Genomic organization of the human rod photoreceptor cGMP-gated cation channel β-subunit gene

Gene 245 (2000) 311–318 www.elsevier.com/locate/gene

Genomic organization of the human rod photoreceptor cGMP-gated cation channel b-subunit gene Michelle D. Ardell a,b, D. Lawrence Bedsole c, Robert V. Schoborg a,d, Steven J. Pittler e,f, * a Department of Pharmacology, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA b Department of Internal Medicine, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA c University of South Alabama College of Medicine, Mobile, AL 36688, USA d Department of Microbiology, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA e Department of Physiological Optics, University of Alabama at Birmingham, Birmingham, AL 35294-4390, USA f Vision Science Research Center, University of Alabama at Birmingham, Birmingham, AL 35294-4390, USA Received 25 August 1999; received in revised form 23 December 1999; accepted 13 January 2000 Received by J.L. Slightom

Abstract We previously reported that the CNGB1 locus encoding the rod photoreceptor cGMP-gated channel b-subunit is complex, comprising non-overlapping transcription units that give rise to at least six transcripts (Ardell, M.D., Aragon, I., Oliveira, L., Porche, G.E., Burke, E., Pittler, S.J., 1996. The beta subunit of human rod photoreceptor cGMP-gated cation channel is generated from a complex transcription unit. FEBS Lett. 389, 213–218). To further understand the transcriptional regulation of this extraordinarily complex locus, and to develop a screen for defects in the gene in patients with hereditary disease, we determined its genomic organization and DNA sequence. The CNGB1 locus consists of 33 exons, which span approximately 100 kb of genomic DNA on chromosome 16. The b-subunit comprises two domains, an N-terminal glutamic acid-rich segment (GARP), and a C-terminal channel-like portion. Two additional exons encoding a short GARP transcript and a truncated channel-like transcript have been identified. A major transcription start point was identified 79 bp upstream of the initiator ATG. To begin analysis of the basis for the generation of multiple transcripts, and to identify promoters driving expression in retina, approximately 2.5 kb of the upstream region were sequenced. Putative cis-elements, which can bind the retina-specific transcription factors Crx and Erx, were found immediately upstream of the transcription start point, and may be important for gene expression in this tissue. From our analysis, a model is reported to account for at least four of the retinal transcripts. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Alternative splicing; Gene structure; Phototransduction; Promoter; Retina

1. Introduction A critical function of the visual transduction cascade of mammalian rod photoreceptors is mediated by nonselective cation channels, which are directly gated by cGMP (Barnstable, 1993; Yau, 1994). These channels function to control the flow of sodium and calcium ions through the plasma membrane in response to lightAbbreviations: CNGB1, cyclic nucleotide-gated cation channel b-subunit gene (previous designations now obsolete are CNCG2 and CNCG3L); GARP, glutamic acid rich protein; LA-PCR, long and accurate PCR; RACE, rapid amplification of cDNA ends; RP, retinitis pigmentosa; tsp, transcription start point. * Corresponding author. Tel.: +1-205-934-6744; fax: +1-205-934-5725. E-mail address: [email protected] (S.J. Pittler)

induced changes in intracellular cGMP levels. In the dark, the elevated levels of cGMP result in a significant number of the channels being maintained in their open state. Adsorption of light by the photoreceptor leads to hydrolysis of cGMP, with a concomitant closure of the channel and hyperpolarization of the cell ( Finn et al., 1996). Mammalian rod photoreceptor cGMP-gated cation channels are multimeric polypeptides comprising a- and b-subunits (Chen et al., 1993, 1994). Cloning and functional co-expression of these subunits in Xenopus oocytes have demonstrated that the a-subunit contains a cGMPbinding domain, six transmembrane segments, and an ion pore domain ( Kaupp et al., 1989; Dhallan et al., 1992; Pittler et al., 1992). While the b-subunit also contains apparent channel-like domains, it does not

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form functional subunits when expressed alone. Coexpression with the a-subunit, however, imparts rapid channel flickering, -cis-diltiazem and Ca2+/ calmodulin sensitivity as observed with the native channel (Chen et al., 1993, 1994; Ko¨rschen et al., 1995). Molecular characterization of the bovine and human rod photoreceptor b-subunits reveals that both comprise a novel bipartite structure (Ardell et al., 1995, 1996; Ko¨rschen et al., 1995; Colville and Molday, 1996). The N-terminal one-third of the channel b-subunit is glutamic acid-rich and is also expressed as a separate protein (GARP), which is highly enriched in rod photoreceptors (Sugimoto et al., 1991; Ardell et al., 1995; Colville and Molday, 1996). The C-terminal two-thirds correspond to the channel-like portion that is homologous to the a-subunit. We previously mapped the CNGB1 locus encoding the GARP and b-subunit transcripts to chromosomal region 16q13, and determined that it is quite complex, giving rise to at least six transcripts in the retina (Ardell et al., 1996). Due to the essential function of the rod photoreceptor cGMP-gated cation channel in the visual transduction pathway, mutations in the genes encoding the channel subunits are good candidate genes for producing retinitis pigmentosa (RP). Indeed, defects in the a-subunit gene have been linked to one form of RP (Dryja et al., 1995). In order to develop a screen for defects in the b-subunit in RP patients, and to begin to dissect the complex transcription pattern generated from this locus, it was first necessary to determine the intron/exon organization. In this report, we show the complete gene structure encoding the human rod photoreceptor b-subunit, a partial characterization of the upstream region, and provide a basis for the complex pattern of gene expression.

2. Materials and methods 2.1. Materials The human leukocyte genomic DNA prepared in lEMBL3-sp6/T7 and the Promoter Finder Kit were from Clontech (Palo Alto, CA); the Expand Long Template PCR System was from Boehringer Mannheim; DNA markers were from MBI Fermentas. Human retinas were obtained as a gift from Dr G. Pena and the Florida Lions Eye Bank, Bascom Palmer Eye Institute (Miami, FL). Retinal RNA was isolated using RNAzol B ( Tel-test, Inc.) according to the manufacturer’s protocol. A yield of approximately 0.7 mg of RNA/g of human retina was obtained. Primer sequences were chosen using Gene Runner primer analysis software (Hastings Software), and the oligonucleotides were either generated in house using a Beckman Oligo 1000 DNA synthesizer or obtained commercially (Life Technologies, Gaithersburg, MD). The sequence of

primers used for DNA sequencing will be provided upon request. Sequencing reactions were carried out on both strands with the fMol cycle sequencing system (Promega, Madison, WI ) as described previously (Ardell et al., 1996). Sequence analysis and manipulations were performed with the Wisconsin Genetics Computer Group (GCG) DNA analysis programs running in UNIX on a SUN workstation, and with Vector NTI 5.0 (InforMax, N. Bethesda, MD) on a PC. 2.2. Determination of intron/exon junctions The genomic DNA library was subjected to multiple rounds of screening with radiolabeled PCR products corresponding to different regions of the b-subunit cDNA sequence (Ardell et al., 1996) using a method previously described (Pittler et al., 1990). Positively hybridizing clones were purified to homogeneity, and DNA was isolated and sequenced directly. Comparison of the genomic sequence to the cDNA sequence was used to confirm the identity of the clones and to ascertain intron/exon boundaries. For regions where overlapping genomic clones could not be isolated, LA-PCR was performed with the Expand System (Boehringer Mannheim). The product PCR-1 ( Fig. 1) was obtained using 50 ng of human genomic DNA as template with the primers 5∞-GAGTGGCAGCAAGAAGGCAATT and 5∞-AGCACCCTCTGGACCCAGCCCA; PCR-2 was obtained with primers 5∞-GAACTGTTGGCTGATTCCCGTG and 5∞-ACGTAGGCTTTGCTGAGGATGG; PCR-3 was obtained with primers 5∞-TCGCTATGACAGGAAAGATGGG and 5∞-CACTTGATGCAACTTGTCGAGC; PCR-4 was obtained with external primers 5∞-GTAAGTCATTTGCTCACCTGTCTGGGAAC, and 5∞-TACAGAGATGCCCAAGAACCAGTCTCAAC, and verified with internal primers 5∞-AAGCCCTCCTTCCTAGCAC and 5∞-GTACAGAGATGCCCAAGAAC. 2.3. Identification of the transcription start point An oligonucleotide primer, 5∞-AGATGCCAACCGCCAGCCAGGAAT, complementary to nt −15 to −38 within the first exon of CNGB1 ( Fig. 2) was synthesized and labeled with [c-32P]ATP using T4 polynucleotide kinase. Unincorporated label was removed by two rounds of ethanol precipitation in 2 M NH Ac, 10 mM 4 MgCl . Two picomoles of the radiolabeled primer were 2 hybridized to either 40 mg of human retina RNA or yeast tRNA in 50 mM Tris–Cl, pH 8.3, 75 mM KCl, 3 mM MgCl at 75°C for 10 min followed by 3 h at 2 50°C. The RNA was then reverse-transcribed in 50 mM Tris–Cl, pH 8.3, 75 mM KCl, 3 mM MgCl , 0.5 mM 2 dNTPs, 10 mM DTT, 2.4 mg of actinomycin D, 40 units of RNAsin (Promega) and 300 units of SuperScript II (Gibco/BRL) reverse transcriptase at 42°C for 1 h. The

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Fig. 1. Structure and functional domains of the human b-subunit gene. (A) Exon distribution of the b-subunit gene. The gene structure was determined by DNA sequence analysis of bacteriophage l genomic clones and PCR products spanning gaps. Introns that were not sequenced in their entirety were size estimated by LA-PCR with flanking primers. The donor and acceptor site sequences ( Table 1) all conform to consensus splice site sequences (Mount, 1982). The b-subunit gene comprises 33 exons spanning 100 kb [the 10 kb scale bar is for panels (A) and (C )]. Exons 12a and 18a represent alternative exons used in the generation of hGARP (1.6 kb) and hRCNC2a (10 kb) transcripts (see Fig. 3), respectively. The 33 exons of the b-subunit gene are numbered above the exon, and the designation below the exons corresponds to previously published nomenclature (Ardell et al., 1995, 1996; Sautter et al., 1998) and as in Fig. 3. (B) Exon distribution of functional and conserved domains in the b-subunit. Domains that are essential or possibly important for calcium calmodulin/regulation of cGMP affinity (Cam) are contained within exons 19 and 32, respectively (Grunwald et al., 1998; Weitz et al., 1998). A cyclic nucleotide binding domain (CNBD) is contained within exons 29–31. Regions highly homologous to the transmembrane (S1–S6) and (P) pore domains found in the a-subunit are encoded within exons 21–26. Two glutamate-rich domains (GRD1 and 2) that are conserved in human, rat, and bovine GARP sequences are encoded by exons 2, 3 and 14. Overall identities in the GARP protein between the three species are less than 60%, yet two conserved domains (CD1 and 2) show >85% identity that are encoded by exons 2 and 11, 12. (C ) Genomic clones and PCR products characterized. Eight human genomic clones (lhCNCG-a to f ) and three PCR products (PCR-1 to 4) were sequenced and compared to the cDNA sequence to determine the gene structure.

RNA was digested for 30 min at 37° C by adding 1 mg of DNase-free RNase (Sigma), and the samples were extracted with phenol/chloroform/isoamyl alcohol (25:24:1). Sodium acetate, pH 5.2, was added to a final concentration of 300 mM, and the cDNAs were precipitated by the addition of two volumes of absolute ethanol in the presence of 1 ml of Pellet Paint (Novagen). Following ethanol precipitation, the pellet was washed with 70% ethanol and dried. The pellet was resuspended in 1× loading buffer (98% deionized formamide, 10 mM EDTA, pH 8.0, 0.01 mg/ml of xylene cyanol, 0.001% w/v Bromophenol Blue) and electrophoresed on an 8% polyacrylamide-urea gel adjacent to a DNA sequencing ladder generated from the human GARP cDNA subclone designated pBS-GAR (Ardell et al., 1995) using the channel-specific oligonucleotide 5∞-GATTCGGTTCTGGTTCCACCTC as the primer. Gels were visualized utilizing autoradiography, and a Molecular Imager phosphoimager running with Quantity One (version 4) software (BioRad, Hercules, CA) was used to determine signal intensity of bands.

performed in a 50 ml final volume with 50 ng each of the Promoter Finder primer AP1 and the b-subunit specific primer GSP1, 5∞-CCTAAATGCCTTTCTAGAGGGAGAAC. Each reaction mixture included 2.5 units of Taq/Pwo DNA polymerases in Buffer 1 from the Expand Long Template PCR System and 1 ml of the library as template. Thirty cycles of amplification were performed on a Perkin Elmer GeneAmp 2400 thermal cycler. Following a 2 min initial denaturation at 94°C, 10 cycles of amplification at 94/60/68°C for 10 s/30 s/10 min were performed. In the 16 subsequent cycles, the extension time was increased by 40 s every other cycle for a total of 26 cycles, followed by a final extension of 7 min at 68°C. One microliter of a 1/50 dilution of the first reaction was then transferred to a second reaction mixture containing 50 ng each of the nested primers AP2 and GSP2 5∞-CCACCTCACCCTGAGTTACCTGCTTAG, and the PCR repeated as described above. The products obtained from this round of PCR were purified and sequenced directly with a fMol cycle sequencing kit (Promega).

2.4. Determination of the upstream sequence

3. Results

The upstream sequence of the b-subunit gene was amplified by modification of the Promoter Finder (Clontech, Palo Alto, CA) method. For each of the human DNA libraries contained in the kit, PCR was

3.1. Organization of the human b-subunit gene The gene structure ( Fig. 1A) was determined by characterizing eight genomic clones (lhCNCG-a to

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A

B

Fig. 2. (A) Determination of transcription start point. A 32P-labelled oligonucleotide was hybridized with human retina total RNA ( lane 1) or yeast tRNA ( lane 2) and extended with reverse transcriptase as described in Section 2. The resulting primer extension products were electrophoresed adjacent to a DNA-sequencing ladder to determine their lengths. The major tsp is marked (). (B) Putative binding sites of transcription factors in the upstream region of the channel b-subunit gene. The sequence obtained from PCR amplification of genomic DNA is shown, where the A of the ATG initiator codon (shown in boldface) is numbered +1 (intron sequence is excluded from numbering). An ~5 kb intron ( intron) separates the 5∞-untranslated region between nt −8 and −9. The major tsp is indicated by an asterisk. The oligonucleotide used for primer extension is double-underlined. Potential sequences of importance for photoreceptor gene expression and their orientations are indicated by arrows except for two Crx binding sites, which are boxed.

lhCNCG-h; Fig. 1C ) isolated from a human leukocyte library by directly sequencing the exons and flanking introns, and by sizing the inserts on agarose gels following excision with the restriction enzyme, SalI. We have previously reported our characterization of the genomic clone lhCNCG-a (Ardell et al., 1995a), which contains 12 common exons encoding the b-subunit and GARP cDNAs, as well as an additional exon specific to GARP (exons 2–12 and 12a, Fig. 1A and B). The additional exon (exon 12a) encodes the C-terminal eight amino acids of hGARP 1.6 and the complete hGARP 1.6 3∞-untranslated region (see Fig. 3). Analysis of the seven other genomic clones revealed the presence of 29 exons spanning the protein-coding region of the b-subunit, but none of the clones isolated contained the complete 5∞ or 3∞ untranslated regions. Further analysis of the clones lhCNCG-e and lhCNCG-f revealed that they were non-overlapping and were missing 138 bp of the published cDNA sequence (Chen et al., 1993; Ardell et al., 1996; Colville and Molday, 1996), indicating the existence of additional uncharacterized exons. PCR amplification with primers flanking the missing 138 bp sequence resulted in the production of an approximately 5 kb product (PCR-2, Fig. 1C ) containing two additional exons and one intron. The exon sequence obtained was identical to the published cDNA sequence (Chen et al., 1993; Ardell et al., 1996; Colville and Molday, 1996). Lambda clones lhCNCG-b and lhCNCG-c do not cover all of intron bb ( Table 1, Fig. 3). The size of this intron was estimated to be 9.4 kb (PCR-4, Fig 1C ) using a nested set of primers for long-amplification PCR (data not shown). To complete the 3∞ end characterization of the gene, PCR amplification with primers spanning the 3∞-untranslated region and the last known intron between exons 32 and 33 ( Fig. 1A) resulted in a 4.3 kb product (PCR-3, Fig. 1C ). The size and DNA sequence of this product are consistent with the absence of additional introns in the published 3∞-untranslated region sequence. We previously reported the likely presence of an additional intron separating the 5∞-untranslated region in the b and GARP transcripts (Ardell et al., 1995, 1996). Comparison of the cDNA 5∞-untranslated sequence with that of genomic clone lhCNCG-a showed identity only for the first 8 nt upstream of the ATG initiator codon and revealed the presence of a consensus acceptor splice site. PCR analysis of the genomic clone, however, failed to amplify a product using cDNAderived primers that flank the putative intron. Additionally, attempts to isolate genomic clones with short probes covering the 49 bp of the putative first exon were not successful. Therefore, to establish the existence of an intron, LA-PCR was performed producing a 5 kb product (PCR-1, Fig. 1C ) that was confirmed by DNA sequence analysis. The 5∞-sequence was identical to our reported 49 bp of upstream sequence (Ardell

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Fig. 3. Map of b-subunit and related transcripts in retina. (A) Representation of exons and introns identified in genomic clones (not drawn to scale). Only exons that are used differentially or spliced to alternate exons are shown. Exon designations above the map and introns below are according to previously published reports (Ardell et al., 1995, 1996; Sautter et al., 1998), and exon designations below the map are as in Fig. 1A. (B) Determined and predicted splicing patterns of related b-subunit transcripts. Complementary DNAs corresponding to hGARP 1.6 (Ardell et al., 1995; Colville and Molday, 1996), hGARP 2.5 (Grunwald et al., 1998), hRCNC2a (Chen et al., 1993), and the full-length b-subunit ( Ko¨rschen et al., 1995; Ardell et al., 1996; Colville and Molday, 1996; Grunwald et al., 1998) have been found in mammalian retina cDNA libraries. (C ) Molecular basis for a reported b-subunit variant. The cDNA sequence for the full length b-subunit reported by Grunwald et al. (1998) differs from the sequences from our laboratory (Ardell et al., 1996) and Colville and Molday (1996) by the absence of 18 nucleotides encoding amino acids 189GAASDP. These 18 nt at the 3∞end of exon 9 (g8) and surrounding sequence are shown. Contained within the exon sequence is a possible alternate donor site (D2), which, when spliced to the acceptor site (A1), would maintain the same reading frame and the Ala codon GCG.

et al., 1996; confirmed in the promoter finder analysis, see Section 3.2) and to the acceptor sequence determined from genomic clone lhCNCG-a. These results confirm

the existence of an additional upstream exon (exon 1, Fig. 1A) that is separated from exon 2 by an ~5 kb intron. From these analyses of the genomic clones and

Table 1 Intron/exon junctions of the rod cGMP-gated cation channel b-subunit gene Exona

Size (nt)

Intron (nt positionb)

Size (kb)

Donor site

Acceptor site

1 12 13 14 15 16 17 18 18a 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

>68 37 160 87 88 163 163 108 >158 158 156 209 51 87 65 123 142 160 98 84 119 148 220 >500

ga (-8) o gb (874) ba (1034) bb (1194) bc (1282) bd (1445) be (1608) bf (1716) b2a (–) 2aa (1874) 2ab (2030) 2ac (2239) 2ad (2290) 2ae (2377) 2af (2442) 2ag (2565) 2ah (2707) 2ai (2867) 2aj (2965) 2ak (3049) 2al (3168) 2am (3316) 2an (3536)

5.0 9.5 1.2 9.4 0.9 8.6 9.8 2.7 0.2 1.3 2.0 1.5 1.0 2.5 1.5 7.0 1.1 2.5 4.0 3.5 0.5 11.0 3.8

gtaactcagg… gtaagtgaca… gtggggagtc… gtaagtcatt… gtatgagaag… gtaggtgtgg… gttacctttt… gtgagacccc… gtaggtctgt… gtgagtctgg… gtgagtcctg… gtgagtcaca… gtgtgtgccg… gtaggactgc… gtgagactcc… gtgagccaca… gtatcggggc… gtaagatggt… gtacaccttt… gtgagctggc… gtcagacgca… gtatgtaact… gtaatgaggt…

…gtctccacag …gtccctgcag …cccaactcag …cgggtcacag …ttctctgcag …tctcccccag …gtgtttgcag …gttcccacag …gttcccacag …catcccacag …ttccccacag …cttctttcag …gggatttcag …ctccctccag …ccttctgcag …ctctctccag …cccttcccag …ttgttcacag …accgggacag …tttctttcag …tctgccccag …tttcttccag …ctgattccag

a Junctions for GARP exons 2–11 and 12a were previously reported (Ardell et al., 1995). Exon numbering is according to Fig. 1A. b Position relative to ATG start codon, intron labels according to Fig. 3 and previous reports (Ardell et al., 1996; Sautter et al., 1998).

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PCR products, we deduced that the human rod channel b-subunit is encoded by 33 exons ( Table 1 and Fig. 1) spanning approximately 100 kb of genomic DNA. 3.2. Determination of the transcription start point We previously reported a putative transcription start point at nt −65 as determined by 5∞ RACE (Ardell et al., 1995). To map the transcription start point more precisely, a primer extension analysis was employed. A major product (70% intensity) of 65 nt was observed (Fig. 2A, lane 1), which, based on the sequencing ladder run alongside the gel, corresponds to 79 bp upstream of the initiation codon ( Fig. 2B). Three minor products occurring at nt −78, −80 and −81 (13, 9 and 9% relative intensity, respectively) were also reproducibly observed. No extension products were observed in the reactions where yeast tRNA was substituted for human retinal RNA (Fig. 2A, lane 2). 3.3. DNA sequence of the upstream region To begin characterization of the putative promoter upstream of the tsp, the DNA sequence of this region was determined. Nested gene-specific antisense primers were designed according to the sequence of PCR product PCR-1 ( Fig. 1C ) for use in Promoter Finder analysis as described in Section 2, resulting in the generation of three overlapping PCR fragments (not shown), the largest of which was 2.5 kb. Shown in Fig. 2A is ~500 bp of upstream sequence determined from the PCR products. The sequence of the 3∞ portion of the fragments exactly matched that previously published for the 5∞ untranslated region of the cDNA (Ardell et al., 1995), confirming the identity of the products. Analysis of this sequence with SignalScan software (Genetics Computer Group, Wisconsin) identifies numerous potential cis-elements. Cis-elements with long consensus sequences and those demonstrated to be important for photoreceptor-specific gene expression are shown. Most notable are the presence of a Ret1/PCE1-like sequence consensus RNNRATTAR, where R is a purine (Mohamed et al., 1998) beginning at nt −94, and two consensus Crx-binding sites (C/TTAATCY, where Y is a pyrimidine) begininning at nt −122 and −81 contained within the Ret1/PCE1 site (Morabito et al., 1991; Furukawa et al., 1997; Chen et al., 1997). Additionally, within this region are potentially important E-box, GC box, and AP-2 elements.

4. Discussion In this report, we demonstrate that the gene encoding the human rod photoreceptor cation channel b-subunit comprises 33 exons ( Fig. 3A) spanning over 100 kb on

chromosome 16q13. In the human retina, seven b-subunit related transcripts of 10, 9.5, 7.4, 6.0, 3.8, 2.5, and 1.6 kb have been identified (Ardell et al., 1996). From the determination of the genomic organization, the exon make-up for three of the transcripts (1.6, 2.5, and 10 kb) can be predicted, as shown in Fig. 3B. All but the 10 kb transcript were identified with an RNA blot probe covering exons 2–12 (exons g1–g11, Fig. 3A) within the GARP region. A probe covering exons 14– 19 (exons b2–b6, and 2a2) only hybridizes to the 9.5, 7.4 and 6.0 kb transcripts, and a probe specific to exon 18a (exon 2a1) only hybridizes to the 10 kb transcript, which is very low in abundance. Thus, the short form of GARP (hGARP2) is encoded by exons 1–12 with splicing to the penultimate exon 12a, and a longer form of GARP (hGARP1) by exons 1–16 and last exon b4b, excluding exon 12a by splicing all of intron gb. The exon organization for the longest 3.8 kb GARP transcript has not been determined. The rod photoreceptor b-subunit transcript is encoded by at least one of the remaining three transcripts (9.5, 7.4, and 6.0 kb) encoding exons 1–33, excluding all alternatively spliced exons. We previously argued that the protein encoded by the hRCNC2a transcript may be functionally important because it is present in retina based on RT-PCR with a 2a specific primer and is detectable by Northern analysis (Ardell et al., 1996), and has been functionally expressed in a heterologous system (Chen et al., 1993). More recently, a bovine counterpart of this transcript was identified in testis that is extremely abundant and can be functionally expressed ( Wiesner et al., 1998). The absence of the critical calmodulin-binding site in this truncated b-subunit suggests that this subunit may be important in calcium independent channel regulation in testis and possibly in retina. The hRCNC2a transcript begins with a unique exon (18a or 2a1) contained within intron bf. Within this 2.8 kb intron may reside an alternate promoter for expression of the 2a transcript. The generation of overlapping and non-overlapping transcripts from a single locus (GARP and 2a transcripts) is novel, suggesting that in some tissues, the locus may function as two separate genes. It remains to be established whether this transcript is present in human testis. In addition to the 2a-like transcript, three other cDNAs (bovine testis CNG4C, CNCb1d, CNCb1e) were identified in bovine testis (Biel et al., 1996; Wiesner et al., 1998) that would all be encoded by exons 15–33, excluding exons 18a and b2a, b4a and b4b, and containing unique first exons that are predicted to reside within intron bb. Another alternate transcript was reported in rat olfactory tissue (Sautter et al., 1998) that begins with unique exon b2a, which is spliced to exons 15–33 (excluding 18a) and is alternatively spliced to b4a that is predicted to be within intron bd. The presence of six potential alternate first exons encoding b-subunit and

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related transcripts indicates that at least six independent promoters may be functional within the locus. The use of multiple alternate promoters to generate protein diversity has been observed in other loci in eukaryotes (Qiu et al., 1991; Qiu and Davis, 1993). Further characterization of the introns and expression patterns in human tissues will be necessary to fully understand the functional diversity of the locus. Of three full-length sequences that have been reported for the human rod b-subunit (Ardell et al., 1996; Colville and Molday, 1996; Grunwald et al., 1998), one differs (Grunwald et al., 1998) by the absence of 18 nt at the 3∞-end of exon 9. The most likely basis for this difference is alternate use of donor sites ( Fig. 3C ). The use of donor site D2 would remove 18 nt encoding amino acids 189–194 but maintain the same open reading frame. The functional significance, if any, of this alternate b-subunit remains to be determined. To begin characterization of the upstream region, a primary tsp was determined 79 bp upstream of the initiator methionine ( Fig 2A). Some stuttering of the RNA polymerase is indicated by the presence of additional minor bands that were reproducibly observed (five separate experiments) in the same location, but we cannot totally rule out the possibility that some of the mRNA has been clipped at the 5∞-end producing ragged ends. The location of the tsp differs by only 14 bp from preliminary determination by 5∞-RACE that we previously reported (Ardell et al., 1996). Characterization of the DNA sequence upstream of the tsp revealed a number of potentially functional cis-elements. While functional analysis will be required to establish their importance, some of the identified elements have been shown to be critical for expression in photoreceptors. The cis-element, Ret-I/PCEI binds a retina-specific transcription factor, Erx (Martinez and Barnstable, 1998), and may be required for photoreceptor-specific expression (Morabito et al., 1991; Kikuchi et al., 1993; Ahmad et al., 1994; Yu et al., 1996). Another protein, Crx, is a retina-specific transcription factor that can transactivate photoreceptor-specific gene promoters in transient transfection assays in non-retinal cells (Boatright et al., 1997; Chen et al., 1997; Furukawa et al., 1997; Kennedy et al., 1998). The presence of Crx and Ret-1/PceI binding sites in the upstream region is consistent with a retinal promoter in this region. Additional elements in the upstream region that have been implicated in the regulation of photoreceptor gene expression are the GC-box (Morabito et al., 1991) and E-box (Ahmad, 1995) elements. The locus encoding the human b-subunit is clearly large and complex. The data presented indicate the use of multiple promoters and alternative splicing that is regulated in a tissue-specific manner. Targeted knockouts of specific regions will be needed to understand the complex nature of gene expression from this locus.

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5. Addendum Since submission of this manuscript, Ko¨rschen et al. (1999) have reported on the presence of four repeats in the GARP portion of the channel that bind other phototransduction proteins. These repeat regions, designated R1–R4 are contained within exons 2, 5, 8, and 11, respectively. GARP1 and GARP2 are the bovine splice variant counterparts of hGARP1 and hGARP2 ( Fig. 3B), respectively.

Acknowledgements We thank D. Shane Pendley, Genevieve I. McLeod and Cliff Knizely for expert technical assistance and Dr Warren Zimmer for advice on the use of the Promoter Finder Kit. This work was supported in part by The Foundation Fighting Blindness, NIH grants RO1-EY09924, F32-EY06579, and the Knights Templar Eye Foundation, Inc.

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