Expression of Visual and American Chameleon Nonvisual Opsins In

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Expression of Visual and Nonvisual Opsins in American Chameleon SHOJI KAWAMURA,* SHOZO YOKOYAMA*? Received 29 May 1996; in revised form 5 November 1996

We previously characterized five visual opsin genes of American chameleon (Anolis carolinensis). Here we report its nonvisual opsin gene orthologous to the chicken pineal gland-specific opsin (P- opsin) gene. In the pure-cone American chameleon retina, all visual opsins including rod opsin are expressed. In both pineal and parietal eye, three visual opsins as well as P-opsin are expressed. Although opsins are detected in the pineal glands of a wide variety of vertebrates, Southern analysis suggests that the P-opsin gene is used mainly by birds and reptiles. © 1997 Elsevier Science Ltd.

P~9_psin Retina Pineal Parietaleye Vertebrates

INTRODUCTION opsin-like proteins have been detected by an immunocy- The periodic nature of biological functions is under direct tochemical study (Foster et al., 1993). Currently known opsins are classified into six major groups (Yokoyama, influence of both a daily light/dark cycle and an 1994, 1995, 1996): (1) the RH1 cluster (consisting of endogenous clock (Sassone-Corsi, 1994). Biological rhodopsins); (2) the RH2 cluster (a mixture of opsins with clocks have been detected in the hypothalamic supra- various absorption sensitivities); (3) the SWS1 cluster chiasmatic nucleus of mammals (Meijer & Rietveld, (blue, violet, and UV opsins); (4) the SWS2 cluster (blue 1989), pineal gland of birds, reptiles, and fish (Takahashi opsins); (5) the LWS/MWS cluster (a mixture of green et al., 1989; Falcon et al., 1989), and the retina of and red opsins); and 6) the P-opsin cluster. We have amphibians, birds, and mammals (Cahill et al., 1991; previously characterized visual opsin genes of the Tosini & Menaker, 1996). The pineal gland of many non- American chameleon rhl ac, rh2ac, swsl ac, SWS2Ac, and mammalian vertebrates is believed to regulate photo- lWSAc that encode RH1, RH2, SWS1, SWS2, and LWS, reception and melatonin synthesis and has been identified respectively (Kawamura & Yokoyama, 1993, 1994, as a major component of the circadian system (Under- 1995, 1996). The lizard has an additional simple but wood, 1990; Korf, 1994). Despite different sites of the highly structured photoreceptor organ, the parietal (third) clock and variation in the pineal anatomy (Korf, 1994), eye, suspected of enhancing the detection of dawn and the core mechanism of the circadian rhythm appears to be dusk (Solessio & Engbretson, 1993). Here we report the fundamentally similar among all vertebrates, involving cloning and sequencing of the P-opsin gene of American opsins or opsin-like proteins (Takahashi, 1993). Such chameleon (denoted PAc). The isolation of PAc and the proteins have been detected in the pineal gland of visual opsin genes provides a unique opportunity for the lampreys, teleosts, frogs, birds, and reptiles (Vigh- comprehensive study of opsin gene expression in the Teichmann et al., 1982; Vigh-Teichmann & Vigh, retina and other photoreceptive organs. 1990; Kalsow et al., 1991; Araki et al., 1992; Foster et al., 1993; Tamotsu et al., 1994; Masuda et al., 1994; MATERIALS AND METHODS Yoshikawa et al., 1994). Recently, the gene encoding the pineal gland-specific opsin, named pinopsin (Okano et Genomic library screening al., 1994) or P-opsin (Max et al., 1995), has been isolated A genomic library was constructed with BamHI- from the chicken. digested lambda EMBL3 vector and Sau3AI partially The pineal gland of the American chameleon (Anolis digested genomic DNA of the American chameleon carolinensis) shows light-dependent daily cycles of (Kawamura & Yokoyama, 1993). We also constructed melatonin synthesis both in vitro and in vivo (Menaker another genomic library using BamHI- and EcoRI- &Wisner, 1983; Underwood & Hyde, 1989), where digested genomic DNA, the long arm of BamHI-digested and the short arm of EcoRI-digested EMBL4 lambda vectors. From these libraries, we isolated 2Ac34 and *Biological Research Laboratories, Department of Biology, Syracuse University, 130 College Place, Syracuse, NY 13244, U.S.A. )~Ac167 containing exons 1-4 and exons 4 and 5 of PAc, ?To whom all correspondence should be addressed [Tel 315-443-9166; respectively, using the bovine rhodopsin cDNA (bd20) Fax 315-443-2012; Email [email protected]]. (Nathans & Hogness, 1983) as the probe. Probe- 1867 1868 S. KAWAMURAand S. YOKOYAMA hybridizing restriction fragments were subcloned into the sapiens), and bovine (Bos taurus), liver of chicken Bluescript SK(-) plasmid vectors. Probe labelling, (Gallus gallus), and muscle of lamprey (Petromyzon plaque hybridization, membrane washing and sequencing marinus), goldfsh (Carassius auratus), Mexican cavefish for both strands were done as described previously (Astyanax fasciatus), and Tokay gecko (Gekko gekko) (Kawamura & Yokoyama, 1993). (Blin & Stafford, 1976). Frog (Xenopus laevis) and coelacanth (Latemeria chalumnae) DNA were gifts from Reverse transcriptase-polymerase chain reaction (RT- Drs. B. Knox and R. DeSalle, respectively, ten micro- PCR) analysis grams (5 #g for American chameleon) per lane of From four animals, approximately 1, 2, and 20 #g of genomic DNA was digested with SstI, electrophoresed total RNA were extracted from the parietal eye, pineal on 0.5% agarose gel, and transferred to a Hybond-N gland, and retina, respectively (Chomczynski & Sacchi, nylon membrane by using the VacuGene vacuum blotting 1987). Total RNA was mixed with the PCR reaction mix system (Pharmacia). The last 170 bp portion of the PAc [10 mM Tris-HCl (pH 9.0), 1.5 mM MgCI2, 50 mM KC1 exon 4 was amplified by PCR using two primers: 5'- 0.1% Triton X-100, 200/tM dNTPs, and primers at 1/~M TCA TGGT( G/C)A TCG( G/C)(T/C)TTCCTI( A/G )T( G/ each], MMLV reverse transcriptase, and Taq polymerase C)TGCTGG(G/C)T-3' and 5'-CTGTYI'GTI'CATGAA- in total volume of 25/d. The samples were placed in a G(A/C)(A/C)(G/A)TAG-3' and was used as the hybridi- thermal cycler at 50°C for 8 min, followed by 35 cycles zation probe. of 92°C for 45 sec, 55°C for 60 sec, and 72°C for 90 sec. The probe was labeled with [:¢_32p] dATP by the Five microlitres each of PCR products was electrophor- random-priming method. Hybridization was carried out esed on 2.5% agarose gel. PCR was also carried out at 65°C following the commercial protocol for Hybond-N without reverse transcriptase for each opsin gene, membrane. The hybridized membrane was washed in resulting in no amplification in all tissues. Southern 1 ×SSC/0.1% SDS at 65°C four times (30 min each), hybridization of RT-PCR products was carried out using which allows ~20% mismatch (Meinkoth & Wahl, mixed radioactive probe containing bovine rhodopsin 1984). (bd20) (Nathans & Hogness, 1983), human blue (hs37) and red (hs7) (Nathans et al., 1986), and chicken P-opsin RESULTS AND DISCUSSION cDNAs (Max et al., 1995). Hybridization was carried out at 65°C following the commercial protocol for Hybond-N Molecular structure of the American chameleon P-opsin membrane (Amersham). Hybridized membrane was gene washed in 1 × SSC/0.1% SDS at 55°C and autoradio- The basic structure of PAc is identical to that of the graphed after 5 hr of exposure. All amplified cDNA chicken P-opsin gene. It has five exons. The position of fragments were sequenced either directly or after being the second intron is displaced 15 nucleotides toward the cloned and were confirmed to match the corresponding 3' direction compared to the equivalent intron in the genomic DNA sequences. visual opsin genes (Max et al., 1995). Splice junction signals (GT/AG) are conserved in all introns. The Zoo blot analysis deduced P-opsins of American chameleon (PAt) and High molecular weight DNA were extracted from the chicken (Pc,g) have two less amino acids than visual blood of the owl (Bubo virginianus), turkey (Meleagris opsins, located between the fourth and fifth putative gallopavo), pigeon (Columba livia), human (Homo transmembrane domains (Okano et al., 1994; Max et al.,

I II PAc M ...... LNG- - Tp- G PFEGpQWPFLA - PRG TYTSVAVLMGLVVVLATVVNGLVI WSVRYKR LRS PLNY I LVNLAVADL LVTSFG S TI SFANN I yGFFVFG PTACEFEG F i01 PGg M ...... S ...... S ...... NS - SQ -A- p - p - NG- - Tp - GPFDGPQWPYQA- PQSTYVGVAVLMGTVVACASVVNGLVI VVS I CYKKLRS PLNY I LVNLAVADLLVTLCG S SVS LSNN I NGFFVFGRRMCE LEG F 109 RHIAc M--N---G ..... T--EG .... Q- -NFYVPMS- - NK - T-GVVR- NPFEY PQYYL- AD P -WQFSALAAYMFLLI LLGF P I NFLTI,FVTI QHKKLRTPLNY I LLNLAVANLFMVLMGFTTTMYTSMNGYF I FG TVGCN IEG F 115 RH2Ac M--N---G .... T--EG .... I--NFYVPLS--NK~T-GLVE-SPFEYPQYYL AEP-WKYKvVCCYIFFLIFTGLPINILT•LVTFKHKKLRQPLNYILVNLAVADLFMACFGFTVTFYTAWNGYFIFG•IGCAIEGF 115 SWSIAc M~-SG ...... Q ..... E- - DFYLF ...... E -NI SSVG PWDG PQYH I -APM - WAFYFQTAFMGFVFFAGTPLNAI I L I VTVKYKKLRQ PLNY I LVNI SFAGFLFCTFSVFTVFMAS SQGYFFFGRHVCAMEAF 110 SWS2Ac MTMQKSRPDS .... R- DNL P - -E - - DFFI PVP- LDV-A-NI TTLS PFLVPQTH L - GN P - S LFMGMAAFMFI LIVLGVP INVLTI FCTFKYKKLRSHLNY I LVNLSVSNLLVVCVGSTTAFY SFSNMYFS LG PTACKIEGF 124 LWSAc M--AGT~WDvAVFAARRRNDEDDT-TRDSLF~TNSNNT~-GPFEGPNYHI-A-~RWVYNITSVwMIFVVIASIFTNGLVLVATAKFKKLRHPLNwILVNLAIADLGETVIASTISVINQISGYFI~GH~MCVLEGY 134 Ill IV V -- PAc MVS L~ IVGLWSLA I LAFERYLVI CKPVGDFRFQQRHAVI GCAF~/LWSLLWTL P PLFGWSSY I PEGLRTRCGPNWYTGGN - - DNNSY I MTLFVTCF I TPLAMI I FSYANLL LTLRAVAAQQKEMATTQQAE REVT~ 239 PGg MVSLTG IVGLWSLAI LALERYVVVCRPLGDFQFQRRHAVSGCAFT~IGWALLWSTPPLLGWS SYVPEGLRTSCGPNWYTGGS - - NNNS Y I LS LFVTCFVLPLSLI LFSY~LLTL~~ R~I 247 RHIAc FATLGGEMSLWSLVVLAVERYWI CKPMSNFRFGETHALIGVSC~WI MADACAG P PLLGWS RY I PIDGMQCSCGVDYYTPTPEVHNESFVIYMFLVHFVT PLTI I FF~G RLV~ESA~E R~ 255 RH2AC FATLGGQVALWSLVVLAIERY IWCKPMGNFRFSATHALMGI SFTWFMSFSCAAP PLLGWSRY I PEGMQCSCG PDYyTLNPDYHNESYVLYMFG~I ~I ~SYG~I ~ESAS~ER~I 255 SWSIAc LGSVAGLVTGWS LAFLAFERy IVI CKPFGNFRFNSKHALLV%rAATWFIGIGVS I PPFFGWSRY I PEGLQCSCGPDWYTVGTKYKSEYYTWFLFI FCF IVPLTLI I FSYSQL~L~V~ESA~ER~S ~ 250 SWS2Ac SATI~MVSLWS LAWAFERYLVI CKPLGNFTFRG"I~HAII GCAV~GLAAS L PPLFGWSRy I PEGLQCSCGPDWYTTE~N~YVI FLFCFCFGVPLSVI I FSYGRLLLTL~VAK~EQSA~ER~ 263 LWSAc TVSTCG I SALWSLAVI SWERWVVVCKPFGNVI~DAKLAVAG IVFSWVWSAVWTAP PVFGWS RYWPHGLKTSCG PDgFSGSDD PG~YM IVLMI TCCFI P~VI LL~ I ~V~SES~K~S~ 274 VI VII PAc T~LVCNLPYASFAMVVATNKDLLIQ PALASLPSYFSKTATVYN P I I YVFMNKQFRSCLLS - TLSCGR - RP - QAAQG ~P~ I SS PRGRTLEGS RN~PSAS~SGND~TS 352 PGg VMVMAFLLCWLPYSTFALWATHKGI I IQPVLASLPSYFSKTATVYNPI I YVFMNKQFQSCLLE- MLCCGy ~ Q P - QRTGKAS PGTFG PHADVTAAGL RNKVMpAH PV 351 RHIAC IMVI SFLVCWVPYASVAFY I FTHQGSDFG PV~I pAFFAKSSAIYNPVI Y ILMNKQFRNCMIM- TLCCGKNPLGDEE- - TSAGT- - - KTETSTVSTSQVS PA 352 RH2AC LM~LLAWTPYAMVAFWl FTNKGVDFSATLMSVPAFFSKSSS LYNP I I YVLMNKQFRNCMI T- TI CCGKNPFGDED-VSSSVSQS - KTEVSSVSS SQVS PA 355 SWSIAc VMvG•FCLCYvPYASLAMYMvNNRDHGLDLRLVTIPAFFSKSSCVYNPIIYCFMNKQFRACILE-TV-CGK-PMSDES-D -VSSSAQKTEVSSVSSSQVSPS 347 SWS2Ac VI~LVCWLpyASFALWVVTHRGEPFDVRLAS I pSVFSKA~TVYNPVI YVLMNKQFRSCMLKLIF - CGKS PFG DED- D- - VSG SSQA~I~VSSVSSSQVS PA 365 LWSAc VMI IAYCFCWGPyTVFACFAAANPGYAFH pLAAALPAYFAKSATI YN PI I yVFMNRQFRNC IMQ- LF- -GK- KV- DDGSE - - LSS TS - RTEVSSVSNSSVS PA 369

FIGURE 1. Alignmentof the deducedamino acid sequences of PAc, PGg (Max et al., 1995) and five visual opsins of American chameleon (Kawamura& Yokoyama,1993, 1994, 1995, 1996). Gaps, denotedby dashes,were introducedto optimize sequence similarity. Putative transmembrane domains l-Vll are indicated by horizontal lines. OPSIN GENE EXPRESSION f86~

American chameleon 1 Expression of visual- and P-opsin genes --~ RH1 Chicken Using opsin gene-specific primers [Fig. 3(A)], we have examined the expression of rhl Ac, rh2Ao sWSl Ac, SWS2Ac, ~ Chicken I RH2 IWSAc and PAc in the retina, pineal gland, and parietal eye of American chameleon by RT-PCR assay. In the retina, im American chameleon 64 all five visual opsin genes are expressed [Fig. 3(B)]. t I American chameleon I SWS1 However, the expression of the rod opsin gene, rhlAc, is totally unexpected because not only is rod opsin not Chicken detected in the retina by immunocytochemical assay 66 Chicken (Foster et al., 1993) but also this species is believed to sws2 have a pure-cone retina (Yu & Fager, 1982; Fowlkes et ~I American chameleon I al., 1984; Walter et al., 1986). This suggests that either Chlcken rod opsin is produced at a low level in some cone I P-opsln photoreceptor cells or this species possesses a small American chameleon number of rod photoreceptors which might have been overlooked in the previous analyses. In the American LWS chameleon retina, three types of cone pigments with EC:[[:n chameleon] maximal absorption (2max) at 625 nm, 503 nm, and 462 nm are known to exist (Provencio et al., 1992), which appear to correspond to LWS, RH2, and SWS2 FIGURE 2. The phylogenetic tree for P-opsins and visual opsins of pigments, respectively (Kawamura & Yokoyama, 1993, American chameleon and chicken. RH1, RH2, SWS1, SWS2, and 1995, 1996). Therefore, expression of two additional LWS opsins of chicken correspond to its rhodopsin, green, violet, blue, visual opsin genes in the retina suggests the presence of and red opsins (Okano et al., 1992). To construct a rooted tree, Rhl ultraviolet-sensitive cones which have been detected in (GnBank K02315), Rh2 (M12896), Rh3 (M17718), and Rh4 (M17719 its closely related Puerto Rican Anolis species (Fleishman and M17739) opsins of Drosophila melanogaster were used as the outgroup. The topology and branch lengths of the phylogenetic tree et al., 1993). were estimated by using the neighbor-joining method (Saitou & Nei, 1987) based on the Poisson-corrected numbers of amino acid substitutions per site. The bootstrap supports were generated by resampling 1000 replications (Higgins et al., 1992) and are indicated (A) beside branch nodes unless they are 100%. opsJn expected size gene forward reverse of cDNA (bp)

PAC 5'-ccgtcattggctgcgctttc-3' 5'-acagctcgcagggtcaagag-3' 235 1995) (Fig. 1). PAc and Pog have 73% amino acid identity rhIAc 5'-cccttattggggtgagttgc-3' 5'-gccgctttcactgtacagac-3' 241 with each other, while they have 45-50% amino acid rh2Ac 5'-ccttgatgggcatttctttt-3' 5'-gcctctcgaactttgcatat-3' 241 identity to the visual opsins in vertebrates. A phyloge- sWSlAc 5'-tacacctgqttcctcttcat-3' 5'~ctgtttgttcatgaagcagtag-3' 330 netic tree constructed from the sequences of a number of swS2Ac 5'-ccatcatcggttgtgctgtg-3' 5'-acagctctcagggtgagaag-3' 241 bird and reptile opsins clearly shows that PAc is more IWSAC 5'-ccgtggctggcattgtcttc~3' 5'-accgcacggatagccaacca-3' 241 closely related to Pog than to the visual opsins (Fig. 2). PAc and Pog are unique at four sites, containing amino 0BI ~ reUrm ~ parietale~e acids F88 (F96 for Pog), D131 (D139), S163 (S171), and N176 (N184), while those at the corresponding sites of the visual opsins are Y, N, R, and D (Fig. 1). Thus, Fig. 2 suggests that the common ancestor of the two opsins might have achieved amino acid replacements Y88F, N131D, R163S, and D176N, following the site number of -240 PAc. When the seven transmembrane model of an opsin (Hargrave et al., 1983) is considered, Y88F, R163S, and D176 are located in the cytoplasmic region, while N131D is in the intradiscal region. Furthermore, Pog shows the FIGURE1 3. RT-PCR assay for the visual- and //"P-opsin gene expression chicken P-opsin-specific amino acid replacements in the retina, pineal gland and parietal eye of American chameleon. (A) Primers used for the RT-PCR analysis. For swslAc and the other opsin K135R and R306Q in the intradiscal region. Since these genes, codons between Y198-Q307 and those between the second amino acid changes probably do not interact with the nucleotide of A139 and that of V217 ofpac are amplified, respectively chromophore in the transmembrane region directly, they (see Fig. 1). (B) RT-PCR amplification of opsin cDNAs from the might have been important in modifying the interaction retina and pineal gland. (C) Southern hybridization of RT-PCR products from the parietal eye with the mixed radioactive probe of the P-opsin with other proteins in the pineal gland. The containing bovine rhodopsin (bd20) (Nathans & Hngness, 1983), exact effects of these changes on the function of the P- human blue (hs37) and red (hs7) (Nathans et al., 1986) and chicken P- opsin remain to be examined. opsin cDNAs (Max et al., 1995). 1870 S. KAWAMURA and S. YOKOYAMA

hybridized to genomic DNA from jawless fish (marine lamprey), bony fishes (goldfish, Mexican cavefish, and coelacanth), frog, birds (chicken, pigeon, great horned owl. and turkey), lizards (American chameleon and Tokay gecko), and mammals (human and bovine) (Fig, (kb) 4). Hybridization signals were detected only fl)r lizards. 23.1 - chicken, pigeon, turkey and lamprey but not tk)r the other

9.4- species (the hybridization data for Mexican cavelish. coelacanth, turkey, and bovine are not shown). Curioush. 6.6- owl DNA showed no hybridization signal. The lack of the 4.4- P-opsin gene is consistent with the observation that the pineal gland of another nocturnal owl (Strir urah'n.sis)is 2.3 partially degenerated and may not play a physiological 2.0- circadian oscillatory role in owls (Taniguchi et al.. 1993). 1.4- The hybridization result strongly suggests that the P- 1.1 - 0.9-- opsin gene is found mainly in some birds and reptiles and that teleosts, amphibians, and mammals do no! possess FIGURE 4. Southern hybridization of vertebrate genomic DNA to the the orthologous gene. exon 4 of PA~.. A mixture of 2 Hindlll and qSX174RF Haelll size Phylogenetic analyses show that P-opsin alrcad\ standards are indicated in kb. Two hybridizing bands tier American existed before the divergence of various verlebratcs chameleon arc consistent with those expected from the restriction maps (Okano el al., 1994: Max el a[., 1995). tto,~evcr. of two allclic forms of P,v, (data not shown). Southern analysis suggests that the P-opsin gcnc disappeared in many vertebrate lineages during evolution. What are the types of opsins used in the pineal In the pineal gland, expression of PA~, and lower levels ghmds of teleosts, amphibians, and even in some of SWS2Ac and lwsA,, expression are detected by staining mannnals (Korf. 1994)? Perhaps these species use visual the agarose gel with ethidium bromide [Fig. 3(B)]. When opsins and/or entirely new, yet unknown, pineal gland- the gel was Southern-blotted and hybridized to mixed specilic opsins for pineal photoreception. radioactive probes (see Materials and Methods), expres- swsl,v, sion of was additionally detected (result not REFERENCES shown). The immunocytochemical study has suggested the presence of RHI (rod) opsin in the pineal gland of Araki. M.. Fukada, 'i'.. Shichida, Y., Yoshizawa. T. & "lokunaga. t (19t)2). l)iffcrentiation of both rod and cone types ol pholorccepl~ls American chameleon (Foster et aL, 1993), but we cannot in the il# i'il'o and iH I'ilro developing pineal glancts ol the qu'

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Yokoyama, and an anonymous 2201-2208. reviewer were greatly appreciated. We thank G. A. Engbretson for his Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new help in isolating RNA from American chameleon and J. Nathans and method for reconstructing phylogenetic tree. Molecular Biology and M. Max for their providing cDNA probes. The work was supported by Evolution, 4, 406~-25. a NIH grant GM42379.