Isolation and sequence determination of the chicken rhodopsin gene

V&n Res. Vol. 28, NO. 4, pp. 471-480, 1988 hinted in Great Britain. All rights reserved

Copyright 0

0042-6989/88 $3.00 + 0.00 1988 Pergamon Press plc

ISOLATION AND SEQUENCE DETERMINATION OF THE CHICKEN RHODOPSIN GENE MASAW

TAKAO,’ AKIRA YASUI’

and FUMIO TOKUNAGA”

‘Department of Physics, Faculty of Science and 2Research Institute for Tuberculosis and Cancer, Tohoku University, Sendai 980, Japan (Received 14 July 1987; in revisedform 21 September 1987) Abstract-A chicken genomic library was screened with a bovine opsin cDNA probe. A clone isolated under high stringency hybridization conditions contained DNA sequences highly homologous to all of the five exons of bovine and human opsin genes. Sequence comparison of the putative open reading frame in the chicken DNA fragment of 4.3 kb with bovine opsin cDNA revealed 82% identity for the nucleotide and 87% for the deduced amino acid sequence, indicating that this DNA fragment contains the complete chicken opsin gene. The position of four introns and amino acid sequences at all putative cytoplasmic loops are exactly conserved in chicken and mammals. Opsin Rhodopsin homology (chicken)

Visual pigment

Genomic

INTRODUCTION

Rhodopsin is a photo-receptor protein which acts as a signal transducer in the rod outer segments of animal eyes (reviewed by Lamb, 1986). The rhodopsin molecule consists of retinal as a chromophore and a single polypeptide, opsin. This molecule is thought to consist of seven helices which transverse the lipid bilayer of the disk membrane (Ovchinnikov, 1982; Hargrave et al., 1983). On absorbing light, the rhodopsin conformation is preferentially altered to one coming into contact with at least three known proteins on the cytoplasmic surface, transducin, rhodopsin kinase and 48k-protein. Although the conformational changes by light have been suggested from the differential susceptibility to proteolysis (Pellicone et al., 1985b), the interacting regions with cytoplasmic proteins have not been determined yet. Besides rhodopsin, animals with color vision possess several kinds of color pigments in the cone outer segments. Nathans et al. (1986) established the amino acid sequences of three kinds of human color pigments which are 4042% homologous to human opsin. In chicken, DNA related to the visual pigment genes has been detected by cross-hybridization with a bovine opsin cDNA probe (Martin et al., 1986). These studies demonstrate a single *To whom correspondence

should be addressed. 471

sequence

Membrane

protein

Sequence

strongly hybridizing fragment and several weakly hybridizing fragments on Southern blots of restricted chicken genomic DNA, presumably genes for opsin and color pigments (Yen and Fager, 1984). To investigate the homology and difference between avian and mammalian opsins as well as between opsin and color pigments, we have cloned a cDNA encoding a part of bovine opsin (Koike et al., 1983) for use as a DNA probe. This paper describes the isolation and sequence analysis of a strongly hybridizing clone of DNA from a chicken genomic library. The amino acid sequence comparison clearly shows that the isolated DNA includes the whole chicken opsin gene. MATERIALS AND METHODS

Chicken genomic

Zibrary and cDNA probe

The chicken genomic DNA library which had been prepared by cloning of Sau3AI partially digested total chicken genomic DNA into a cosmid vector pKY2662. This was kindly provided by S. Mizuno (Tohoku University). An incomplete cDNA for bovine opsin was cloned in pBR322 previously by Koike et al. (1983). To prepare a probe for screening, a DNA fragment, BO-IP, generated by PstI digestion of the plasmid containing cDNA was recovered from low temperature-melting point agarose gel (Bio-Rad), and labeled with

412

MASASHITAKAO ct ai

[a-32P]dCTP (Multiprime labeling system, Ame&am). ~yb~dization was performed under the condition described below. Co/any hybridization and Southern blot analysis According to Grunstein and Hogness (1975), in situ colony hybridization was carried out. Total chicken DNA from whole blood and human DNA from cultured cell (HMV-I) were blotted on a filter (Nylon, Amersham) after digestion with restriction enzymes. Southern blots or in situ colony DNA blots were incubated overnight at 42°C with the 32P-labeled probe in hybridization buffer containing 5 x SSC (1 x SSC = 0.15 M NaCI, 0.015 M sodium citrate), 20 mM sodium phosphate (pH 6.5), 10% dextran sulfate, 50% formamide, 100 pg/ml sheared salmon DNA and 1 x Denhardt’s solution (0.2% each of BSA, polyvinylpyrrolidone and Ficoll 400). For the low stringency hybridization, the membrane filter was washed with 2 x SSC containing 0.1% SDS at 42°C. After autoradiography with an X-ray film (Kodak XARS), the same membrane filter was rewashed with 0.2 x SSC containing 0.1% SDS at 62°C to obtain a signal resulting from hybridization under high stringency conditions. Subcloning and nucleotide sequencing

Isolated DNA clones were digested with restriction enzymes and each restriction fragment was subcloned into pUC9 or pUC18. After confirming insertion of the fragment, double stranded DNA was denatured and used as a template for dideoxy sequencing method (Sanger et al., 1977; Chen and Seeburg, 1985). When sequencing results were ambiguous (probably due to the presence of GC-rich segments), 7-deaza-dGTP (Boehringer Mannheim) was used instead of dGTP in the polymerization step to avoid this problem (Mizusawa et al., 1986). The nucleotide sequences of almost all subclones were dete~in~ in both directions. The sequences of all subclones could be unequivocally ordered by overlapping sequences. RESULTS AND DISCUSSION

Rhodopsin genes from cattle, human and fruit fly have been cloned and sequenced in several laboratories (reviewed by Applebury and Hargrave, 1986). As expected from the conservation of their functional properties, their sequences are well conserved. Our strategy to isolate the chicken visual pigment gene, there-

fore, was based on the assumed high homology in nucleotide sequence between mammalian and avian opsin genes. An incomplete cDNA of bovine opsin cloned previously in our laboratory (Koike et ul., 1983) contains two fifths of the coding region upstream from the stop codon (434 bp) and the 3’-untranslated region (377 bp) [Fig. l(A)]. Most of the non-coding region was removed from the coding region by Pstl digestion. With respect to the opsin gene structure (Nathans and Hogness, 1983) the resulting fragment (BO-IP) is to contain the complete exon 4 and 5 and a part of exon 3. Using the BO-IP as a nucleotide probe [Fig. I(A)], eight positive clones were isolated from 5 x lo4 transfo~ant colonies by colony hybridization under low stringency conditions. Six of them were still positive after the high stringency wash. Their electrophoretic patterns of several restriction endonuclease digests revealed that all clones were identical to each other except one. which came from one of the two weakly hybridizing clones. The restriction pattern of this DNA did not show any band common to those of the other seven clones. We further investigated one of the six strongly hybridizing clones, designated gAR 13 1. To confirm that gARl31 includes segments homologous to BO-IP, digests with several endonuclease were analyzed by Southern hybridization. As seen in Fig. l(B), the probe could detect only one fragment from each digest. Subclones, gAR131B, gAR131H and gAR131P were constructed from the positive fragments of gAR131 digested with BamHI, Hind III, and PstI, respectively. Mapping of the restriction site of these subclones showed that they contained a common 1.6 kb segment in a BamHI/HindIII fragment recognized by the probe BO-IP. Starting with the common segment, a 4285bp sequence stretching to both sides of the f .6 kb segment was determined as shown in Fig. 2 and compared with those of bovine and human opsin genes reported by Nathans and Hogness (1983, 1984). Five highly homologous segments indicated by open blocks in Fig. 2 were found to correspond to exons 1 to 5 of the mammalian opsin gene, They are interrupted by four segments bounded by consensus sequences of the intron-splicing junction (Table 1). We thus infer that gAR131 includes the chicken opsin gene and that the five segments homologous to both the human and bovine genes are exons and four

I

BamHI

Bgl II

Hind IU

PM

m

475

Isolation of chicken rhodopsin gene JCommon

1.6kb segment -J, gARl31H

I

1 kb

gAR131P 1

5%

gAR131B Sp S ScPvScPvPvH

- --zz?._.L- a -_= 77rr-7

-- -1c--

L L--__\-Lz‘--x---L - > -vr---7

Bg

7

PHScPPSc

F

B

5 \_

Fig. 2. Sequence strategy of gARl31. Relative position of inserts of subclones gARl31H, gARl31P and gARl31B is shown in the upper panel. Five exons of chicken opsin predicted by the sequencing and the comparison with bovine opsin are indicated as open blocks in a middle graph. BumHI( BgfII(Bg), HincII(Hc), Hi&III(H), PsrI(P), SacI( pVuII(Pv), SmaI(S), SphI(Sp) and TuqI(T). Not all sites for PvuII and TaqI are indicated. A bar beneath them is the segment used as a probe in Fig. 4. The sequenced segments are shown as horizontal arrows in a lower graph.

non-homologous segments are introns. This inference can be justified as described below. Assuming that the splicing sites are similar to those of mammalian opsin genes, the putative open reading frame in the sequenced region of gAR13 1 consists of 1053 bp which generates a polypeptide of 351 amino acid residues (Fig. 3). Lengths of exons 14 are exactly the same as those of the mammalian opsin genes. The fifth exon is 9 nucleotides longer than mammalian ones. When comparing the homology for these exons between chicken and bovine, the exons l-4 gave 83-86% identity for the nucleotide sequence and 8493% for the amino acid sequence. In contrast, relatively weak homology was found in exon 5, 67% for nucleotide sequence and 69% for amino acid sequence. Similar values were obtained by pairwise comparison of chicken and human. Although the lengths of introns are quite similar between human and bovine opsin genes (Nathans and Hogness, 1984), the first and second introns of the chicken gene were much shorter than those of the human one (834 bp and 75 bp vs 1783 bp and 1205 bp, respectively), but the third and fourth introns of the chicken

Table 1. Sequence in the splice junction 5’ exon

intron

Consensus

ZAG GT&AGT--------(Py

Intron Intron Intron Intron

GCG ACG CAG GAG

1 2 3 4

3’exon > 1l)NFAG

G

GTGAGT----
Sequences of the putative splice sites are listed and compared with consensus sequence reported by Mount (1982). Py = C, T. Pu = A, G.

gene were comparable in length to that of the human gene (130 bp and 876 bp vs 116 bp and 833 bp, respectively). None of the four introns showed any appreciable homology to the human introns except sequences near splicing junctions. Since the deduced amino acid sequence encompassing these four splice junctions consists of more than 9 consecutive residues identical to the equivalent stretches in human and bovine opsin, and since alternative splicing sites were not found (except ones which resulted in an inclusion of a nonsense codon in the reading frame, a frame-shift or a significant loss of homology), we concluded that the above assumed positions for splicing sites were correct. Previous analysis on the chicken genome by Martin er al. (1986) showed that bovine opsin cDNA probe could detect bands of chicken DNA digests with the size of 12 kb (,&RI), 4.4 kb (HindIII), 4 kb (BumHI) or 2 kb (PstI) in high stringency hybridization experiments which were similar to our conditions. The latter three fragments in their study were consistent in size with the inserts of the subclone gARl31H (4.2kb), gARl3lB (4.3 kb) and gAR13lP (2.1 kb), respectively. Although the BO-IP probe contains the whole sequence of exon 5 and therefore it should detect 2 bands in, the Southern blot of Hind111 or BgZII digests of gARl31, only one band is visible [Fig. l(A) and (B)]. This may be due to the weak homology between sequences of exon 5 in the bovine and chicken genes. We found a unique 12 kb EcoRI fragment in chicken total DNA by Southern hybridization using a fragment from gAR 131 as a probe (Fig. 4), suggesting that the isolated gene is a single copy in the chicken genome.

MASASHITAKAOet al.

Fig. 3. Sequence of gAR131. Position of the nucleotide (upper) and amino acid residue (lower) is counted from the first nucleotide determined and the first Met, respectively. In the coding region, boxed Asn residues are two potential sites for N-glycosylation. Putative chromophore-attachment site Lys 296 is also boxed. To save space, the determined sequences for each intron are omitted (see also Table I). The complete sequence is available on request. The underlined bases with arrows in the 3’-flanking represent complementary elements described in the text. The boxed bases at the position 553 and 3788 are putative TATA box and polyadenylation signal, respectively.

When comparing the sequences upstream from the coding region among opsin genes of chicken, human and cattle, several conserved segments were found as boxed in Fig. 5. A putative TATA box (Corden et al., 1980) is well conserved within a 13 bp stretch (region VI), while CCAAT homology (Jones et al., 1985) inferred in mammals is altered to CTAAT in chicken (region IV). Between these putative TATA and CCAAT elements, a GC-rich element (region V) seems to be conserved in the three sequences. Further upstream of the CCAAT homology, a TATA-like GATTAATA sequence (region III) is common to chicken and mammals and a sequence of 10 nucleotides harboring the CCAAT sequence is present at region II in human and chicken. A sequence GTGTCACCT common in both species was

also found (region I). Since the opsin gene is expressed specifically in retinal rod cells (Brann and Young, 1986), these highly conserved sequences might be related to the cell type specific expression in both organisms. In contrast to the upstream part, the sequence of chicken opsin downstream from the 3’ end of the coding region does not show any significant homology with those of the bovine or human gene. In the chicken gene, a putative polyadenylation signal ATTAAA is located at 128 bp downstream from the stop codon. Within this untranslated region, two complementary sequences which form the hairpin structure were detected, as in the bovine gene (Nathans and Hogness, 1983). The deduced amino acid sequence of chicken opsin gives a hydropathy profile (Kyte and

kb

-2.5

Fig. 4. Detection of the sequence of gARl31 in genomic DNA. EcoRI-restricted total DNA of chicken (lane I) or human (lane 2) is analyzed by Southern hybridization. Each 8 pg DNA was mn on 0.5% agarose gel and hybridized under low stringency conditions with a probe shown in Fig. 2. Note that a 10 kb fragment in the human DNA coincides with the EcoRI-restricted fragment of the human opsin gene (Nathans and Hogness, 1984).

477

B 310 Human opsin Bovine oprin Chicken oprin Human blue Human green /red

320

330

WyOfRnCIUTTICCCKWPLOQDEASA-TVSMTPT-----SQVAPA DEASt-T 5%. - _ TTL s”,“r_g -KM -QLFGKtVDDGSEL--S-O

340

350 -SWAPA TswSPA ST&GPN S--VSPA

Fig. 6. Structural model of the chicken rhodopsin and sequence comparison of the C-terminal segment of rhodopsins and color pigments. (A) The chicken rhodopsin amino acid sequence is arranged to represent a membrane topology, based on the model of Hargrave et al. (1983). The position at which the conservative changes (M:V:I:L, A:G, F:Y, S:T:Q:N, K:R and E:D) occur between chicken and bovine are indicated by shaded circles. The other substitutions are denoted with black circles. Arrows are the positions of introns. (B) The sequence of 42 amino acids from the C-terminal end of the chicken opsin is compared with the counterparts of human and bovine opsins and color pigments, human blue and human green/red. The number on top is that of the chicken sequence.

479

Isolation of chicken rhodopsin gene

Chicken Human

Bovine

*....

G~i-i-CCTGAGTACC ----TCTCC-T&i.&--CTCA&TTCCTCCT

________~__~________~~~~__________r__~~~~~~~~~~~~~~~~~~~~~

Fig. 5. The 5’-flanking ref@onsof chicken and mammalian genes. Sequences of chicken, human and bovine genes are aligned to optimize the nucleotide identity. Segments highly conserved between chicken and mammals are boxed. Single and double dots represent the nucleotide identical between chicken and human and between chicken and both mammals, respectively.

Doolittle, 1982) similar to that of bovine or human opsin (not shown). We arranged the primary sequence of chicken opsin according to the proposed structural model for bovine rhodopsin (Hargrave er al., 1983) as shown in Fig. d(A), Amino acid substitutions in seven transmembrane regions were found at up to 20 positions. However, 14 of these 20 residues are the results of conservative changes (see legend of Fig. 6) and the substitution does not occur in charged and proline residues. Two asparagine residues which are sites of glycosylation in bovine rhodopsin (Asn2, Asn 15) and the lysine which bears the chromophore in bovine rhodopsin (Lys296) are conserved in the identical position in chicken rhodopsin. Regions highly conserved between the chicken and mammalian sequences were found in three cytoplasmic loops and the first half of the C-terminal segment which is exposed to the cytoplasm. Since bovine rhodopsin can activate chicken transducin to the same degree as bovine transducin (McMurray et al., 1985) and since the chemical cleavages at loops III/IV, V/VI and C-terminal segment reduce the interaction with transducin (Pellicone et al., 1985a), some of these conserved segments may play an important role for the interaction with transducin. When compa~ng the C-terminal segment between rod pigments (chicken, bovine and human opsin) and cone pigments (human blue, green and red) [Fig. 6(B)J, the first half of the C-terminal segment (3 10-332) shows excellent homology. In the latter half of the C-terminal segment (333-351), the insertion and deletion of a few amino acids was observed in chicken opsin as compared to mammalian opsins. This feature

was found between human color pigments and mammalian opsins. In spite of these variations, this region consists of conserved blocks in all visual pigments. Therefore, the latter half of both rod and cone pigments may participate in a common function. Since the amino acid sequence of this part shares the potential phosphorylation sites, serine and threonine residues, this very C-terminal segment may be a domain interacting with rhodopsin kinase which is present in both rod and cone outer segments (Walter et al., 1986). Acknowledgements-We thank Professor Shigeki Mizuno for providing the genomic library and Professor Atsushi Oikawa for continuous support as well as helpful discussion. This work was supported in part by Grants-in-Aid for the Special Research Project on the “Molecular Basis of Biological Evolution” (58212002, 59106004) to F.T.

REFERENCES Applebury M. L. and Hargrave P. A. (1986) Molecular biology of the visual pigments. Vision Res. 26, 1881-1895. Brann M. R. and Young W. S. iii (1986) Localization and quantitation of opsin and transducin mRNAs in bovine retina by in situ hybridization hystochemistry. FEBBS Lett. zoo, 275-278. Chen E. J. and Seeburg P. H. (1985) Supercoil sequencing: a fast simple method for sequencing plasmid DNA. DNA 4, 165-170. Corden J., Wasylyk B., Buchwalder A., Sassone-Corsi P., Kedinger C. and Chambon P. (1980) Promoter sequences of eukaryotic protein-coding genes. Science, N.Y. 2@9, 14061414. Grunstein M. and Hogness D. (1975) Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc. natn. Acad. Sci. U.S.A. 72, 3961-3965. Hargrave P. A., McDowell J. H., Curtis D. R., Wang J. K., Juszczak E., Fong S-L., Mohanna Rao J. K. and Argos

480

MASASHITAKAOet al.

P. (1983). The structure of bovine rhodopsin. Biophys. Struct. Mech. 9, 235-244. Jones K. A., Yamamoto K. R. and Tjian R. (1985) Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro. Cell 42, 559-512. Koike S., Nabeshima Y., Ogata K., Fukui T., Ohtsuka E., Ikehara M. and Tokunaga F. (1983) Isolation and nucleotide sequence of a partial cDNA clone for bovine opsin. Biochem. biophys. Res. Commun. 116, 563-567. Kyte J. and Doolittle R. F. (1982) A simple method for displaying the hydropathic character of a protein. J. molec. Biol. 157, 105-132. Lamb T. D. (1986) Transduction in vertebrate photoreceptors. TINS 9, 224-228. Martin R. L., Wood C., Baehr W. and Applebury M. L. (1986) Visual pigment homologies revealed by DNA hybridization. Science, N.Y. 232, 12661269. McMurray M. M., Hansen J. S., Haley B. E., Takemoto D. J. and Takemoto L. T. (1985) Interspecies conservation of retinal guanosine 5’-triphosphatase. Biochem. J. 225, 227-232. Mizusawa S., Nishimura S. and Seela F. (1986) Improvement of the dideoxy chain termination method of DNA sequencing by use of deoxy-7-deaza-guanosin triphosphate in place of dGTP. Nucleic Acids Res. 14, 1319-1324. Mount S. M. (1982) A catalogue of splice junction sequences. Nucleic Acids Res. 10, 459-472. Nathans J. and Hogness D. S. (1983) Isolation, sequence

analysis, and intron-exon arrangement of the gene encoding bovine rhodopsin. Cell 34, 807-8 14. Nathans J. and Hogness D. S. (1984) Isolation and nucleotide sequence of the gene encoding human rhodopsin. Proc. natn. Acad. Sci. U.S.A. 81, 4851-4855. Nathans J., Thomas D. and Hogness D. S. (1986) Molecular genetics of human color vision: the genes encoding blue, green and red pigments. Science, N.Y. 232, 2033210. Ovchinnikov Y. A. (1982) Rhodopsin and bacteriorhodopsin: structure-function relationships. FEBBS Lett. 148, 179-189. Pellicone C., Cook N. J., Nullans G. and Virmaux N. (1985a) Light-induced conformational changes in rhodopsin detected by modification of G-protein binding, GTPyS binding and cGMP phosphodiesterase activation. FEBBS Lett. 181, 184-188. Pellicone C., Nullans G., Cook N. J. and Virmaux N. (1985b) Light-induced conformational changes in the extradiscal regions of bovine rhodopsin. Biochem. biophys. Res. Commun. 127, 81&821. Sanger F., Nicklen S. and Coulson A. (1977) DNA sequencing with chain-terminating inhibitors. Proc. natn. Acad. Sci. U.S.A. 74, 5463-5467. Walter A. E., Shuster T. A. and Farber D. B. (1986) Light-induced phosphorylation of proteins from the allcone retina of the lizard, Anolis carolinensis. Invest. Ophthal. visual Sci. 27, 16091614. Yen L. and Fager R. S. (1984) Chromatographic resolution of the rod pigment from the four cone pigments of the chicken retina. Vision Res. 24, 15551562.