Mice homozygous for the rd mutation display hereditary retinal degeneration and the classic rd lines serve as a model for human retinitis pigmentosa. In affected animals the retinal rod photoreceptor cells begin degenerating at about postnatal day 8, and by four weeks no photoreceptors are left. Degeneration is preceded by accumulation of cyclic GMP in the retina and is correlated with deficient activity of the rod photoreceptor cGMP-phosphodiesterase. We have recently isolated a candidate complementary DNA for the rd gene from a mouse retinal library and completed the characterization of cDNAs encoding all subunits of bovine photoreceptor phosphodiesterase. The candidate cDNA shows strong homology with a cDNA encoding the bovine phosphodiesterase beta subunit. Here we present evidence that the candidate cDNA is the murine homologue of bovine phosphodiesterase beta cDNA. We conclude that the mouse rd locus encodes the rod photoreceptor cGMP-phosphodiesterase beta subunit.

Retinal degeneration in the rd mouse is inherited as an autosomal recessive trait and is caused by a defect in the gene encoding the ,B subunit of cGMP phosphodiesterase. Recently, a close genetic association of the rd gene with an endogenous xenotropic murine leukemia virus unpublished observations).A potential cause for the transcriptional defects of the rd gene can be inferred from genetic linkage analysis of endogenous xenotropic murine leukemia viruses (Xmv) in inbred strains of mice that showed that an integrated provirus, Xmv-28, is closely linked to the rd locus on chromosome 5 (7). Perfect concordance between Xmv-28 and the rd locus was observed in all inbred, backcross, and congenic mouse strains tested (7,8). Although the majority of murine leukemia proviruses (MLVs) appear to be silent, some have been shown to cause specific mutations by integrating within and disrupting normal expression of genes. At least two spontaneous mutations have been shown to be caused by endogenous proviruses: dilute (d) (9) and hairless (hr) (10).Given the precedent for insertional mutagenesis, we examined the position ofXmv-28 with respect to the rd locus on chromosome 5. We show that the Xmv-28 provirus is integrated within the first intron of the rd 3-PDE gene and that it is oriented in the 3' -5' direction with respect to the sense strand of the ,B-PDE gene.** METHODS Mice. A breeding stock ofC57BL/6J rd/rd (B6rd) and +1+ (B6) mice was maintained by sibling matings at the Jules Stein Eye Institute vivarium. Inbred and wild mouse strains used to check for cosegregation of rd and Xmv-28 were obtained from T. Roderick (The Jackson Laboratory) and C. Kozak (National Institutes of Health), who also provided the NFS/N mice.

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The inherited retinal degeneration of the rd mouse results in the exclusive loss of one cell type, the photoreceptors. We took advantage of this visual-cell loss to devise a strategy for the isolation of photoreceptor-specific cDNAs based on the use of subtractive and differential hybridizations. The resulting pool of photoreceptor-specific cDNAs was screened for a candidate cDNA for the rd gene, and a putative rd cDNA that maps to mouse chromosome 5, the chromosome to which the rd gene has been assigned, was identified. On Northern blots the candidate rd cDNA hybridizes a 3.3-kilobase RNA species from 9-to 11-day-old developing normal retina and, much more faintly, a 3.6-kb RNA species from agematched rd retina. The 0.3-kilobase difference in the size of the mRNAs hybridized suggests that a structural alteration in the gene corresponding to the candidate rd cDNA has occurred in the rd mouse. This was further supported by the detection of polymorphisms between rd/rd and +/+ mouse genomic DNA after digestion with restriction endonucleases and probing with the candidate rd cDNA. Expression of mRNAs hybridized by the candidate rd cDNA is detected in normal and diseased retinas at postnatal day 1 but the signal intensity is considerably lower in the rd retina. To our knowledge, this is the earliest molecular defect reported in the rd retina that is observed prior to any phenotypic signs of photoreceptor degeneration.The primary lesions affecting most inherited retinal diseases are unknown; however, several studies of animal models for the human disease retinitis pigmentosa have revealed that biochemical defects related to cyclic nucleotide metabolism are associated with some of these disorders (1-3). The rd mouse, the most thoroughly investigated animal model, is affected with an autosomal-recessive retinal degeneration characterized by an early onset and a rapid progression. The rd mutation has been localized to mouse chromosome 5 (4) and its expression is restricted to the photoreceptor cell layer (5, 6). Elevated levels of cGMP present in the rd retina by postnatal day 6, 2 days before pathological signs are observed (1), lead to the degeneration of the visual cells (7) and are the result of deficient cGMP phosphodiesterase activity (8). Photoreceptor maturation in the rd retina appears to parallel development in the normal retina until postnatal day 8, when ultrastructural changes become evident (9-12). By 20 postnatal days the rd photoreceptor layer consists of a single row of cone nuclei (13) and by day 30 the rd retina is virtually devoid of rod photoreceptors, whereas the small cone population (3% of the total visual cells) has been reduced to between one-third and one-half of the original number (13,14). The inner cell layers of the retina remain intact and apposed to the pigment epithelium. We took advantage of this phenotypic expression of the disease to devise a strategy for the isolation of a candidate cDNA for the rd gene. MATERIALS AND METHODScDNA Library Preparation. Care of mice used for all...