Removal of phosphorylation sites of gamma subunit of phosphodiesterase 6 alters rod light response.

The phosphodiesterase 6 gamma (PDE6 gamma) inhibitory subunit of the rod PDE6 effector enzyme plays a central role in the turning on and off of the visual transduction cascade, since binding of PDE6 gamma to the transducin alpha subunit (T alpha) initiates the hydrolysis of the second messenger cGMP, and PDE6 gamma in association with RGS9-1 and the other GAP complex proteins (G beta 5, R9AP) accelerates the conversion of T alpha GTP to T alpha GDP, the rate-limiting step in the decay of the rod light response. Several studies have shown that PDE6 gamma can be phosphorylated at two threonines, T22 and T35, and have proposed that phosphorylation plays some role in the physiology of the rod. We have examined this possibility by constructing mice in which T22 and/or T35 were replaced with alanines. Our results show that T35A rod responses rise and decay more slowly and are less sensitive to light than wild-type (WT). T22A responses show no significant difference in initial time course with WT but decay more rapidly, especially at dimmer intensities. When the T22A mutation is added to T35A, double mutant rods no longer showed the prolonged decay of T35A rods but remained slower than WT in initial time course. Our experiments suggest that the polycationic domain of PDE6 gamma containing these two phosphorylation sites can influence the rate of PDE6 activation and deactivation and raise the possibility that phosphorylation or dephosphorylation of PDE6 gamma could modify the time course of transduction, thereby influencing the wave form of the light response.

Conserved transcriptional regulation of a cone phototransduction gene in vertebrates.

cGMP-phosphodiesterase (PDE) is a key component in visual phototransduction. Rod and cone photoreceptors each produce their unique cGMP-PDE subunits. The alpha' catalytic subunits are believed to be cone-specific. In this study, we report that transfection of the -132 to +139 sequence in the upstream region of the human alpha'-PDE gene fused to luciferase cDNA gives the highest level of reporter gene transcription in cultured retinoblastoma Y79 cells. Transgenic Xenopus laevis carrying this sequence fused to green fluorescent protein (GFP) expressed GFP in cones, suggesting a conserved regulatory mechanism for alpha'-PDE transcription in both human and frog.

CORD9 a new locus for arCRD: mapping to 8p11, estimation of frequency, evaluation of a candidate gene.

PURPOSE: To determine the locus of the mutant gene causing autosomal recessive cone-rod dystrophy (arCRD) in a consanguineous pedigree, to evaluate a candidate gene expressed in retina that maps to this locus, and to estimate the percentage of arCRD cases caused by mutations in this gene. METHODS: DNAs from family members were genotyped for markers covering the entire genome at an average spacing of approximately 9 centimorgans (cM). The data were input into a pedigree computer program to produce output files used to calculate lod scores. Significant linkage was revealed at 8cen, prompting the genotyping of a number of additional markers. Exons of a candidate gene were sequenced directly by standard fluorescent dideoxy methods. Haplotype analysis was performed with markers in this locus in 13 multiplex and 2 simplex CRD families in which neither parent had disease. RESULTS: Four-point linkage analysis gave a maximum lod score of approximately 7.6 at both D8S1769 and GATA101H09 in the large consanguineous family. Recombination events defined an interval of 8.7 cM between D8S1820 and D8S532 within which the gene must lie. This 8p11 locus (CORD9) is immediately distal to but distinct from the RP1 autosomal dominant RP (adRP) locus. Two islands of homozygosity were found in this locus: The alleles of 6 of 10 markers in one of the islands and 2 of 4 in the other were homozygous. The UniGene cluster Hs.8719 (UniGene System, provided by the National Center for Biotechnology Information and available at http://www.ncbi.nlm.nih.gov/UniGene), which tags a gene with significant homology to Dual Specificity Phosphatase 3, maps within the CORD9 interval and is highly expressed in the retina. To evaluate this gene as a potential disease candidate, intron-exon structure was determined, and exons were screened in the consanguineous family. No variants were found that could be related to disease. Haplotype analysis of 15 other families with CRD, using markers at CORD9, excluded this locus in 9 of 15. CONCLUSIONS: A new arCRD locus (CORD9) has been identified corresponding to a yet unidentified gene in the 8.7-cM interval D8S1820-D8S532. No mutations were found in one candidate gene in affected members of the primary study family. Haplotype analysis of a cohort of 13 multiplex and 2 simplex families with CRD ruled out the CORD9 gene in 9 of 15 of the families. To date, a total of 126 loci carrying gene mutations causing various forms of retinal degeneration have been mapped, and the mutant gene has been identified in 64 of them. However, only 2 loci for arCRD have been documented. This is the report of a third.

Nrl and Sp nuclear proteins mediate transcription of rod-specific cGMP-phosphodiesterase beta-subunit gene: involvement of multiple response elements.

cGMP-phosphodiesterase (PDE) is the key effector in rod photoreceptor signal transduction. Mutations in the gene encoding its catalytic beta-subunit (beta-PDE) cause retinal degenerations leading to blindness. We report that the short -93 to +53 sequence in the upstream region of this gene is sufficient for beta-PDE transcription in both Y79 human retinoblastoma cells and Xenopus embryo heads maintained ex vivo. This sequence also functions as a minimal rod-specific promoter in transgenic Xenopus tadpoles. The Nrl transcription factor binds in vitro to the betaAp1/NRE regulatory element located within this region and transactivates it when overexpressed in nonretinal 293 embryonic kidney cells. We also found a G/C-rich activator element, beta/GC, important for promoter activity in Y79 retinoblastoma cells and Xenopus embryos. Both the ubiquitous Sp1 and the central nervous system-specific Sp4 transcription factors are expressed in retina and interact with this element in vitro. Electrophoretic mobilities of beta/GC-Y79 nuclear protein complexes are altered by antibodies against Sp1 and Sp4. Thus, our results implicate Nrl, Sp1, and Sp4 in transcriptional regulation of the rod-specific minimal beta-PDE promoter. We also conclude that Xenopus laevis is an efficient system for analyzing the human beta-PDE promoter and may be used to study other human retinal genes ex vivo and in vivo.

In vivo studies of the gamma subunit of retinal cGMP-phophodiesterase with a substitution of tyrosine-84.

The inhibitory rod cGMP phosphodiesterase gamma subunit (PDEgamma) is a major component of the photoresponse and is required to support rod integrity. Pdeg(tm1)/Pdeg(tm1) mice (which lack PDEgamma owing to a targeted disruption of the Pdeg gene) suffer from a very rapid and severe photoreceptor degeneration. The Y84G (Tyr(84)-->Gly) allele of PDEgamma has previously been shown in experiments carried out in vitro to reduce the regulatory control of the PDE catalytic core (PDEalphabeta) exerted by the wild-type gamma subunit. To determine the effects of this mutation on in vivo function, the murine opsin promoter was used to direct expression to the photoreceptors of +/Pdeg(tm1) mice of a mutant Y84G and a wild-type PDEgamma control transgene. The transgenic mice were crossed with Pdeg(tm1)/Pdeg(tm1) mice to generate animals able to synthesize only the transgenic PDEgamma. Our results showed that wild-type PDEgamma and Y84G transgenes could complement the Pdeg(tm1)/Pdeg(tm1) mutant for photoreceptor survival. The mutation caused a significant biochemical defect in PDE activation by transducin. However, the Y84G mutation did not fully eliminate the control of PDEgamma on the PDE catalytic core in vivo; the expression of the mutant subunit was associated with only a 10-fold reduction in the amplitude of the a-wave and a 1.5-fold decrease in the b-wave of the corneal electroretinogram. Unexpectedly, the mutation caused a much 'milder' phenotype in vivo than was predicted from the biochemical assays in vitro.

Transient transfection of human retinoblastoma cells : application to the analysis of the regulatory regions of photoreceptor-specific genes.

Retinoblastoma (Rb) is an intraocular tumor usually diagnosed in children under four years of age (1). The tumor rises when both alleles of the Rb tumor suppressor gene become inactivated in a retinal precursor cell during development (2,3). The first retinoblastoma cell line to be established in culture, Y-79 (4), has been shown to originate from neuroectodermal cells that express both neuronal and glial cell markers (3). Both Y-79 cells and Rb tumor cells produce mRNAs encoding several proteins unique to the photoreceptors (5), including different subunits of cone- and rod-specific cGMP-phosphodiesterases (6). Therefore, cultured Y-79 cells, which have a human retinal origin, could be particularly useful for studying the regulatory mechanisms of photoreceptor-specific gene expression (7).

The human cGMP-PDE beta-subunit promoter region directs expression of the gene to mouse photoreceptors.

PURPOSE: We previously demonstrated that 350 bp of the human rod cGMP phosphodiesterase beta-subunit (beta-PDE) gene promoter are sufficient to direct high levels of gene expression in human Y-79 retinoblastoma cells in vitro. In this study the cell specificity and expression pattern conferred by the short beta-PDE 5' flanking sequence in vivo were examined. METHODS: A construct containing the bacterial LacZ gene driven by a fragment of the beta-PDE 5' flanking region (-297 to +53) was used to generate transgenic mice. Gene expression was analyzed by measuring beta-galactosidase activity in tissue homogenates or visualizing enzymatic activity or protein production at a cellular level by in situ histochemistry or immunocytochemistry. RESULTS: Three independently derived transgenic lines were generated carrying the -297 to +53 beta-PDE 5' flanking region fragment. Within the retina, the reporter gene was specifically expressed in photoreceptors, consistent with the localization of endogenous beta-PDE. Significant expression of LacZ was not observed in other ocular or peripheral tissues. CONCLUSIONS: Photoreceptor-specific reporter gene expression is driven in vivo by a 350-bp segment of the beta-PDE 5' flanking sequence. This study demonstrates the utility of the human beta-PDE promoter for directing the expression of foreign genes to photoreceptors and suggests that the -297 to +53 beta-PDE 5' flanking region fragment may have important implications for therapeutic gene delivery to the visual cells.

A homozygous deletion in RPE65 in a small Sardinian family with autosomal recessive retinal dystrophy.

PURPOSE: We have been engaged in an ongoing study to screen candidate genes for mutations in small families with various forms of autosomal recessive retinal dystrophy. Here we report the screening of a cohort of 14 families from Sardinia for mutations in the genes encoding the alpha- and beta-subunits of cGMP-phosphodiesterase and RPE65 (PDE6A, PDE6B, and RPE65). METHODS: Haplotype analysis was performed on each family using simple sequence repeat markers closely flanking or within each of the three gene candidates. For families in which a gene could not be ruled out from segregating with disease, exons of the gene from proband DNAs were screened for mutations by SSCPE (single strand conformation polymorphism electrophoresis). All variants found by SSCPE were sequenced directly. RESULTS: By haplotype analysis, 6/14, 11/14, and 4/13 families were ruled out for PDE6A, PDE6B, and RPE65, respectively. A few variants were found in the proband DNAs of the remaining families, but only one was significant: a 20 bp deletion in exon 4 of RPE65. The deletion co-segregated with disease in one family and caused a frame shift that produces a stop codon downstream. It was absent from the other Sardinian families that we tested, and from Sardinian and North American controls. Results of studies of phenotype in homozygotes and heterozygotes in this Sardinian family are compared with those from a non-Sardinian family recently reported to have the same RPE65 mutation. CONCLUSIONS: This RPE65 mutation, which appears to be quite restricted in its occurrence in Sardinia, leads to childhood onset severe retinal dystrophy or Leber congenital amaurosis. Affecteds of the other 13 plus two additional families were diagnosed with arRP. This family lived in an area of Sardinia where none of the others lived suggesting different ancestral origins.

Retinoschisin, the X-linked retinoschisis protein, is a secreted photoreceptor protein, and is expressed and released by Weri-Rb1 cells.

X-linked retinoschisis is characterized by microcystic-like changes of the macular region and schisis within the inner retinal layers, leading to visual deterioration in males. Many missense and protein-truncating mutations of the causative gene RS1 have now been identified and are thought to be inactivating. RS1 encodes a 224 amino acid protein, retinoschisin, which contains a discoidin domain but is of unknown function. We have generated a polyclonal antibody against a peptide from a unique region within retinoschisin, which detects a protein of approximately 28 kDa in retinal samples reduced with dithiothreitol, but multimers sized >40 kDa under non-reducing conditions. A screen of human tissues with this antibody reveals retinoschisin to be retina specific and the antibody detects a protein of similar size in bovine and murine retinae. We investigated the expression pattern in the retina of both RS1 mRNA (using in situ hybridization with riboprobes) and retinoschisin (using immunohistochemistry). The antisense riboprobe detected RS1 mRNA only in the photoreceptor layer but the protein product of the gene was present both in the photoreceptors and within the inner portions of the retina. Furthermore, differentiated retinoblastoma cells (Weri-Rb1 cells) were found to express RS1 mRNA and to release retinoschisin. These results suggest that retinoschisin is released by photo-receptors and has functions within the inner retinal layers. Thus, X-linked retinoschisis is caused by abnormalities in a putative secreted photoreceptor protein and is the first example of a secreted photo-receptor protein associated with a retinal dystrophy.

Disease expression of RP1 mutations causing autosomal dominant retinitis pigmentosa.

PURPOSE: To determine the disease expression in heterozygotes for mutations in the RP1 gene, a newly identified cause of autosomal dominant retinitis pigmentosa (adRP). METHODS: Screening strategies were used to detect disease-causing mutations in the RP1 gene, and detailed studies of phenotype were performed in a subset of the detected RP1 heterozygotes using electroretinography (ERG), psychophysics, and optical coherence tomography (OCT). RESULTS: Seventeen adRP families had heterozygous RP1 changes. Thirteen families had the Arg677ter mutation, whereas four others had one of the following: Pro658 (1-bp del), Ser747 (1-bp del), Leu762-763 (5-bp del), and Tyr1053 (1-bp del). In Arg677ter RP1 heterozygotes, there was regional retinal variation in disease, with the far peripheral inferonasal retina being most vulnerable; central and superior temporal retinal regions were better preserved. The earliest manifestation of disease was rod dysfunction, detectable as reduced rod ERG photoresponse maximum amplitude, even in heterozygotes with otherwise normal clinical, functional, and OCT cross-sectional retinal imaging results. At disease stages when cone abnormalities were present, there was greater rod than cone dysfunction. Patients with the RP1 frameshift mutations showed similarities in phenotype to those with the Arg677ter mutation. CONCLUSIONS: Earliest disease expression of RP1 gene mutations causing adRP involves primarily rod photoreceptors, and there is a gradient of vulnerability of retinopathy with more pronounced effects in the inferonasal peripheral retina. At other disease stages, cone function is also affected, and severe retina-wide degeneration can occur. The nonpenetrance or minimal disease expression in some Arg677ter mutation-positive heterozygotes suggests important roles for modifier genes or environmental factors in RP1-related disease.

A QTL on distal chromosome 3 that influences the severity of light-induced damage to mouse photoreceptors.

C57BL/6J-c(2J) (c2J) albino mice showed much less damage to their photoreceptors after exposure to prolonged light than BALB/c mice and seven other albino strains tested. There were no gender differences, and preliminary studies suggested that the c2J relative protective effect was a complex trait. A genome-wide scan using dinucleotide repeat markers was carried out for the analysis of 194 progeny of the backcross (c2J x BALB/c)F(1) x c2J and the thickness of the outer nuclear layer (ONL) of the retina was the quantitative trait reflecting retinal damage. Our results revealed a strong and highly significant quantitative trait locus (QTL) on mouse Chromosome (Chr) 3 that contributes almost 50% of the c2J protective effect, and three other very weak but significant QTLs on Chrs 9, 12, and 14. Interestingly, the Chrs 9 and 12 QTLs corresponded to relative susceptibility alleles in c2J (or relative protection alleles in BALB/c), the opposite of the relative protective effect of the QTLs on Chrs 3 and 14. We mapped the Rpe65 gene to the apex of the Chr 3 QTL (LOD score = 19.3). Northern analysis showed no difference in retinal expression of Rpe65 message between c2J and BALB/c mice. However, sequencing of the Rpe65 message revealed a single base change in codon 450, predicting a methionine in c2J and a leucine in BALB/c. When the retinas of aging BALB/c and c2J mice reared in normal cyclic light were compared, the BALB/c retinas showed a small but significant loss of photoreceptor cells, while the c2J retinas did not. Finding light damage-modifying genes in the mouse may open avenues of study for understanding age-related macular degeneration and other retinal degenerations, since light exposures may contribute to the course of these diseases.

A deletion in a photoreceptor-specific nuclear receptor mRNA causes retinal degeneration in the rd7 mouse.

The rd7 mouse, an animal model for hereditary retinal degeneration, has some characteristics similar to human flecked retinal disorders. Here we report the identification of a deletion in a photoreceptor-specific nuclear receptor (mPNR) mRNA that is responsible for hereditary retinal dysplasia and degeneration in the rd7 mouse. mPNR was isolated from a pool of photoreceptor-specific cDNAs originally created by subtractive hybridization of mRNAs from normal and photoreceptorless rd mouse retinas. Localization of the gene corresponding to mPNR to mouse Chr 9 near the rd7 locus made it a candidate for the site of the rd7 mutation. Northern analysis of total RNA isolated from rd7 mouse retinas revealed no detectable signal after hybridization with the mPNR cDNA probe. However, with reverse transcription-PCR, we were able to amplify different fragments of mPNR from rd7 retinal RNA and to sequence them directly. We found a 380-nt deletion in the coding region of the rd7 mPNR message that creates a frame shift and produces a premature stop codon. This deletion accounts for more than 32% of the normal protein and eliminates a portion of the DNA-binding domain. In addition, it may result in the rapid degradation of the rd7 mPNR message by the nonsense-mediated decay pathway, preventing the synthesis of the corresponding protein. Our findings demonstrate that mPNR expression is critical for the normal development and function of the photoreceptor cells.

Estrogen receptor in the human eye: influence of gender and age on gene expression.

PURPOSE: Many epidemiologic studies indicate an increased incidence of certain vision threatening conditions in postmenopausal women. These data suggest that changes in sex steroid homeostasis may affect the physiology of the eye. To provide support to this hypothesis, the expression of estrogen receptor alpha (ERalpha) in human eye tissues was investigated. METHODS: Complementary studies including RNA analysis by reverse transcription polymerase chain reaction, western blot analysis, and immunocytochemistry were used to provide evidence of ERalpha expression. Protein detection was carried out using a mouse monoclonal antibody raised against an epitope located in the ligand binding domain of the human receptor. Cellular localization was studied on formalin-fixed paraffin-embedded eye sections using conventional immunohistochemical techniques. RESULTS: Gender and age differences in ERalpha mRNA expression were observed in retina. The 65-kDa ERalpha protein was detected in the retina and retinal pigment epithelium (RPE) of young female eyes but not in eye tissues dissected from men and postmenopausal women. Immunocytochemistry corroborated ERalpha staining of a young female neurosensory retina and RPE. In addition, ERalpha could be detected in the ciliary body, in the iris, and in the epithelium of the lens. CONCLUSIONS: The presence of the ERalpha in the human eye suggests that the sex steroid hormone axis may play a role in the pathogenesis of certain ocular diseases.

Photoreceptor cell damage by light in young Royal College of Surgeons rats.

PURPOSE: To determine the effects of genetic background and light rearing conditions on intense-light-mediated retinal degeneration in young RCS rats. MATERIALS AND METHODS: Albino rats, homozygous or heterozygous for the rdy gene were bred and born in dim cyclic light. At P7 they were moved to a dark environment, and maintained there until exposure to intense visible (green) light at P18 or P25. Other rats remained in the dim cyclic light environment. At various times between P11 and P40 rats were killed for determinations of rhodopsin and photoreceptor cell DNA levels, western transblot analysis of retinal S-antigen (arrestin) and alpha-transducin, or northern slot blot analysis of their respective mRNA levels. RESULTS: At P18, unexposed dark maintained homozygous RCS rats and their phenotypically normal heterozygous counterparts have nearly equivalent rhodopsin levels and photoreceptor cell DNA. Intense light exposure at this age, to 8 hours of continuous light or 3 hours of intermittent light, did not lead to a loss of either rhodopsin or retinal DNA when compared with their respective unexposed controls. At P25 rhodopsin levels were higher than at P18, while photoreceptor cell DNA was essentially the same as in the younger rats. However, intense light exposure at P25 resulted in substantial losses of rhodopsin and photorecptor cell DNA and the losses were greater in homozygous rats than in heterozygous animals. Light damage of P25 rats maintained in dim cyclic light was essentially the same as in dark maintained homozygous rats, but no damage was found in the heterozygous animals. By western analysis, alpha-transducin levels in the retina increased with time in darkness, while retinal S-antigen levels either remained the same or decreased during the period P15-P35. For rats in the cyclic light environment S-antigen expression was greater than alpha-transducin at all ages. Slot blot analysis of mRNAs for the two proteins generally followed the patterns seen by western analysis. S-antigen mRNA was expressed at an earlier age and at higher levels than alpha-transducin in both types of rats from both light rearing conditions. Peak expression of S-antigen most often occurred at P18 in both the heterozygous and homozygous rats. CONCLUSIONS: The relative expressions of S-antigen and alpha-transducin in P18 and P25 rats correlates with their relative resistance to retinal light damage at P18 and their enhanced susceptibility at P25. Rats homozygous for the rdy gene also exhibit more damage than heterozygous animals when photoreceptor cell DNA is used to estimate the extent of retinal light damage.

A nonsense mutation in a novel gene is associated with retinitis pigmentosa in a family linked to the RP1 locus.

Retinitis pigmentosa (RP) represents a group of inherited human retinal diseases which involve degeneration of photoreceptor cells resulting in visual loss and often leading to blindness. In order to identify candidate genes for the causes of these diseases, we have been studying a pool of photoreceptor-specific cDNAs isolated by subtractive hybridization of mRNAs from normal and photoreceptorless rd mouse retinas. One of these cDNAs was of interest because it mapped to proximal mouse chromosome 1 in a region homo-logous to human 8q11-q13, the locus of autosomal dominant RP1. Therefore, using the mouse cDNA as probe, we cloned the human cDNA (hG28) and its corresponding gene and mapped it near to D8S509, which lies in the RP1 locus. This gene consists of four exons with an open reading frame of 6468 nt encoding a protein of 2156 amino acids with a predicted mass of 240 kDa. Given its chromosomal localization, we screened this gene for mutations in a large family affected with autosomal dominant RP previously linked to the RP1 locus. We found an R677X mutation that co-segregated with disease in the family and is absent from unaffected members and 100 unrelated controls. This mutation is predicted to lead to rapid degradation of hG28 mRNA or to the synthesis of a truncated protein lacking approximately 70% of its original length. Our results suggest that R677X is responsible for disease in this family and that the gene corresponding to hG28 is the RP1 gene.

Screening of the gene encoding the alpha'-subunit of cone cGMP-PDE in patients with retinal degenerations.

PURPOSE: To screen the exons of the gene encoding the alpha'-subunit of cone cyclic guanosine monophosphate (cGMP>phosphodiesterase (PDE6C) for mutations in a group of 456 unrelated patients with various forms of inherited retinal disease, including cone dystrophy, cone-rod dystrophy, macular dystrophy, and simplex/multiplex and autosomal recessive retinitis pigmentosa. METHODS: The 22 exons of the PDE6C gene were screened for mutations either by denaturing gradient gel electrophoresis and single-strand conformation polymorphism electrophoresis (SSCP) or by SSCP alone; variants were sequenced directly. RESULTS: Although many sequence variants were found, none could be associated with disease. CONCLUSIONS: The results show that PDE6C was not the site of the amutations responsible for the types of inherited retinal degenerations analyzed in the large population of patients 'in the present study. The types of degeneration included those that predominantly affect cone-mediated function (cone and cone-rod dystrophies) or rod-mediated function (retinitis pigmentosa) or that have a predilection for disease in the macula (macular dystrophies).

Genetic and physical maps of the mouse rd3 locus; exclusion of the ortholog of USH2A.

The rd3 retinal degeneration gene was previously mapped 10 +/- 2.5 cM distal to Akp1 on mouse Chromosome (Chr) 1 (Chang et al., 1993), a region that may be homologous to the locus of the human USH2A gene, which carries mutations responsible for Usher IIa retinal degeneration/hearing loss syndrome. An intercross from an Rb(11, 13)4Bnr(rd3/rd3) x C57BL/6J mating was set up, 428 F2 meioses were analyzed, and the rd3 gene was placed between the markers D1MIT292/D1MIT209 and D1MIT510, a distance of 1.40 +/- 0.57 cM. These flanking markers and the mouse ortholog of USH2A (Mush2a) were mapped in the T31 mouse radiation hybrid (RH) panel, with the result that D1MIT292/D1MIT209 and D1MIT510 were 7.9 cR3000 apart ( approximately 800 kb), and Mush2a was > 30 cR3000 proximal to the pair, excluding it from the rd3 locus. A contig spanning the rd3 locus and consisting of 2 YACs and one BAC was generated, and Mush2a was absent from it, confirming its exclusion from the locus. Comparison of adjacent marker pairs in the Whitehead genetic map and our genetic map showed some discrepancies in order of markers and genetic distances. Comparison of our genetic map and the RH map showed some highly skewed relationships between genetic and physical distances.

The mouse X-linked juvenile retinoschisis cDNA: expression in photoreceptors.

Retinal photoreceptor cells are particularly vulnerable to degenerations that can eventually lead to blindness. Our purpose is to identify and characterize genes expressed specifically in photoreceptors in order to increase our understanding of the biochemistry and function of these cells, and then to use these genes as candidates for the sites of mutations responsible for degenerative retinal diseases. We have characterized a cDNA, a fragment of which (SR3.1) was originally isolated by subtractive hybridization of adult, photoreceptorless rd mouse retinal cDNAs from the cDNAs of normal mouse retina. The full-length sequence of this cDNA was determined from clones obtained by screening mouse retinal and eye cDNA libraries and by using the 5'- and 3'-RACE methods. Both Northern blot analysis and in situ hybridization showed that the corresponding mRNA is expressed in rod and cone photoreceptors. The gene encoding this cDNA was mapped to the X chromosome using an interspecific cross. Based on the nucleotide and amino acid sequences, as well as chromosome mapping, we determined that this gene is the mouse ortholog (Xlrs1) of the human X-linked juvenile retinoschisis gene (XLRS1). Analysis of the predicted amino acid sequence indicates that the Xlrs1 mRNA may encode a secretable, adhesion protein. Therefore, our data suggest that X-linked juvenile retinoschisis originates from abnormalities in a photoreceptor-derived adhesion protein.