|Molecular Vision 1998;
Received 7 August 1998 | Accepted 24 August 1998 | Published 4 September 1998
Molecular Characterization of the Mouse Gene Encoding Cellular Retinaldehyde-binding Protein
Kennedy,1 Jing Huang,2 John C. Saari,2
John W. Crabb1
1Adirondack Biomedical Research Institute, Lake Placid, NY, 12946; 2Departments of Ophthalmology and Biochemistry, University of Washington School of Medicine, Seattle, WA, 98195
Correspondence to: Dr. John W. Crabb, Protein Chemistry, Eye Institute,
Cleveland Clinic Foundation, 9600 Euclid Avenue, Cleveland, OH 44195;
Phone (216) 444-5832; email:
Dr. Kennedy's present address is: Center for Transgene Research, Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, 46556.
Purpose: To clone and characterize the mouse gene encoding cellular retinaldehyde-binding protein (CRALBP). CRALBP appears to modulate enzymatic generation and processing of 11-cis-retinol and regeneration of visual pigment in the vertebrate visual cycle. Mutations in human CRALBP segregate with autosomal recessive retinitis pigmentosa.
Methods: A genomic clone encompassing the 5' end of the CRALBP gene through exon 6 was isolated from a mouse 129/Sv genomic DNA library. Exons 7 and 8 were PCR amplified from mouse eye cDNA and 129/SvJ genomic DNA. The gene structure was determined by automated DNA sequence analysis.
Results: The sequence of 6855 nucleotides was determined, including all 8 exons, 3 introns plus 3932 and 629 bases from the 5'- and 3'-flanking regions, respectively. The lengths of introns 3-6 were determined by PCR amplification. Northern analysis identifies a ~2.1 kb transcript in mouse eye; Southern analysis supports a single copy gene.
Conclusions: The mouse CRALBP gene is similar to the human gene; the coding sequence is ~87% identical, the non-coding sequence ~65% identical. In contrast to the human gene, the mouse gene contains a consensus TATA box. One of two photoreceptor consensus elements important for CRALBP expression in human retinal pigment epithelium is also present in the mouse gene. Additional conserved and species-specific consensus sequences are identified. The mouse CRALBP genomic clones and structure provide valuable tools for developing an in vivo model to study protein function and gene regulation.
Cellular retinaldehyde-binding protein (CRALBP) serves as a substrate carrier protein for enzymes of the mammalian visual cycle in vitro, modulating whether 11-cis-retinol is stored as an ester in the retinal pigment epithelium (RPE) or oxidized by 11-cis-retinol dehydrogenase to 11-cis-retinal for visual pigment regeneration . Recent evidence suggests that CRALBP also stimulates the isomerohydrolase responsible for generation of 11-cis-retinol . CRALBP is strongly expressed in RPE and Müller cells of the retina, where the binding protein carries endogenous 11-cis-retinol and/or 11-cis-retinal . In addition, the protein is expressed in ciliary body, cornea, pineal gland, optic nerve, brain, and transiently in iris. In at least brain and optic nerve, CRALBP lacks 11-cis-retinoid ligands and apparently serves functions unrelated to visual pigment regeneration . Notably, a missense mutation in human CRALBP that destroys retinoid-binding capability has been genetically linked with autosomal recessive retinitis pigmentosa .
RLBP1, the gene encoding human CRALBP, has been localized to human chromosome 15q26  and its structure reported . Previously, we demonstrated that RLBP1 domains -2089 to -1539 bp, -243 to +80 bp, and two photoreceptor consensus elements (PCE1) between RLBP1 positions -165 to -140 direct CRALBP expression in human RPE cultures but not in non-ocular cell cultures . As part of ongoing efforts to develop an in vivo model for studying CRALBP function and gene regulation in transgenic mice, we have cloned and characterized the mouse CRALBP gene (mouse Rlbp1). Here we report over 6 kb of Rlbp1 sequence, including 5' and 3' flanking regions and the entire coding sequence. Conserved and species-specific consensus sites of potential regulatory elements are identified and the deduced mouse CRALBP protein structure discussed.
Materials and Reagents
A female mouse 129/Sv genomic library in lambda FIX II (Stratagene, Inc., La Jolla, CA) was used for cloning part of the mouse CRALBP gene. C57BL6 mice were sacrificed by cervical dislocation and eyes dissected and frozen in dry ice/ethanol within 60 s of death . 129/SvJ genomic DNA was purchased from Jackson Labs, Bar Harbor, ME. RNA and DNA molecular weight markers were from Novagen, Inc. (Madison, WI) and Life Technologies, Inc. (Gaithersburg, MD).
Mouse Genomic Library Screening and Characterization of the Mouse CRALBP Gene
Approximately 1 X 107 recombinant phage from the 129/Sv mouse genomic DNA library (1.0 X 1010 pfu/ml) were plated on Escherichia coli strain BB4(LE392). Plaques were screened with human (1317 bp) and bovine (1173 bp) CRALBP cDNA probes . Several positive clones were plaque-purified and one, designated [lambda]A2, was selected for further analysis. Plasmid subclones mp3.2SK and mp2.8SK were created from [lambda]A2 by EcoR1 restriction digestion and subclone mp3.6CRII by the polymerase chain reaction (PCR). 129/SvJ mouse genomic DNA was PCR amplified using primers designed to mouse and human CRALBP and 10-100 ng of genomic DNA or plasmid DNA as template. Standard PCR cycle conditions were 94 °C/30-45 s, 55-63 °C/30-45 s and 72 °C/min per kb of expected product for 35 cycles. The amplification systems used were Taq polymerase (Fisher, Pittsburgh, PA) for plasmid DNA and the Expand Long Template PCR system (Boehringer Mannheim, Indianapolis, IN) for genomic DNA templates.
Mouse Eye cDNA Preparation and Analysis of CRALBP cDNA
RNA was isolated from C57BL6 mouse eyes with the RNA Ultraspec II Isolation system (Cinna-Biotecx, Houston, TX). First strand cDNA synthesis was performed according to the Superscript preamplification System (Life Technologies) using primer NNT20 (N=G,A,T or C), a modified oligo-dT primer with elevated melting temperature. Mouse CRALBP cDNA was amplified with primers specific to mouse or human CRALBP using the standard PCR conditions. To completely sequence mouse CRALBP exons 7-8, nested 5' primers were successively used in combination with primer NNT20 to specifically amplify mouse C57BL6 eye cDNA. Aliquots of synthesis products were serially diluted 1:20 in consecutive Touchdown-PCR reactions . Touchdown-PCR cycle conditions were 94 °C/30 s, 55 °C/30 s and 72 °C/2 min for 10 cycles followed by 94 °C/30 s, 48 °C/30 s and 72 °C/2 min for 20 cycles.
Northern Blot Analysis
Total RNA was isolated from mouse eyes, liver and kidney, electrophoresed on 1% agarose gels containing 6.6% formaldehyde, blotted to Zeta-Probe GT membranes (Bio-Rad, Hercules, CA) in 20 x SSC, crosslinked to the membrane by UV irradiation (Stratalinker, Stratagene, Inc.) and hybridized with mouse CRALBP cDNA probes. Blocking and hybridization were carried out in the presence of Hybrisol II (Oncor, Gaithersburg, MD) and 20% formamide at 52 °C; final high stringency washing was at 63 °C with 0.1X SSC/0.1% SDS. Two mouse CRALBP cDNA probes (240 bp and 290 bp) were used in Northern analyses. The 240 bp probe spanned sequence in exons 5 and 6 and was generated by PCR with forward primer (5'-AGGCTCTCCGCTGCACTATC-3') and reverse primer (5'-GCATGTCCACCATCTTCTTGAG-3'). The 290 bp probe spanned sequence in exons 6 and 7 and was generated by PCR using forward primer (5'-GGAGAAACTGCTGGAAACTGAGG-3') and reverse primer (5'-CAAGAAGGGCTTGACCACATTGTAGG-3').
Southern Blot Analysis
Mouse genomic DNA samples were subjected to agarose electrophoresis using Tris acetate buffers and transferred to Zeta-Probe GT positively charged nylon membranes (Bio-Rad) by downward alkaline capillary transfer . A 230 bp probe spanning the exon/intron 5 boundary was generated by PCR with forward primer 5'-AGGCTCTCCGCTGCACTATC-3', reverse primer 5'-TGCTGGAAAGATGCTGACTACC-3'. In a modification of the random-primer labeling protocol  primers specific to the probe were substituted for random primers (Specific-primer labeling). The labeled probe was purified from unincorporated nucleotides using Sephadex G-25 chromatography . The denatured probe was incubated with the membrane in hybridization solution (6X SSC, 5X Denharts buffer, 0.5% SDS and 1% sheared salmon sperm DNA) at 68 °C overnight. Membranes were washed under moderate stringency conditions (0.2X SSC/ 0.1% SDS at 42 °C) prior to autoradiography.
DNA Sequencing and Oligonucleotide Synthesis
Oligonucleotide primers were designed using PRIMER DESIGNER version 2.0 software (Scientific and Educational Software). Plasmid DNA templates for sequencing were purified using the Wizard Mini-Preps System (Promega, Madison, WI). For direct sequencing, PCR products were isolated from Sea Plaque low melting point agarose (FMC Bioproducts, Rockland, ME) and purified with the Wizard PCR Preps System (Promega). DNA Sequencing was performed using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, Foster City, CA) with AmpliTaq DNA Polymerase, FS. Synthesis of oligonucleotides and collection of automated DNA sequencing results were performed in the Molecular Biology Core Facility, Adirondack Biomedical Research Institute, using Perkin Elmer, Applied Biosystems Division instrumentation and reagents (model 392 DNA/RNA Synthesizer, model 373, DNA Sequencer). All mouse CRALBP DNA sequences were determined in both directions at least twice. Sequence results were interpreted and aligned using SEQUENCHER 3.0 software (Gene Codes, Ann Arbor, MI) and GeneWorks v 2.45N (Intelligenetics, Mountain View, CA) computer software. Sequence upstream of the presumptive translational start-site in the mouse CRALBP gene was inspected for consensus transcription factor binding sites using SIGNAL SCAN and Mat Inspector.
Copy Number and Transcript Size of the Mouse CRALBP Gene
Northern analysis reveals a predominant CRALBP mRNA species of about 2.1 kb in mouse eye RNA (Figure 1A) but not in mouse liver or kidney RNA (not shown). Similar size human CRALBP mRNAs are predicted (2046 bp and 2269 bp) from the human gene sequence . Southern analysis of mouse 129/SvJ genomic DNA digested with different enzymes and hybridized with a 230 bp probe from the mouse CRALBP gene revealed a banding pattern totaling less than 20 kb/digest and consistent with a single copy gene (Figure 1B). Somatic cell hybridization previously localized the mouse CRALBP locus to a single site on mouse chromosome 7 .
Isolation of Mouse CRALBP Genomic Clones and cDNA Sequences
Using human and bovine CRALBP cDNA probes, a [lambda]-phage clone designated [lambda]A2 was isolated from a mouse 129/Sv genomic DNA library and found to contain an ~13 kb insert that included the 5' end of the CRALBP gene through exon 6 (Figure 2). Three plasmid subclones were prepared from [lambda]A2 DNA and designated mp3.2SK, mp2.8SK and mp3.6CRII (Figure 2). The structures of mouse CRALBP exons 7 and 8 were obtained by direct sequence analysis of PCR products generated from mouse eye cDNA.
Characterization of the Mouse CRALBP Gene
The genomic organization of the mouse CRALBP gene is shown in Figure 2 and the DNA sequence of the gene in Figure 3. The sequence of the mouse CRALBP DNA was determined by automated analysis of genomic subclones and PCR products. About 5.3 kb of contiguous sequence was determined from overlapping subclones mp3.6CRII and mp2.8SK (putative gene positions -4300 to + 1000), including 4 kb of 5'-flanking sequence, exons 1-3 and introns 1-2. The sequences of exon 4 (205 bp) and exon 5 (179 bp) were determined from subclone mp3.2SK. The sequence of 1297 nucleotides of the mouse CRALBP cDNA was determined by direct analysis of PCR products generated from C57BL6 mouse eye cDNA. Mouse 129/SvJ genomic DNA was amplified with primers designed from mouse C57BL6 CRALBP cDNA, and direct sequence analysis of PCR products yielded the genomic sequences of exons 6-8, intron 7 (177 bp) and 692 bp of 3'-flanking sequence. The lengths of mouse CRALBP intron 3 (1.8 kb), intron 4 (1.5 kb), intron 5 (2.6 kb) and intron 6 (1.2 kb) were determined by PCR amplification using as a template either the 13 kb [lambda]-phage clone or mouse 129/SvJ genomic DNA (Figure 4).
The structure of the mouse CRALBP gene was determined from a clone isolated from a 129/Sv mouse genomic library and from PCR amplification of mouse eye cDNA and mouse 129/SvJ genomic DNA. The mouse CRALBP gene is similar to the human gene in organization and size and contains eight exons and seven introns. In both genes, exon 1 is entirely untranslated, and the start (initiation) and stop (termination) codons are located in partially untranslated exons 2 and 8. A total of 6855 nucleotides of sequence was determined, including 3932 bases from the 5'-flanking region, 629 bases from the 3'-flanking region, all 8 exons and 3 introns. All mouse CRALBP exon/intron boundaries follow standard vertebrate GT/AG splicing convention . Moreover, the 5' splice site of all introns conforms with the consensus 6 bp binding sequence of the spliceosome component U1 snRNP . Exons range in size from 111 to 848 bases and introns vary from 178 to 2600 bases. Based on Southern blot analysis and localization to a single site on mouse chromosome 7 , the mouse CRALBP gene (Rlbp1) exists in single copy in the mouse genome. A search of the Mouse Genome Informatics (MGI) Resource at The Jackson Laboratory (Bar Harbor, ME) shows that no known mouse retinal diseases map to the CRALBP locus. The transcription start site of the mouse gene is predicted based on homology with the human CRALBP gene and is about 841 bases upstream of the ATG translation initiation signal. This putative transcription start site is consistent with the mouse CRALBP mRNA species detected by Northern analysis (~2.1 kb), a size within experimental error of the non-polyadenylated mRNA species (1868 bases) predicted from the mouse gene sequence. In the mouse gene, a consensus polyadenylation signal (AATAAA) and a terminator sequence (YGTGTTYY), separated by 12 bases, were found beginning 663 bases downstream from the translation stop (Figure 3). In the mouse CRALBP cDNA, a poly-A tail was found immediately downstream of the terminator sequence. Mouse and human CRALBP 3' untranslated regions are ~60% conserved. Unlike the gene encoding human RPE65, an RPE-specific protein also thought to be associated with vitamin A metabolism, the mouse and human CRALBP genes do not contain multiple ATTTA motifs implicated in transcript instability . Overall, coding DNA in the mouse and human CRALBP genes exhibits ~87% sequence identity and non-coding DNA (exons 1-2) exhibits ~65% identity.
As an approach to detecting cis-elements that regulate CRALBP expression, evolutionary conserved sequences in the promoter region of the mouse and human genes have been sought. Alignment of ~4 kb of mouse and human CRALBP gene sequences upstream of exon 3 reveals two homologous domains of ~1.4 kb and ~1.8 kb (approximate gene positions -2.9 kb to -1.5 kb and 0.7 kb to +1.1 kb, respectively), separated by the human-specific Alu repetitive sequence (Figure 5). Previously we reported that PCE1 sites in the human proximal promoter bind RPE nuclear factors that appear to interact with the distal promoter region -2089 to -1539 to drive high levels of CRALBP expression in the RPE . The PCE1 consensus sequence CAATTAG (designated RCS1 in Drosophila) was originally identified in several mammalian photoreceptor genes and in Drosophila rhodopsin genes and proposed to direct photoreceptor cell-specific gene expression . The two PCE1 between human RLBP1 positions -165 and -140 exhibit significant homology with Ret-1 in the rat opsin gene, which has been reported to direct in vivo gene expression in both rod photoreceptors and brain . One of the PCE1 sites is conserved in the mouse proximal promoter (Figure 6A), supporting the hypothesis that PCE1 influences CRALBP gene expression. Notably, two identical sequences (GCAGGA) flanking the PCE1 in human RLBP1 and important for complex formation with human PCE1 binding proteins  are not conserved in the mouse gene. A consensus TATA box exists in the mouse gene  while the human proximal promoter contains an apparent Initiator (Inr) element, a 46 bp insert containing a Ying-Yang 1 (YYI) consensus site , and two RPE65 related sequences . Other conserved proximal promoter consensus elements shown in Figure 6A include (i) RET-2, which in the rat opsin promoter specifically binds retinal nuclear factors ; and (ii) STRD, associated with high levels of arrestin expression in the retina and more broadly recognized by the vitamin D3, steroid, thyroid hormone and retinoic acid receptors [17,23]. The significance of these sites remains to be determined.
Conserved sequences in the mouse and human distal promoter region (Figure 6B) may be associated with putative enhancer activity in the human CRALBP gene between -2089 and -1539 . Conserved distal promoter consensus sites include: (i) the Cone rod homeobox (Crx)-binding element that binds transcription factor Crx and transactivates several pineal/photoreceptor-specific genes [24,25,26]; (ii) activator protein 1 (AP-1) a common transcriptional activator ; (iii) Brn-2, associated with development and survival of the hypothalamus and pituitary [28,29]; (iv) MZF-1, important in tissue-specific expression of myeloid cells ; and (v) Glass-like (human CRALBP -2484 to -2465), implicated in photoreceptor cell development . Further studies are required to determine the significance of these consensus sites and the most highly conserved sequence between RLBP1 positions -1930 to -1900 bp and -1892 to -1852 bp.
The mouse, human and bovine CRALBP protein sequences each encode 316 amino acids and overall are about 87% identical (Figure 7). Important amino acid residues conserved in all three species include retinoid-binding pocket components Gln-210 and Lys-221  and Arg-150, which if substituted with Gln (i. e., R150Q) can result in retinal degenerations associated with retinitis pigmentosa . As previously described for bovine and human CRALBP, mouse CRALBP shares limited homology (Figure 8) with [alpha]-tocopherol transfer protein, protein tyrosine phosphatase and phosphatidyl inositol-transfer protein . Contrary to an earlier report , CRALBP is also distantly related with squid retinaldehyde-binding protein as shown in Figure 8. Structure function studies with bovine and human recombinant CRALBP have characterized ligand interactions [3,32,34,35,36] and established in vitro evidence for a substrate carrier function in RPE [1,2,37].
This study represents initial work toward developing an in vivo model for studying CRALBP function and gene regulation in transgenic mice. The current results and clones have facilitated the construction of a mouse CRALBP gene targeting vector containing the neomycin resistance gene inserted into exon 3. Efforts to disrupt the mouse CRALBP gene by homologous recombination are in progress and homozygous knockout mice will be used for morphological, electrophysiological and retinoid metabolism studies. The current results also establish a foundation for identifying in vivo the minimal CRALBP promoter required for high level RPE expression in mice. Identification of the CRALBP minimal promoter will enhance gene therapy approaches for retinal diseases associated with CRALBP and other RPE expressed genes.
This study was supported in part by USPHS grants EY06603, EY02317, EY01730 and by Research to Prevent Blindness, Inc (RPB). JCS is a Senior Scientific Investigator of RPB. We gratefully acknowledge the technical assistance of S. Goldflam, P. S. Adams and A. Moquin and V. Oliver, and M. LaDuke in manuscript preparation. BK also thanks C. Morrissey and Drs. D. Eisinger, F. Martin and M. Tenniswood for useful discussions.
A preliminary report of this work was presented at the 70th Annual Meeting of the Association for Research in Vision and Ophthalmology, May 1998, Ft. Lauderdale, FL.
This work includes thesis research submitted by BNK in partial fulfillment of the requirements for the doctoral degree in the Cell and Molecular Biology Program jointly administered by the University College Dublin, Ireland and the Adirondack Biomedical Research Institute, Lake Placid, NY.
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