July 2005
Volume 46, Issue 7
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Glaucoma  |   July 2005
Molecular Cloning and Expression Profiling of Optineurin in the Rhesus Monkey
Author Affiliations
  • Tayebeh Rezaie
    From the Molecular Ophthalmic Genetics Laboratory, Surgical Research Center, Department of Surgery, and the
  • David M. Waitzman
    Department of Neurology, University of Connecticut Health Center, Farmington, Connecticut; the
  • Jennifer L. Seeman
    Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin; and
  • Paul L. Kaufman
    Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin; and
    The Wisconsin National Primate Research Center, Madison, Wisconsin.
  • Mansoor Sarfarazi
    From the Molecular Ophthalmic Genetics Laboratory, Surgical Research Center, Department of Surgery, and the
Investigative Ophthalmology & Visual Science July 2005, Vol.46, 2404-2410. doi:10.1167/iovs.04-1243
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      Tayebeh Rezaie, David M. Waitzman, Jennifer L. Seeman, Paul L. Kaufman, Mansoor Sarfarazi; Molecular Cloning and Expression Profiling of Optineurin in the Rhesus Monkey. Invest. Ophthalmol. Vis. Sci. 2005;46(7):2404-2410. doi: 10.1167/iovs.04-1243.

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      © 2015 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. It has been shown that mutations in the optineurin (OPTN) gene are involved in the etiology of adult-onset primary open-angle glaucoma (POAG). In view of close similarities between human and nonhuman primate ocular development and function, the rhesus monkey is considered a suitable model for human visual system research. Therefore, this study was conducted to clone the orthologue of the human OPTN gene in the rhesus monkey (Rh-OPTN) and to determine its genomic organization. A further purpose was to establish Rh-OPTN protein expression profiles and tissue distribution in the rhesus anterior segment, retina, and optic nerve.

methods. The Rh-OPTN gene was cloned and its genomic structure determined. The mRNA expression pattern was examined by Northern blot analysis. The protein’s cellular localization, ocular expression, and tissue distribution were established by immunolabeling.

results. The Rh-OPTN gene has 13 exons and encodes for a 571-amino-acid protein. Both cDNA and amino acid sequences are 96% identical with the human OPTN. Northern blot analysis revealed prominent expression of two different transcripts in heart, brain, kidney, lung, spleen, skeletal muscle, and small intestine. Cellular and tissue distribution of Rh-OPTN protein were highly similar to its human and mouse homologous proteins.

conclusions. The optineurin gene and protein are evolutionary conserved between humans and the rhesus monkey. High similarity of ocular expression and tissue distribution between the two optineurin proteins suggests that this nonhuman primate is a suitable model for the pathophysiology and treatment of human glaucomatous optic neuropathy.

Glaucoma is one of the leading causes of blindness worldwide. 1 2 3 4 5 The disease is caused by gradual degeneration of retinal ganglion cells and secondary loss of axons that carry visual information from the eyes to the brain. The reported glaucoma prevalence differs significantly, depending on race, age, and the population studied. 1 2 3 4 6 7 8 9  
Primary open-angle glaucoma accounts for more than half of all cases worldwide and far more than that in the Western world. POAG is a bilateral disease of slow onset that is characterized by atrophy of the optic nerve fibers (the retinal ganglion cell axons) as they pass through the region of optic disc and lamina cribrosa. This condition is frequently associated with elevated intraocular pressure (IOP). In low-pressure glaucoma (LPG), also known as normal-pressure glaucoma (NPG), glaucomatous optic nerve damage and visual field defects occur in association with IOPs within the normal range, although the progression of the damage is probably still IOP dependent. 10 LPG prevalence may account for between 10% and 50% of all POAG cases in persons over the age of 40, with the highest prevalence reported in Japan. 11 12 13 14  
Molecular studies of glaucoma-affected families have identified seven genetic loci for adult-onset POAG 15 16 17 18 19 20 and three other loci for primary congenital glaucoma (Stoilov I, et al. IOVS 2002;43:ARVO E-Abstract 3015). 21 22 However, until very recently, when we discovered optineurin (OPTN) 23 and WD40-repeat 36 (WDR36) 24 genes for adult-onset POAG, only two other glaucoma-causing genes had been identified: cytochrome P4501B1 (CYP1B1) for primary congenital glaucoma 25 and myocilin (MYOC) for POAG. 26 In 2002, we reported that mutations in the optineurin gene are involved mainly in low-pressure adult-onset POAG. 23 The molecular mechanisms of OPTN involvement in the development of glaucoma are not as yet understood. 27 We have shown secretion of the optineurin protein by trabecular meshwork (TM) and nonpigmented ciliary epithelium (NPCE) cell lines and its presence in aqueous humor. 23 We have also detected expression of this protein in human ocular tissues, with strong immunoreactivity in the ciliary body, ciliary muscle, cornea, iris, and retina. 27 Furthermore, we have mapped and cloned the OPTN counterpart in mouse and investigated the cellular and tissue localization of the protein in mouse eyes. 28 Our recent expression profiling of optineurin in the mouse revealed that this protein is expressed in very early stages of eye development. 28 In this report, we present molecular cloning and genomic organization of the Rh-OPTN gene and the localization and distribution of its protein in various ocular tissues of the rhesus monkey. 
Materials and Methods
Tissues and Antibodies
Eyes and other tissues used in this study were obtained from three rhesus monkeys (Macaca mulatta). All experiments were conducted in accordance with the guidelines given in the ARVO Statement for Use of Animals in Ophthalmic and Vision Research and under the auspices of the animal care and use committees of the University of Connecticut Health Center and University of Wisconsin-Madison. The animals were deeply anesthetized and perfused with 4% paraformaldehyde and 0.1% glutaraldehyde, as described previously. 29 The eyes were enucleated and processed for immunohistochemistry studies. A thin layer of rhesus monkey skin was used to establish a dermal fibroblast cell line. These cells were maintained in DMEM-F12 (Invitrogen-Gibco, Grand Island, NY), supplemented with 10% FBS, and 1% antimycotic (Invitrogen-Gibco) in an incubator at 37°C in 5% CO2. Whole blood was taken from one rhesus monkey, and the genomic DNA was extracted. 
The primary antibody used in this study is an anti-OPTN antibody that has been raised against a polypeptide at the C terminus of the optineurin protein that is 100% conserved between human and monkeys. 
cDNA Cloning and Genomic Organization of the Rh-OPTN Gene
Total RNA was isolated from a cultured rhesus monkey dermal fibroblast cell line (TRIzol; Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol. Preparation of cDNA was performed by reverse transcriptase (RT)-PCR using a commercial kit (SuperScript First-Strand Synthesis System; Invitrogen-Gibco). Rhesus monkey full-length cDNA was amplified by using primers designed based on human OPTN cDNA. PCR products were cloned into a TOPO-TA cloning vector (Invitrogen) and transformed into chemically competent Escherichia coli cells supplied by the manufacturer. Several clone inserts were sequenced in their entirety. The genomic DNA was isolated from a rhesus monkey blood sample with a kit (Gentra Systems, Inc., Minneapolis, MN). The exons and flanking DNA sequences of the Rh-OPTN gene were PCR amplified by using primers originally designed based on human genomic sequences (GenBank accession number AL355355; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD). By using the dye terminator chemistry (BigDye; Applied Biosystems, Inc. [ABI], Foster City, CA), all the DNA and cDNA sequencing experiments were performed bidirectionally, by using either an automated DNA sequencer (model 377; ABI) or a genetic analyzer (model 3100; ABI). Sequence analyses and alignments were performed with publicly available programs (i.e., BLAST 2 sequences at the National Center for Biotechnology Information, Bethesda, MD; http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html and ClustalW: Multiple Sequence Alignment at the Baylor College of Medicine (Houston, TX; http://searchlauncher.bcm.tmc.edu/multi-align/multi-align.html). The Rh-OPTN gene sequences obtained during the course of this study are now available in GenBank accession numbers AY228373 and AY228374 for the full-length cDNA, accession numbers AY749110 to AY749122 for genomic sequences of exons 1 to 13 and accession number AH014152 for the entire genomic sequence). The 13 coding exons in the rhesus monkey showed a very high degree of similarity and fully conserved intron–exon boundaries with the human OPTN gene. PCR primers and conditions are available on request. 
Northern Blot Analysis
A rhesus monkey tissue Northern blot analysis (normalized by amount of mRNA) blot from Biochain Institute (Hayward, CA) was used for this study. The blot contained mRNA samples from seven tissues of heart, brain, kidney, lung, spleen, skeletal muscle, and small intestine of monkey as well as human adult placenta as a control. A cDNA probe covering amino acids 107-412 of the Rhesus monkey OPTN was amplified using forward primer of (5′-TTGAAGGAAGAGCTTGGAAAA-3′) and the reverse primer of (3′-CCACTTTTTCTGACTCTTTTCTTG-5′). The cDNA probe was labeled with fluorescein-12-dUTP (1 mM; Roche Diagnostics, Indianapolis, IN). The blot was prehybridized (FastHyb solution; Biochain) for 3 hours at 65°C and subsequently hybridized with the solution with the probe overnight. Membrane was washed with 1× SSC/0.1% SDS at 65°C for 15 minutes and incubated with blocking solution at room temperature for 30 minutes. Membrane was incubated with antibody solution (anti-fluorescein alkaline phosphatase conjugates; 1:5000) for 30 minutes. After it was washed three times, the membrane was incubated with detection reagent at room temperature for 4 minutes and then was exposed to x-ray film for 3 minutes. The equality and integrity of RNA were confirmed by probing a membrane from the same lot with a fluorescein-labeled human β-actin cDNA probe. 
Immunocytochemistry Analysis
Rhesus monkey dermal fibroblast cells were grown on glass coverslips in six-well plates for 48 hours. After they were washed with phosphate-buffered saline (PBS), the cells were fixed with 4% paraformaldehyde on ice and permeabilized in 0.1% Triton X-100. Nonspecific hybridization was blocked by incubation with 4% bovine serum albumin in PBS. Cells were incubated with primary antibody at 1:200 (optineurin anti-peptide antibody raised in chicken), diluted in blocking solution for 1 hour, and rinsed in PBS. The secondary antibody, goat anti-chicken IgG (H+L; Alexa 488; Molecular Probes, Eugene, OR), was diluted 1:500 in blocking solution and applied for 45 minutes. Nuclei were stained with TO-PRO-3 iodide (Molecular Probes) at 1:200 for 30 minutes. After a brief wash in PBS, the coverslips were mounted on slides (Prolong Antifade medium; Molecular Probes) to preserve fluorescence and visualized under a laser scanning confocal microscope (model 410; Carl Zeiss Meditec, Dublin, CA). 
Immunohistochemistry Analysis
Eyes from a perfused rhesus monkey were postfixed for 2 hours in 4% paraformaldehyde in PBS at 4°C and subsequently processed for paraffin embedding. For immunohistochemistry experiments, specimens were sectioned into 5-μm sections and mounted on poly-l-lysine-coated glass slides. Tissue sections were deparaffinized with xylene and rehydrated in a decreasing ethanol series. Nonspecific binding sites were blocked with incubation in blocking solution (Power Block; BioGenex, San Ramon, CA) for 10 minutes. The sections were subsequently incubated overnight at 4°C with primary anti-optineurin antibody (1:500 dilution in 0.1% BSA in PBS). After three brief washes with PBS, the sections were incubated for 45 minutes with the secondary antibody goat anti-chicken IgG (H+L; Alexa 594; Molecular Probes) diluted 1:400 in 0.1% BSA in PBS. After a wash with PBS, sections were incubated with TO-PRO-3 iodide (Molecular Probes) at 1:400 for 30 minutes. After washing in PBS, the specimens were mounted (Prolong Antifade; Molecular Probes) to preserve fluorescence and visualized under the laser scanning confocal microscope. Control experiments for nonspecific staining included replacement of the primary anti-OPTN antibody with preimmune serum from the same chicken in which the antibody was raised. 
Results
Isolation and Characterization of the Rhesus Monkey Optineurin Gene
The Rh-OPTN full-length cDNA was amplified with primers originally designed from the 3′ and 5′ ends of the human optineurin gene. A PCR product of approximately 2.0-kb was cloned into the TOPO-TA cloning vector, and several insert clones were subsequently sequenced in their entirety. Two lines of clones were identified and designated variants 2 and 3, as they were compatible with the human OPTN isoforms 2 and 3, respectively. Similar to human optineurin cDNA, these two variants also resulted from different splicing of the noncoding exons at the 5′ untranslated region of the Rh-OPTN gene. The nucleotide and deduced amino acid sequences for Rh-OPTN variant 2 are presented in (Fig. 1)
The obtained 1925- and 2076-bp sequences of rhesus monkey OPTN variants 2 and 3 are 96% identical with their counterparts of human isoforms 2 and 3, respectively. Both of these Rh-OPTN sequences translate into a 571 amino acid protein that is 96% identical with the human OPTN protein (Fig. 2)
Genomic Organization of the Rhesus Monkey OPTN Gene
An orthologue to the human optineurin gene was PCR amplified from rhesus monkey genomic DNA by using human oligonucleotide primers. Alignment of the obtained sequences for the Rh-OPTN cDNA with its human orthologue revealed the presence of 13 coding exons ranging in sizes from 74 to 203 bp (Table 1) . The location and sequence of the intron–exon boundaries were fully conserved with its human counterpart for all the 13 coding exons, except for the splicing acceptor site at exon 5. In rhesus monkey, the first six amino acids of exon 5 were spliced out due to a different acceptor-splicing site that has been created by a single base sequence change. All the observed splice acceptor and donor sites were in accordance with the consensus GT-AG rule (Table 1 ; see also GenBank accession number AH014152). The predicted Rh-OPTN protein contains conserved coiled-coil and C2H2-Zn finger domains, as has been reported for the human OPTN. 
Northern Blot Analysis
The tissue distribution and expression profiling of monkey optineurin was examined by Northern blot analysis of an mRNA multiple tissue blot (BioChain). As shown in Figure 3 , two different transcripts showed prominent expression (arrows) that is consistent with presence of two OPTN cDNA variants in rhesus monkey (GenBank Accession numbers of AY228373-4). The highest expression of monkey OPTN was observed in skeletal muscle, which is very similar to its human counterpart as reported previously. Expression of OPTN in heart, brain, kidney, lung, and skeletal muscle is comparable among human, mouse, and monkey. 28 30  
Immunocytochemistry Analysis
Rhesus monkey dermal fibroblasts showed a strong positive staining for the optineurin protein in immunocytochemistry analysis with the anti-optineurin antibody (Fig. 4) . The endogenous optineurin is associated with vesicular structure near the nucleus. Control experiments were performed by replacement of the primary antibody with nonimmune serum at the same concentration as the antibody. No labeling was observed in any of the control experiments. 
Expression Profiling of Rh-OPTN Protein in Ocular Tissues
Tissue distribution of the Rh-OPTN protein was determined by immunohistochemical labeling of paraffin-embedded sections of rhesus monkey eyes and optic nerve using an anti-optineurin antibody. Intense staining was observed in iris anterior epithelium, constrictor muscle, and endothelial cells of the iris blood vessels (Fig. 5A) . In the anterior segment, positive immunoreactivity was observed in trabecular meshwork, nonpigmented ciliary epithelium, ciliary muscle, and Schlemm’s canal endothelium (Fig. 5B) . In the retina, prominent staining was detected in the nerve fiber; ganglion cell, and inner and outer plexiform layers; the cell bodies of rods and cones (Fig. 5C) ; and, most intensely, in the retinal pigmented epithelium. Axons of the optic nerve ganglion cells, astrocytes, glial cells, and endothelial cells of the optic nerve blood vessels also stained for optineurin (Figs. 5D 5E 5F) . No labeling was observed in any of the control sections tested. 
Discussion
Our previous studies determined an association between mutations in the human OPTN gene and adult-onset POAG. 23 27 We also mapped, cloned, and characterized the mouse Optn gene, its protein products and determined its ocular expression patterns. 28 Before this study, organization and expression of the OPTN gene in primates other than humans was unknown. In the present study, the complete cDNA sequence, genomic structure, mRNA, and protein expression profiles of the monkey optineurin gene were established. The Rh-OPTN gene is nearly identical with its human counterpart in both genomic structure and amino acid sequence. The mRNA expression pattern of Rh-OPTN is highly similar to previous reports for its homologous genes in human 30 and mouse. 28 Identification of two highly expressed transcripts (Fig. 3A)is in full agreement with the two observed isoforms by cDNA cloning. The mRNA expression patterns of OPTN in heart, brain, kidney, lung, and skeletal muscle are consistent in human, monkey, and mouse. 28 30 Similar to the human gene, the highest expression was observed in skeletal muscle. 
This study revealed that intracellular localization and ocular expression profiles of Rh-OPTN protein is consistent with its homologous protein in mouse and human. 23 27 28 The Rh-OPTN gene shares 96% homology in cDNA and predicted amino acid sequences with its human counterpart. OPTN has now been shown to be an evolutionarily conserved gene, as determined by our molecular cloning and expression profiling of its protein products in mouse, 28 monkey, and human. 23 27 OPTN conservation is also confirmed in pigs (Obazawa M, et al. IOVS 2003;44:ARVO E-Abstract 1115) which shows a high homology (84%) between both polypeptide sequences and transcript expressions in trabecular meshwork cells and astrocytes, as previously described for its human homologous gene. 23 27  
The expression pattern of OPTN protein in ocular tissues of the rhesus monkey is highly similar to our observation in human eyes 27 (and unpublished data). Similar to this study, in the human eye, positive staining for optineurin protein has been observed in vascular endothelium, trabecular meshwork, Schlemm’s canal, iris constrictor muscle, ciliary muscle, nonpigmented ciliary epithelium of the anterior segment, nerve fiber layer, ganglion cell layer, inner and outer plexiform layers, and retinal pigmented epithelium (manuscript in preparation). 
Although the function of human OPTN protein still remains to be determined, the presence of identical functional protein domains, high conservation of the gene structures and sequences, similar ocular expressions and localization of OPTN in rhesus monkey and human are all consistent with the role of this gene in the etiology of glaucoma, as previously reported for several mutations in the human OPTN gene. 23 31 32 33 34  
The rhesus monkey has been used extensively as a model for human diseases and drug trials because of its similarities to the human. Great similarities in eye development and function between monkey and human makes this animal a suitable model for human visual research and pharmacological evaluation of glaucoma therapy. A recent microarray analysis 35 identified a high homology and identity (95%) between monkey and human coding genes. Similarly, a high degree of gene conservation exist between cynomolgus monkey and human. 36 Several studies of an induced-glaucoma model in primates have illustrated common characteristic features with its human phenotype. 37 38 39 40 A gene expression analysis of retina in a monkey glaucoma model showed that most of the genes investigated had expression profiles similar to those of the human and only 0.7% of the 9182 genes analyzed were either up- or downregulated. 41 Cloning and characterization of the rhesus monkey OPTN gene facilitates comparison and conservation of amino acid residues that have been mutated in various forms of glaucoma. This study may also contribute to better understanding of optineurin gene expression, function, and molecular mechanisms that eventually lead to the glaucoma phenotype. 
 
Figure 1.
 
Nucleotide sequence for Rh-OPTN cDNA. The predicted amino acid sequence is shown below the open reading frame. The ATG initiation and the two in-frame stop codons at 3′ and 5′ends are underlined.
Figure 1.
 
Nucleotide sequence for Rh-OPTN cDNA. The predicted amino acid sequence is shown below the open reading frame. The ATG initiation and the two in-frame stop codons at 3′ and 5′ends are underlined.
Figure 2.
 
Amino acid alignment homologies of optineurin in human, rhesus monkey, and mouse.
Figure 2.
 
Amino acid alignment homologies of optineurin in human, rhesus monkey, and mouse.
Table 1.
 
Exon–Intron Size and Boundaries of the Rhesus Monkey OPTN Gene
Table 1.
 
Exon–Intron Size and Boundaries of the Rhesus Monkey OPTN Gene
Exon No. Position in cDNA* Exon Size (bp) Splice Acceptor Exon Boundary Splice Donor GenBank Accession No.
1 79-255 177 ttttcctcag GAACTTCTGC—CAGCTGAAAG gtaagcaggg AY749110
2 256-458 203 cctcctgcag AAGCCATGAA—GTCATCTGAG gtgagcagac AY749111
3 459-641 183 tcatctccag GACCCCACTG—TAGGATGGCT gtaagttttt AY749112
4 642-715 74 gtttttacag GAAGGAGAAG—CCATTGGCAC gtatgtgaag AY749113
5 716-850 135 ctaaatatag GAGCAGATCT—CCAAAGAAAG gtatgaaatc AY749114
6 851-953 103 gattttccag AGTTTCAGAT—CCCAGAGACT gtgagtccta AY749115
7 954-1069 116 cgtgtgatag GTTGGAAGCG—TTCAAGAAAA gtaagaatgc AY749116
8 1070-1219 150 cctttcttag GTGTCAGGCC—AGGATGAAAA gtgagtgtgc AY749117
9 1220-1313 94 tttttaatag GTCCAAATTA—AAGAAAAGAG gtattcaccg AY749118
10 1314-1472 159 tctttttcag TCAGAAAAAG—CAGGGCTCAG gtgaggcacc AY749119
11 1473-1603 131 cctctaacag ATGGAAGTTT—ATGGAGGCAG gtaaggaaaa AY749120
12 1604-1683 80 atcccggcag GCAGTCCTTG—GTTCAAAGAG gtaagtcctg AY749121
13 1684-1805 122 ttactcacag GAACTGAGGA—CATCATTTAG gtgttgatgt AY749122
Figure 3.
 
Expression of optineurin in rhesus monkey tissues. (A) Blot of seven monkey tissues and a control human adult placenta was hybridized with an OPTN probe. Numbers on the left are sizes in kilobases. (B) Membrane hybridized to a human β-actin probe as a control.
Figure 3.
 
Expression of optineurin in rhesus monkey tissues. (A) Blot of seven monkey tissues and a control human adult placenta was hybridized with an OPTN probe. Numbers on the left are sizes in kilobases. (B) Membrane hybridized to a human β-actin probe as a control.
Figure 4.
 
Cellular localization of optineurin protein in dermal fibroblast cells of a Rhesus monkey. OPTN in green and cells nuclei in blue.
Figure 4.
 
Cellular localization of optineurin protein in dermal fibroblast cells of a Rhesus monkey. OPTN in green and cells nuclei in blue.
Figure 5.
 
Immunoreactivity of the rhesus monkey optineurin protein in various ocular tissues. Red: OPTN; blue: cell nuclei. (A) Iris constrictor muscle (arrow) and endothelial cells of the iris vessels (arrowheads) showing intense staining. (B) Positive immunolabeling in trabecular meshwork and Schlemm’s canal (arrow), ciliary muscle (CM), nonpigmented ciliary epithelium (arrowheads). (C) Positive staining in nerve fiber layers, ganglion cells, the inner and outer plexiform layers, and, most intensely, in retinal pigmented epithelium (arrow). (DF) Axons bundles of optic nerve ganglion cells, astrocytes, glial cells, and endothelial cells of the optic nerve vessels also stained for optineurin.
Figure 5.
 
Immunoreactivity of the rhesus monkey optineurin protein in various ocular tissues. Red: OPTN; blue: cell nuclei. (A) Iris constrictor muscle (arrow) and endothelial cells of the iris vessels (arrowheads) showing intense staining. (B) Positive immunolabeling in trabecular meshwork and Schlemm’s canal (arrow), ciliary muscle (CM), nonpigmented ciliary epithelium (arrowheads). (C) Positive staining in nerve fiber layers, ganglion cells, the inner and outer plexiform layers, and, most intensely, in retinal pigmented epithelium (arrow). (DF) Axons bundles of optic nerve ganglion cells, astrocytes, glial cells, and endothelial cells of the optic nerve vessels also stained for optineurin.
Authors thank Nancy Ryan at the Histology Core Services and Susan Krueger at the Confocal Microscopy Facility (University of Connecticut Health Center) for technical assistance. 
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Figure 1.
 
Nucleotide sequence for Rh-OPTN cDNA. The predicted amino acid sequence is shown below the open reading frame. The ATG initiation and the two in-frame stop codons at 3′ and 5′ends are underlined.
Figure 1.
 
Nucleotide sequence for Rh-OPTN cDNA. The predicted amino acid sequence is shown below the open reading frame. The ATG initiation and the two in-frame stop codons at 3′ and 5′ends are underlined.
Figure 2.
 
Amino acid alignment homologies of optineurin in human, rhesus monkey, and mouse.
Figure 2.
 
Amino acid alignment homologies of optineurin in human, rhesus monkey, and mouse.
Figure 3.
 
Expression of optineurin in rhesus monkey tissues. (A) Blot of seven monkey tissues and a control human adult placenta was hybridized with an OPTN probe. Numbers on the left are sizes in kilobases. (B) Membrane hybridized to a human β-actin probe as a control.
Figure 3.
 
Expression of optineurin in rhesus monkey tissues. (A) Blot of seven monkey tissues and a control human adult placenta was hybridized with an OPTN probe. Numbers on the left are sizes in kilobases. (B) Membrane hybridized to a human β-actin probe as a control.
Figure 4.
 
Cellular localization of optineurin protein in dermal fibroblast cells of a Rhesus monkey. OPTN in green and cells nuclei in blue.
Figure 4.
 
Cellular localization of optineurin protein in dermal fibroblast cells of a Rhesus monkey. OPTN in green and cells nuclei in blue.
Figure 5.
 
Immunoreactivity of the rhesus monkey optineurin protein in various ocular tissues. Red: OPTN; blue: cell nuclei. (A) Iris constrictor muscle (arrow) and endothelial cells of the iris vessels (arrowheads) showing intense staining. (B) Positive immunolabeling in trabecular meshwork and Schlemm’s canal (arrow), ciliary muscle (CM), nonpigmented ciliary epithelium (arrowheads). (C) Positive staining in nerve fiber layers, ganglion cells, the inner and outer plexiform layers, and, most intensely, in retinal pigmented epithelium (arrow). (DF) Axons bundles of optic nerve ganglion cells, astrocytes, glial cells, and endothelial cells of the optic nerve vessels also stained for optineurin.
Figure 5.
 
Immunoreactivity of the rhesus monkey optineurin protein in various ocular tissues. Red: OPTN; blue: cell nuclei. (A) Iris constrictor muscle (arrow) and endothelial cells of the iris vessels (arrowheads) showing intense staining. (B) Positive immunolabeling in trabecular meshwork and Schlemm’s canal (arrow), ciliary muscle (CM), nonpigmented ciliary epithelium (arrowheads). (C) Positive staining in nerve fiber layers, ganglion cells, the inner and outer plexiform layers, and, most intensely, in retinal pigmented epithelium (arrow). (DF) Axons bundles of optic nerve ganglion cells, astrocytes, glial cells, and endothelial cells of the optic nerve vessels also stained for optineurin.
Table 1.
 
Exon–Intron Size and Boundaries of the Rhesus Monkey OPTN Gene
Table 1.
 
Exon–Intron Size and Boundaries of the Rhesus Monkey OPTN Gene
Exon No. Position in cDNA* Exon Size (bp) Splice Acceptor Exon Boundary Splice Donor GenBank Accession No.
1 79-255 177 ttttcctcag GAACTTCTGC—CAGCTGAAAG gtaagcaggg AY749110
2 256-458 203 cctcctgcag AAGCCATGAA—GTCATCTGAG gtgagcagac AY749111
3 459-641 183 tcatctccag GACCCCACTG—TAGGATGGCT gtaagttttt AY749112
4 642-715 74 gtttttacag GAAGGAGAAG—CCATTGGCAC gtatgtgaag AY749113
5 716-850 135 ctaaatatag GAGCAGATCT—CCAAAGAAAG gtatgaaatc AY749114
6 851-953 103 gattttccag AGTTTCAGAT—CCCAGAGACT gtgagtccta AY749115
7 954-1069 116 cgtgtgatag GTTGGAAGCG—TTCAAGAAAA gtaagaatgc AY749116
8 1070-1219 150 cctttcttag GTGTCAGGCC—AGGATGAAAA gtgagtgtgc AY749117
9 1220-1313 94 tttttaatag GTCCAAATTA—AAGAAAAGAG gtattcaccg AY749118
10 1314-1472 159 tctttttcag TCAGAAAAAG—CAGGGCTCAG gtgaggcacc AY749119
11 1473-1603 131 cctctaacag ATGGAAGTTT—ATGGAGGCAG gtaaggaaaa AY749120
12 1604-1683 80 atcccggcag GCAGTCCTTG—GTTCAAAGAG gtaagtcctg AY749121
13 1684-1805 122 ttactcacag GAACTGAGGA—CATCATTTAG gtgttgatgt AY749122
Copyright 2005 The Association for Research in Vision and Ophthalmology, Inc.
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