFHRGFALGNTWCQIFA 130

hG 208 hR 20 hB 189 hRh 192 AR 190 Rhl 205 Rh2 212 Rh3 212 Rh4 208

l l l tt *** l * l * hG SGSSYPGVQS YMIVLMVTCCITPLSIIVLCYLQVWLAI-- RAVAKQQKESESTQ------KAEKEVTR----- 268 hR SGSSYPGVQS YMIVLMVT CCIIPLAIIMLCYLQVWLAI-- RAVAKQQKESESTQ------KAEKEVTR----- 268 hB TVGTKYRSES YTWFLFIFCFIVPLSLICFSTYQLLRAL-- KAVAAQQQESATTQ------KAEREVSR----- 249 hRh TLKPEVNNES FVIYMFWHFTIPMIIIFFCYGQLVFl-+- KFAAAQQQESATTQ------KKEVTR----- 252 AR CDFFTNQAYA --IASSIVSFYVPLWMVFVYSR-VFQVAK RQLQKIDKSEGRFHSPNLGQVEQDGR-SGHGLRRSSKFCL 266 Rhl ERDWNPRSYL --IFYSIFVYYIPLFLICYSYWFII~VSA HEKAMREQAKKMNV-KSLRSSEDAEK-SAEGKLAK----- 276 Rh2 TRMWNPRSYL --ITYSLFVYYTPLFLICYSYWFIIAAVAA HEKAMREQAKKMNV-KSLRSSEDCDK-SAEGKLAK----- 283 Rh3 TDNFDTRLFV --ACIFFFSFVCPTTMITYYYSQIVGHVFS HEKALRDQAKKMNV-ESLRSNVDKNKETAEIRIAK----- 284 Rh4 280

hG 336 hR 336 hB 318 hRh 322 AR 342 Rhl 345 Rh2 352 Rh3 354 Rh4 350

** * hG FGKKVDDGSEL-SSASKTEVSSVSSVSPA 364 hR FGKKVDDGSEL-SSASKTEVSSVSSVSPA 364 hB CGKAMTDESDTCSS-QKTEVSTVSSTQVGPN 348 hRh CGKNPLGDDEASATVSKTETSQVAPA 348 AR RRSSSKAYGNGYSSNGNGKTYMGEASGCQLGQEKESERLSQGRNCSTNDSPL 418 Rhl CCVFGKVDD-GKSSDAQSQ-ATASEABSKA 373 Rh2 MCVFGNTDE-PKPDAPASDTETTSEADSKA 381 Rh3 -WLALNEKA-PESSAVAS-TSTTQEPQQTTAA 383 Rh4 -WLGVNEKSGEISSAQS---TTMEQQQTTAA 378 Figure 8. Amino acid sequence homologies between the /3-adrenergic receptor and the human and Drosophila opsins. Colinear alignment of the deduced amino acid sequences of the 4 human visual pigments (Nathans and Hogness, 1984; Nathans et al., 1986), the hamster P-adrenergic receptor (Dixon et al., 1986), and the 4 Drosophila opsins (O’Tousa et al., 1985; Zuker et al., 1985; Cowman et al., 1986; Monte11 et al., 1987). Amino acids are designated by their single letter codes. Alignment has been optimized for the largest number of identities with the least number of gaps. Boxed areas show the position of the 7 putative transmembrane domains of Drosophila opsins. Stars above the sequence indicate homologies between the fl-adrenergic receptor and a minimum of 4 opsin genes. The arrowhead indicates the position of the lysine residue to which the retinal chromophore is thought to be bound. 1556 Zuker et al. * Drosophila Opsin Genes

Figure 9. Phylogeny of visual pigments and the P-adrenergic receptor. This phylogenetic tree relating the different animal visual pigments and the fl-adrenergic receptor was constructed ha-p -RR on the basis of the principle of minimal Dm-Rhl mutation distances (parsimony principle). The data presented in Figure 8 were Dm-Rh2 subjected to phylogenetic analysis by Dm-Rh3 the Farris (1972) method. The most parsimonious tree is shown. Branch Dm-Rh4 lengths (number of mutational events) are indicated in the scale below the tree. ,-h-Green Dm-Rhl stands for the Drosophila ninaE gene, Dm-Rh2, Dm-Rh3, and Dm-Rh4 refer to the Drosophila Rh2, Rh3, and Rh4 opsin genes. The hamster fl-adrenergic receptor (Dixon et al., 1986) is referred to as ha-@-AR. h-Green, h-Red, and h-Blue refer to the human I I I I 1 I I color opsins, and h-Rhod to human 2&l 200 80 40 0 rhodopsin (Nathans and Hogness, 1984; 240 160 120 Nathans et al., 1986). Average number of amino acid substitutions speciesthat accumulatesin the late pupaand peaksafter eclosion correspondingregions of the ninaE and Rh2 opsins(see Fig. 5). (Fig. 6). This RNA is present in the headsof wild-type flies or However, only the first of these loops is conserved between in flies carrying the oru mutation (data not shown). However, Drosophila and vertebrate opsins (residues 87-94; Fig. 4; the RNA is greatly reduced in flies carrying the sevenlessmu- KXLRXPXN). The third cytoplasmic loop (Figs. 4 and 5, res- tation (either sevor sevoru). The small amount of hybridization idues 25 l-284) contains a 10 amino acid insertion that is com- observed in the heads of sev and sev ora flies may represent mon to all Drosophila opsins analyzed to date (O’Tousa et al., expressionof this gene in other cell types, as this RNA is also 1985; Zuker et al., 1985; Cowman et al., 1986; Monte11et al., present in the headsof mutant flies lacking all photoreceptor 1987)but is absent from all vertebrate opsins(Ovchinnikov et cells of the eye (glass;data not shown). The spatial distribution al., 1982; Hargrave et al., 1983; Nathans and Hogness,1983, of Rh3 transcripts was directly determined by hybridizing the 1984; Nathans et al., 1986). radiolabeled Rh3 gene-specificprobe to tissuesections of wild- We previously suggestedthat the first cytoplasmic loop of type adult heads.Figure 7 showsthat RNAs homologousto Rh3 rhodopsin isinvolved in the interaction of rhodopsin with trans- are localized to the distal region of the retina; this is the location ducin (Zuker et al., 1985). Figure 8 presentssequence data in- of the UV-sensitive R7 photoreceptor cells (see Fig. 1). This dicating that this region also shows amino acid conservation result, taken together with the severe reduction of Rh3 tran- betweenopsins (Drosophila and vertebrate) and the p-adrenergic scripts in mutants carrying the sev mutation (Fig. 6), indicates receptor (P-AR), another signal-transducingmolecule that in- that the Rh3 opsin is transcribedin the central R7 photoreceptor teracts with a G-protein (Lefkowitz et al., 1983). Dixon et al. cells of the compound eye. (1986) have recently shownthat bovine rhodopsinand the ham- The spectral and dichroic properties of the Drosophila R7 ster P-AR sharesignificant amino acid sequencehomology, par- photoreceptor cells have not been studied in detail. Thesecells ti&larly in the transmembrane domains. Functional conser- contain a bistable pigment system with sensitivity in the UV vation of catalytic components usedfor signal transduction in (330-350 nm) and metarhodopsin forms in the blue region of the amplification of the visual responseand in the activation of the spectrum (seeHardie, 1983; Hillman et al., 1983). We be- adenylate cyclase-coupled/3-ARs has recently beendemonstrat- lieve the differencesbetween the primary sequencesof the ninaE ed, in that rhodpsin and ,&AR can be properly phosphorylated and Rh2 opsins,and the much more divergent Rh3 opsin, reflect by the other’s kinase (Benovic et al., 1986). Sequenceanalyses the different spectral properties of these photopigments (see of the additional Drosophila opsins (seeMonte11 et al., 1987) Monte11et al., 1987). and the recent isolation of the human color opsingenes (Nathans et al., 1986) have allowed us to place all of these proteins in a Drosophila opsinscontain amino acid sequencedomains that single phylogenetic tree. The phylogeny shown in Figure 9 was are highly conservedwith other signal-transducing proteins constructed on the basis of the principle of minimal mutation Vertebrate and invertebrate rhodopsinsinteract with at least 2 distances(Wilson, 1985; PAUP program, Illinois Natural His- cytoplasmic proteins: a G-protein (transducin) and rhodopsin tory Survey, version 2.4.0). The most parsimonious tree favors kinase(reviewed by Kiihn, 1984; Stieve, 1986). The interaction the branching order shown. Remarkably, the vertebrate P-AR between light-activated rhodopsin and transducin results in a is equally related to Drosophila opsins as it is to those of ver- cascadeof effectsthat give rise to the photoreceptor potential. tebrates. The similarity in the number of mutations neededto Evolutionary conservation of a transducin binding site has been account for the divergence of these sequencessuggests that a postulated, as vertebrate transducin can be activated by verte- singleprimordial “opsin-like” moleculewas present in an ances- brate or invertebrate rhodopsin (Vandenbergand Montal, 1984). tor common to vetebrates and invertebrates, and that it gave On the cytoplasmic face, the first and the third cytoplasmic loop rise to vertebrate opsins,invertebrate rhodopsins,and the P-AR. of the Drosophila Rh3 opsin showa significant similarity to the Future studies involving the isolation and characterization of The Journal of Neuroscience, May 1987, 7(5) 1557 the invertebrate homolog to the P-AR and other sensory recep- Maniatis, T., R. C. Hardison, E. Lacy, J. Lauer, C. O’Connell, D. Quon, tors will help us dissect and understand similarities in signal- G. K. Sim, and A. Efstra.tiadis (1978) The isolation of structural genes from libraries of eukaryotic DNA. Cell 15: 687-701. transduction mechanisms. Maniatis. T.. E. F. Fritsch. and J. Sambrook (1982) Molecular Cloning: A Laboratory Manual, ‘Cold Spring Harbor Laboratory, Cold Spring References Harbor, NY. Bankier, A. T., and B. G. Barrel1 (1983) Shotgun DNA sequencing. Montell, C., K. Jones, C. Zuker, and G. Rubin (1987) A second opsin In Techniques in Nucleic Acid Biochemistry, vol. B5, R. A. Flavell, gene expressed in the ultraviolet-sensitive R7 photoreceptor cells of ed., pp. l-33, Elsevier, Amsterdam. Drosophila melanogaster. J. Neurosci. 7: 1558-1566. Benovic, J. L., F. Mayor, R. L. Somers, M. G. Caron, and R. J. Lelkowitz Nathans, J., and D. S. Hogness (1983) Isolation, sequence analysis, (1986) Light-dependent phosphorylation of rhodopsin by p-adrener- and intron-exon arrangement of the gene encoding bovine rhodopsin. gic receptor k&e. Nature 321: 869-872. Cell 34: 807-8 14. Blumbera. D.. and H. F. Lodish (1980) Comnlexitv of nuclear and Nathans, J., and D. S. Hogness (1984) Isolation and nucleotide se- polysomal RNAs in growing Di‘ctyostelium discoideum cells. Dev. quence of the gene encoding human rhodopsin. Proc. Natl. Acad. Sci. Biol. 78: 268-284. USA 81: 4851-4855. Chung, S., S. M. Landfear, D. D. Blumberg, N. S. Cohen, and H. F. Nathans, J., D. Thomas, and D. S. Hogness (1986) Molecular genetics Lodish (198 1) Synthesis and stability of developmentally regulated of human color vision: The genes encoding blue, green and red pig- Dictyostelium mRNAs are affected by cell-cell contact and CAMP. ments. Science 232: 193-202. Cell 24: 785-797. O’Hare, K., R. Levis, and G. M. Rubin (1983) Transcription of the Cowman, A. C., C. S. Zuker, and G. M. Rubin (1986) An opsin gene white locus in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA expressed in only one photoreceptor cell type of the Drosophila eye. 80: 6917-6921. Cell 44: 705-7 10. Oliver, D. V., and J. P. Phillips (1970) Technical note. Drosophila Dixon, R., B. Kobilka, D. Strader, J. Benovic, H. Dohlman, T. Frielle, Information Service 45: 58. - M. Bolanowski, C. Bennet, E. Rands, R. Diehl, R. Mumford, E. Slater, Ostrov. S. E.. M. Wilson. and W. L. Pak (1974) Drosovhila rhodovsin: I. Sigal, M. G. Caron, R. J. Lelkowitz, and C. D. Strader (1986) Photochemistry, extraction and differences’ in the -norpAp1z photo- Cloning of the gene and cDNA for mammalian @-adrenergic receptor transduction mutant. Biochem. Biophys. Res. Commun. 59: 960- and homology with rhodopsin. Nature 321: 75-79. 966. Fain, G. L., and J. E. Lisman (198 1) Membrane conductances of O’Tousa, J. E., W. Baehr, R. L. Martin, J. Hirsh, W. L. Pak, and M. DhOtOrCCCDtOrS. Pros. Biouhvs. Mol. Biol. 37: 91-147. L. Applebury (1985) The Drosophila ninuE gene encodes an opsin. Farris, J. C.^ (1972) Estimating phylogenetic trees from distance ma- Cell 40: 839-850. trices. Am. Natur. 106: 645-668. Ovchinnikov, Y. A. (1982) Rhodopsin and bacteriorhodopsin: Struc- Fein, A., and E. Szuts (1982) Photoreceptors: Their Role in Vision, ture-function relationships. FEBS Lett. 148: 179-l 89. Cambridge U. P., Cambridge, UK. Ovchinnikov, Y. A., N. G. Abdulaev, M. V. Feigina, I. D. Artamonov, Hafen, E., M. Levine, R. L. Garber, and W. J. Gehring (1983) An A, S. Zolotarev, A. I. Miroshnikov, U. I. Martynov, M. B. Kustina, imnroved in situ hvbridization method for detection of cellular RNAs A. B. Kudelin, and A. S. Bogachuk (1982) The complete amino acid in Drosophila tissue sections and its application for localizing tran- sequence of visual rhodopsin. Bioorg. Khim. 8: 10 1 l-l 0 14. scripts of the homeotic Antennupedia gene complex. EMBO J. 2: 6 17- Pak, W. L. (1979) Study of photoreceptor function using Drosophila 623. mutants. In Neurogenetics, Genetic Approaches to the Nervous System, Hardie, R. C. (1983) Functional organization of the fly retina. In X. D. Breakfield, id., pp 67-99, Elsevier North-Holland, New York. Progress in Sensory Physiology 5, D. Ottoson, ed., pp. l-79, Springer, Pavvin. D. J. C.. and J. B. C. Findlav (1984) Seauence variability in New York. ihe retinal attachment domain of mammalian rhodopsins. Biochkm. Hargrave, P. A. (1982) Rhodopsin chemistry, structure and topog- J. 217: 605-613. raphy. In Progress in Retinal Research, N. Osborne and G. Chader, Rubin, G. M. (1985) P transposable elements and their use as genetic eds., pp. 1-51, Oxford U. P., Oxford, UK. tools in Drosophila. Trends Neurosci. 8: 231-233. Hargrave, P. A., J. H. McDowell, D. R. Curtis, J. K. Wang, E. Juszczak, Sanger, F., S. Nicklen, and A. R. Coulson (1977) DNA sequencing S.-L. Fong, J. K. Mohanna Rao, and P. Argos (1983) The structure with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: of bovine rhodopsin. Biophys. Struct. Mech. 9: 235-244. 5463-5467. Harris, W. A., W. S. Stark, and J. A. Walker (1976) Genetic dissection Scavarda, N. J., J. O’Tousa, and W. L. Pak (1983) Drosophila locus of the photoreceptor system in the compound eye of Drosophila mel- with gene-dosage effects on rhodopsin. Proc. Natl. Acad. Sci. USA anoguster. J. Physiol. (Land.) 256: 4151439. 80: 4441-4445. Hillman. P.. S. Hochstein. and B. Minke (1983) Transduction in in- Stieve, H. (1986) The Molecular Mechanism of Photoreception, (Dah- vertebrate photoreceptors: Role of pigment b&ability. Physiol. Rev. lem Konferenzen 1986) H. Stieve, ed., pp l-10, Springer, Berlin. 63: 668-772. Strver. L. (1983) Transducin and the cvclic GMP vhosvhodiesterase: Kuhn, H. (1984) Interactions between photoexcited rhodopsin and Amplify& proteins in vision. Cold Spring Harbor Symp. Quant. Biol. light-activated enzymes in rods. In Progress in Retinal Research, vol. 48: 841-852. 3, N. Osborne and J. Chader, eds., pp. 124-156, Oxford U. P., Oxford, Stryer, L. ( 1985) Molecular design of an amplification cascade in vi- UK. sion. Biopolymers 24: 29-47. Kvte, J., and R. F. Doolittle (1982) A simple model for displaying the Tomlinson, A. (1985) The cellular dynamics of pattern formation in hydropathic character of a protein. J. Mol. Biol. 157: 105-l 32. the eye of Drosophila. J. Embryol. Exp. Morphol. 89: 313-331. Lancer-Sofer. P. R.. M. Levine. and D. C. Ward (1982) Immunoloaical Vandenberg, C., and M. Montal (1984) Light-regulated biochemical method for mapping genes on Drosophila pblytene chromosomes. events in invertebrate photoreceptors. Biochemistry 23: 2339-2347. Proc. Natl. Acad. Sci. USA 79: 438 l-4385. Wilson, A. C. (1985) The molecular basis of evolution. Sci. Am. 253: Letkowitz, R. L., J. M. Stadel, and M. G. Caron (1983) Adenylate 164-173. cyclase-coupled beta-adrenergic receptors: Structure and mechanisms Zuker, C. S., A. F. Cowman, and G. M. Rubin (1985) Isolation and of activation and desensitization. Annu. Rev. Biochem. 52: 159-186. structure of a rhodopsin gene from D. melanoguster. Cell 40: 851- Levy, L. S., R. Ganguly, N. Ganguly, and J. E. Manning (1982) The 858. selection, expression, and organization of a set of head-specific genes in Drosophila. Dev. Biol. 94: 451-464.