Structural characterization and transcriptional pattern of two types of carp rhodopsin gene

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Structural characterization and transcriptional pattern of two

types of carp rhodopsin gene

Chih-Ying Su, Jormay Lim, Huai-Jen Tsai *

Institute of Fisheries Science, National Taiwan Uni6ersity, Section4,1Roose6elt Road, Taipei106, Taiwan, ROC Received 19 May 1999; received in revised form 30 August 1999; accepted 1 September 1999

Abstract

This work characterizes the genomic structures of two types of carp (Cyprinus carpio) rhodopsin (cRh) gene, i.e. type I (cRh-I) and type II (cRh-II). Two types of cRh gene share only 45.6% polynucleotide identity in the upstream region from nucleotide − 3436 to + 97. However, three conserved regions are found. Homologies to the consensus recognition sites for transcription factors, Crx and Nrl, which are involved in photoreceptor-specific expression, are also observed in cRh genes. With specific polymerase chain reaction (PCR) primers, the two types of cRh gene can be clearly discriminated from each carp genome. Most carps exhibit both types of cRh gene, however, there are still carps possessing either cRh-I or cRh-II. Both cRh-I and cRh-II mRNAs are expressed at an approximately equal level in both eyes extracted from a carp carrying both types of cRh gene. © 2000 Elsevier Science Inc. All rights reserved.

Keywords:Cyprinus carpio; Fish; Genomic structure; PCR; Photoreceptor; Rhodopsin; Sequence comparison; Type discrimination

1. Introduction

Eyes are photoreceptor organs that contribute to image formation. Photoreceptor cells consist of rods and cones. Rods are sensitive to dim light, while cones contribute to daytime vision and color perception. Each photoreceptor consists of a seven-transmembrane protein, opsin, to which its chromophore, 11-cis-retinal, is attached (Rose et al., 1989). Previous investigations have reported on the presence of rhodopsin cDNA in extremely broad varieties, such as bovine (Nathans and Hogness, 1983), humans (Nathans and Hogness, 1984), chickens (Takao et al., 1988), mice (Baehr et al., 1988), rats (Morabito et al., 1991), lizards (Kawamura and Yokayama, 1994), canines

(Pe-terson-Jones et al., 1994), old world monkeys (Nickells et al., 1995), salamanders (Chen et al., 1996), Xenopus (Saha and Grainger, 1993; Batni et al., 1996), squids (Hall et al., 1991; Hara-Nishimura et al., 1993), lamprey (Hisatomi et al., 1991), goldfish (Johnson et al., 1992), marine lam-prey (Zhang and Yokoyama, 1997), skates (O’Brien et al., 1997), carps (Tsai et al., 1994; Lim et al., 1997) and marine fish, John Dory (Archer and Hirano, 1998). These studies include terres-trial animals, amphibians and aquatic animals. Nevertheless, genomic structure of rhodopsin was studied primarily in Xenopus (Batni et al., 1996), chickens (Takao et al., 1988) and mammals, such as bovine (Nathans and Hogness, 1983), humans (Nathans and Hogness 1984; Bennett et al., 1995), mice (Baehr et al., 1988), and rats (Morabito et al., 1991). Those investigations demonstrate that rhodopsin gene contains four or five introns. In-terestingly, Fitzgibbon et al. (1995) indicated that

* Corresponding author. Tel.: + 886-2-2366-1540; fax: + 886-2-2363-8483.

E-mail address:hjtsai@ccms.ntu.edu.tw (H.-J. Tsai)

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the rhodopsin gene of bony fish is intron-free, although the genomic structures of those bony fish have not been completely characterized. Among all the species studied, only one type of rhodopsin gene has been reported except Xenopus and the one studied in a previous investigation, the common carps (Lim et al., 1997).

In light of above discussion, this study demon-strates the rhodopsin genomic structures of the most primitive vertebrate, fish. The entire molecu-lar sequences corresponding to two types of carp rhodopsin (cRh) cDNAs are elucidated. In addi-tion, type discriminations and transcription pat-terns of cRh genes are studied as well. Comparing the polynucleotide sequences of the upstream re-gion and investigating the transcriptional patterns of type I cRh (cRh-I) and type II cRh (cRh-II) genes facilitate an understanding of the rhodopsin gene regulation of fish. More thoroughly under-standing rhodopsin genes of diverse species will provide further insight into visual systems.

2. Materials and methods

2.1. Genomic library construction, screening and

subcloning

Genomic DNA extracted from carp blood (Sambrook et al., 1989) was utilized to construct the genomic DNA library by using Lamda FIX®

II/XhoI Partial Fill-In Vector Kit and Gigapack®

III Gold Packaging Extract (Stratagene). Approx-imately 60 000 phages were hybridized by a radio-labeled probe (1.3 kb fragment of cRh-I cDNA) overnight at 42°C. The membranes were washed and then exposed to X-ray films. Each putative phage clone (100 ml) was heated and vigorously vortexed as a template for polymerase chain reac-tion (PCR). Type-specific primers were used to detect the cRh-I and cRh-II genes. PCR was performed for 25 cycles in a 20 ml solution con-taining 7ml of phage lysate, 0.2 mM of dNTP, 0.5 mM of primers, 1× buffer and 2.5 U VioTaq DNA polymerase (Viogene). Each amplification cycle consisted of denaturation at 94°C for 1 min, annealing at 62°C for 1 min, and extension at 72°C for 1 min.

Insert fragments were excised from recombi-nant phage DNA by SalI digestion and purified by gel extraction. The 5% and 3% flanking regions of

cRh genes were determined by PCR using the

upstream- and downstream-specific primers (5%-GGCTGCGGTTGGATGTTGT-3%, 5%-CTGAT-AAGTGCCAGATATG C-3%, 5%-CCCCGGG-AAAGAGCAACCA-3% and 5%-CATGCCTCTA-CAATCACAACAAA-3%). Insert fragments were then subcloned into pBluescript® _{II SK ( + / − )}

vector.

2.2. The primer extension assay

Total retinal RNA from adult carps dissected during the daytime was extracted with TRI Reagent (Molecular Research Center, Inc). The [g-33_{p]ATP-labeled primers were hybridized by}

retinal RNA at 50°C for 22 h, and then precipi-tated by ethanol. The pellet was resuspended in reverse transcription buffer and reversely tran-scribed with SuperScript™ II RNaseH− _Reverse

Transcriptase (Gibco BRL). Finally, the extension reaction was loaded along with the sequencing reaction of the subcloned DNA sequenced with the same primer on a 6% polyacrylamide/8 M urea gel.

2.3. Sequence analysis

Sequencing was performed with Dye Termina-tor Cycle Sequencing Ready Reaction Kit (ABI) and analyzed on ABI PRISM™ 310 Genetic Ana-lyzer. Polynucleotide sequences were read at both ends and analyzed with PC/GENE® _Program

(IntelliGenetics).

2.4. Type discrimination

Fifty nanograms of genomic DNA extracted from carp blood (Sambrook et al., 1989) was used as template to perform PCR reaction with type-specific primers as above description. The PCR products were digested by TfiI for differentiating

cRh-I and cRh-II genes. In total, 100 carps

ran-domly selected from the local aquacultural ponds (Tzu-Pei, located in northern Taiwan and Lu-Kang, located in middle Taiwan) were analyzed for the distribution frequency of cRh-I and cRh-II genotypes.

2.5. Transcription pattern

Total retinal RNA of each eye of an adult carp carrying both cRh-I and cRh-II genes was ex-tracted and reversely transcribed as above

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descrip-Fig. 1. Comparison of the upstream (A) and downstream (B) polynucleotides of the type I (cRh-I) and type II (cRh-II) rhodopsin genes of carp. Numbers on the right side of each column represent the nucleotide position relative to transcription start site ( + 1), which is boxed. The transcribed region is underlined. TATA and CAAT boxes of cRh-I and cRh-II genes are indicated above and below, respectively. Identical bases are denoted by vertical bars. Gaps, denoted by broken lines, are introduced to maximize identities. Regions containing greater than 75% identity between the upstream region of two types of cRh genes are shaded. The polyadenylation signals found in cDNA clones are in boldface and italics.

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tion. The RNA was removed by RNaseH diges-tion. PCR was performed with type-specific primers as described above except that retinal cDNA was used as a template.

3. Results and discussion

3.1. Isolation of recombinant phages containing

cRh genes

A genomic DNA library of titer 1011 _pfu/ml

was constructed. Two cRh-I clones and three

cRh-II clones were isolated from the library.

Thereafter, recombinant phages contained 14 kb of cRh-I insert and 10 kb of cRh-II insert were obtained, respectively. After restriction mapping and subcloning, DNA fragments around 3.4 and 0.3 kb containing upstream and downstream flanking regions of cRh genes, respectively, were sequenced (Fig. 1). These sequences are submit-ted to the EMBL/GeneBank/DDBJ databases under Accession No. AJ 012013, AJ 012009, AJ 012014 and AJ 012010.

3.2. Characterization of both types of cRh gene The primer extension assay revealed that the

transcription initiation site ( + 1) of cRh was 94 bp upstream of the start codon (data not shown). A TATA box was observed at nucle-otide (nt) − 33 −36 in both I and

cRh-II genes. As generally known, this nt position is

relatively conserved among all known species. Similar to the rhodopsin gene of mice (Baehr et al., 1988), the cRh-II gene had a CAAT box at nt − 121 to − 118. However, the cRh-I gene had a CAAT box at nt − 235 to − 238, which was 120 bp ahead of that of cRh-II gene (Fig. 1A). Both types of cRh gene were intronless. This finding correlates with Fitzgibbon et al. (1995), which contended that rhodopsin gene of bony fish lacks intron.

3.3. Upstream flanking sequences analysis

Compared to the cRh-II gene, the cRh-I gene shared only 45.6% polynucleotide identity in the upstream region ranging from nt − 3434 to + 97 of I or from nt − 3435 to + 97 of

cRh-II. However, three relatively highly conserved

regions were observed at nt − 1262 to − 614, − 539 to − 419 and − 166 to + 97, with 78.0, 79.3 and 92.3% identities, respectively (Fig. 1A).

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Fig. 2. Nucleotide identities among cRh and other known vertebrates rhodopsin upstream sequences. Sequence alignments of the290 immediate upstream nucleotides of cRh-I, cRh-II, Xenopus (Xen), chickens (Chk), Chinese hamsters (Ham), rats (Rat), mice (Mus), humans (Hum) and bovine (Bov) rhodopsin genes is shown. Numbers on the right side of each column represent the nucleotide position relative to transcription start site ( + 1), which is underlined. Gaps, indicated by broken lines, are introduced to maximize identities. Shaded areas represent sequences that are identical in all mammalian species; where the sequence of carp, Xenopus or chicken is identical, it is also shaded. Positions of the TATA box, Ret 4, NRE, BAT-1 and Ret 1 of mammals are indicated below the alignment. Boxed regions indicate consensus recognition sites of Crx, which binds to C/TTAATCC, Ret 4, BAT-1 and Ret 1 (Kumar et al., 1996), and Nrl, which binds to NRE (Chen et al., 1997; Rehemtulla et al., 1996). Arrow indicates the translation initiating codon.

In Fig. 2, the proximal upstream flanking se-quences of cRh-I and cRh-II genes are compared with those of known vertebrates (Batni et al., 1996; Chen et al., 1996, 1997). Consensus recogni-tion sites for Crx and Nrl are observed in the corresponding regions of cRh genes. Crx and Nrl are trans-factors that work synergically to bind to and transactivate the genes for several photore-ceptor-specific proteins (Chen et al., 1997). Crx binds independently to three sites in the rhodopsin proximal promoter region of bovine, which are Ret 1, Ret 4 and BAT-1, and C/ TTAATCC of other photoreceptor-specific genes (Chen et al., 1997); and Nrl binds to NRE (Ku-mar et al., 1996; Rehemtulla et al., 1996). In cRh genes, homologies of the binding core regions of BAT-1 and NRE were observed in corresponding

sites. In addition, sequence sharing homology to C/TTAATCC was also found at nt − 46 −52 in cRh genes (Fig. 2). These findings suggest that Crx and Nrl may also regulate cRh genes.

Ret 1 core region (CTAATTAG) and its facili-tated flanking sequences (GGCCCC) (Morabito et al., 1991; Yu et al., 1996) and Ret 4 (Chen and Zack, 1996) have been proven to play a critical role in driving photoreceptor-specific gene expres-sion in rats. Nevertheless, no corresponding se-quence was observed in either cRh-I or cRh-II gene. In addition, no significant matches were found in cRh genes with the known rhodopsin enhancer region, which is a highly conserved re-gion located at 1.52 kb upstream of mam-malian rhodopsin genes (Nie et al., 1996).

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Except for the consensus recognition sites for Crx and Nrl, the upstream flanking regions of both types of cRh gene shared relatively low identity with those of other terrestrial verte-brates. This evidence implies the existence of other regulatory elements in cRh genes. It is speculated that those potential regulatory ele-ments might locate within the well-conserved up-stream regions between cRh-I and cRh-II genes. According to transgenic studies of rhodopsin up-stream regulatory regions, the proximal upup-stream portion (300 bp) is prerequisite and sufficient for photoreceptor-specific expression of the re-porter gene. Meanwhile, sequences of more distal regions are responsible for accurate expression patterns and higher expression levels (Batni et al., 1996; Knox et al., 1998; Nie et al., 1996; Zack et al., 1991). Hence, it was postulated that the most proximally conserved region of cRh up-stream sequence may play a prominent role in controlling the photoreceptor-specific expression of fish rhodopsin. And, the other two more dis-tally conserved regions may regulate the spatial or temporal expression patterns or function as an enhancer.

3.4. Downstream flanking sequences analysis The downstream untranscribed regions of

cRh-I and cRh-cRh-IcRh-I genes were sequenced from nt 1555

to 1915 and from nt 1653 to 1885, respectively. Sequence alignment of the downstream region and a portion of cDNA sequences between the two types of cRh gene revealed a highly con-served region with a 86.1% polynucleotide iden-tity, which is located at nt 1466 – 1753 of cRh-I gene or nt 1476 – 1763 of cRh-II gene. Three of the four polyadenylation signals of cRh-II cDNA and one of the cRh-I cDNA were also indicated (Fig. 1B). Interestingly, corresponding sequences of the two excess polyadenylation signals of

cRh-II cDNA were found in the downstream flanking

region of cRh-I genomic DNA. In addition, the excess 88 bp of cRh-II cDNA shared 83% iden-tity with a corresponding region of cRh-I gene. This finding suggests that the polyadenylation signals may be alternatively chosen while tran-scribing rhodopsin mRNA.

3.5. Type discrimination of cRh in genome Two types of cRh gene were differentiated

us-ing TfiI to restrict PCR products generated by type-specific primers (Fig. 3A). Type I specific primers generated a cRh-I PCR product of 272 bp. Notably, this fragment was not cut by TfiI. However, a cRh-II PCR product of 281 bp gen-erated by type II specific primers was cleaved by

TfiI into two fragments of 168 and 113 bp (Fig.

3B,C). Ninety carps from the local pond (Tzu-Pei) were examined for the distribution of two types of cRh gene. The results showed that only two fish (2.2% of total samples) exhibited merely

cRh-I genotype, and 19 fish (21.1%) exhibited

merely cRh-II genotype. However, most of them (76.7%) carried both cRh-I and cRh-II geno-types. Similar results were also observed with carps from a geographically different pond (Lu-Kang). Furthermore, the distribution of two types of cRh gene was irrelevant to the sex or size of fish. And there seemed to be no notice-able differences in terms of morphological ap-pearance between carps showing only one cRh type and the ones showing both. Since not every carp carries both cRh-I and cRh-II genes, the two types of cRh gene are less likely to be two distinct genes but two rhodopsin alleles in the carp population.

The common carp is a phylogenetically te-traploid fish. In addition to the fact that there is only one rhodopsin gene in diploid vertebrates, the existence of two types of cRh gene may be attributed to tetraploidy, which is a similar event of the two myc genes of carp (Zhang et al., 1995). Subsequent to the original tetraploidiza-tion event, one of the two duplicate genes could have been silenced in their expression (Ferris and Whitt, 1977). In this study, RT-PCR was per-formed using retinal total RNA to determine whether or not one of the duplicated genes en-coding rhodopsin has been silenced in a carp carrying both cRh-I and cRh-II genes. These re-sults demonstrated that both cRh-I and cRh-II mRNAs were expressed at an approximately equal level in both eyes (Fig. 4). Related investi-gations have indicated that the tetraploid X.

lae6is has two types of rhodopsin corresponding

to two morphologically distinct rod cells (Rohlich et al., 1989; Batni et al., 1996). In con-trast, the rod cells of carp carrying both types of

cRh gene were not morphologically differentiated

into two types under light microscopic observa-tion (data not shown).

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Fig. 4. Transcriptional patterns of cRh-I and cRh-II genes of a carp carrying both types cRh gene. Retinal RNAs extracted from left (L) and right (R) eyes of a carp were subjected to RT-PCR in the presence (A) and absence (B) of reverse transcriptase using cRh-I (I) and cRh-II (II) specific primers. Lane M: molecular weight marker, HaeIII-digested_fX174.

Fig. 3. Determination of two types of cRh gene via polymerase chain reaction (PCR) and restriction analysis. (A) Sequences of type-specific primers. Specific primers for cRh-I gene: for-ward RHO1-f and reverse RHO1-r; for cRh-II gene: forfor-ward RHO2-f and reverse RHO2-r. The different nucleotides be-tween the two types of primers were underlined and in bold-face. (B) Schematic of the expected molecular sizes of PCR products generated by type-specific primers. (C) Agarose gel electrophoresis of PCR products of cRh-I (I) and cRh-II (II) genes, and their TfiI restriction patterns. Lane M: molecular weight marker, HaeIII-digestedfX174.

4. Conclusions

(1.) Molecular structures of two types of carp rhodopsin gene, namely cRh-I and cRh-II, have been determined. Comparison of the upstream flanking sequences of the two types of cRh gene reveals three highly conserved regions, which are at nt − 1262 to − 614, − 539 to − 419 and − 166 to + 97, with 78.0, 79.3 and 92.3% identi-ties, respectively.

(2.) The sequence of the proximal upstream flanking region of cRh genes shows homologies to consensus recognition sites for transcription fac-tors, Crx and Nrl, which are involved in photore-ceptor-specific expression.

(3.) Two types of cRh gene are clearly discrimi-nated from each carp via PCR and restriction analysis. Most carps carry both types of cRh gene, however, there are still carps possessing either

cRh-I or cRh-II genotype.

(4.) In a carp carrying both types of cRh gene, none of the genes is silenced but are expressed equally in both eyes.

Acknowledgements

The authors wish to thank Mr Kuo-Ching Ma for assisting in multiple sequence alignments, and Mr Hung-Kuang Peng, director of Tzu-Pei branch, Taiwan Fisheries Research Institute (TFRI), and Mr Jong-Yih Lai, director of

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44

Kang branch, TFRI, for providing fish. This work was supported by the National Science Council, R.O.C., under NSC 87-2611-B-002-003.

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