Expression of cone photoreceptor cGMP-phosphodiesterase alpha' subunit in Chinese hamster ovary, 293 human embryonic kidney, and Y79 retinoblastoma cells.

Purpose: A functional protein is required for structure/function analysis of cone photoreceptor cGMP-phosphodiesterase alpha' subunit (PDEalpha'). The purpose of this study was to express enzymatically active PDEalpha'.

Methods: Three expression vectors were constructed for transient and stable expression of PDEalpha': pC57 (transient) was obtained by subcloning bovine PDEalpha' cDNA into the pCIS2 expression vector; pNC57 (stable) was constructed by inserting the neo gene controlled by the mouse phosphoglycerate kinase-1 gene promoter into the pC57 vector; and pFC57 (transient) was generated by fusing the sequence encoding the FLAG peptide to the 5' end of the coding region of PDEalpha' cDNA.

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.

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.

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.

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.

Canine cone transducin-gamma gene and cone degeneration in the cd dog.

Purpose: To characterize the cDNA and the organization of the gene encoding the cone-specific gamma subunit of transducin (Tgamma c) and to examine this gene as a candidate for the recessively inherited cone photoreceptor degeneration in the cd dog.

Methods: Canine Tgamma c cDNA was cloned and sequenced. Polymerase chain reaction (PCR) was used to define the Tgamma c gene structure, northern blot analysis to examine the level of expression of Tgamma c mRNA in control and cd-affected retinas, and immunocytochemistry to determine the presence and localization of Tgamma c in normal and cd retinas.

Defective RNA splicing resulting from a mutation in the cyclic guanosine monophosphate-phosphodiesterase beta-subunit gene.

Purpose: The authors have previously reported a 3' splice site mutation in intron 2 of the rod cyclic guanosine monophosphate-phosphodiesterase (cGMP) beta-subunit (beta-PDE) gene in a patient with compound heterozygous autosomal recessive retinitis pigmentosa. The purpose of this study is to determine whether this mutation interferes with RNA splicing, what products it generates, and whether the resultant mRNA is able to support the synthesis of the native protein.

Methods: Two expression constructs were prepared by subcloning genomic DNA fragments (one from the control subject DNA and the other from the patient's DNA) to the pCIS2 expression vector.

Structure and analysis of the transducin beta3-subunit gene, a candidate for inherited cone degeneration (cd) in the dog.

The cDNA for the beta3-subunit of cone-specific transducin (Tbeta3) was cloned and characterized from wild type dogs, and used in linkage studies as a candidate gene for cone degeneration. Sequence analysis of the Tbeta3 cDNA revealed an open reading frame of 1020 bp, potentially coding for a protein of 340 amino acids (aa). The deduced aa sequence of canine Tbeta3 shares 97% identity with the previously identified human Tbeta3, and 82% identity with bovine rod-specific transducin (Tbeta1).

Screening of the PDE6B gene in patients with autosomal dominant retinitis pigmentosa.

Each of the 22 exons and 140 bp of the 5' untranslated region of the gene encoding the beta-subunit of cGMP-phosphodiesterase (PDE6B) were screened by denaturing gradient gel electrophoresis for mutations in the DNAs of 54 unrelated individuals with autosomal dominant retinitis pigmentosa. Six different sequence variants were found in seven patients. Four of the sequence variants did not segregate with disease in the families of the respective probands and/or were present in control DNAs.

Isolation and characterization of a cDNA encoding the alpha' subunit of human cone cGMP-phosphodiesterase.

A human cDNA (alpha-PDE) encoding the alpha' catalytic subunit of cone photoreceptor cGMP-phosphodiesterase has been isolated and characterized. The nucleotide sequence of 2980 bp contains an open reading frame encoding an 859-amino-acid (aa) protein with a calculated molecular mass of 99 kDa. Northern blot analysis of human retinal mRNA hybridized with the alpha'-PDE cDNA revealed a signal corresponding to a 3.

Gene structure and amino acid sequence of the human cone photoreceptor cGMP-phosphodiesterase alpha' subunit (PDEA2) and its chromosomal localization to 10q24.

The genomic organization and nucleotide structure of the human cone photoreceptor cGMP phosphodiesterase alpha'-subunit (alpha'-PDE) gene (PDEA2) as well as its chromosomal localization have been determined. This gene, which spans about 48 kb, consists of 22 exons and codes for an 858-amino-acid protein. The alpha'-PDE gene maps to human chromosome 10q24.

Cloning and characterization of the gene encoding the cGMP-phosphodiesterase gamma-subunit of human rod photoreceptor cells.

Rod photoreceptor cyclic GMP-phosphodiesterase (cGMP-PDE) is one of the key enzymes of the visual phototransduction cascade in the vertebrate retina. The holoenzyme is a heterotetrameric complex, consisting of two large catalytic subunits, alpha (88 kDa) and beta (84 kDa), and two identical inhibitory subunits, gamma (11 kDa). Here we present the complete nucleotide sequence of the gene (cGMP-PDE gamma) encoding the cGMP-PDE gamma-subunit from human rod photoreceptors.

Rod photoreceptor cGMP-phosphodiesterase: analysis of alpha and beta subunits expressed in human kidney cells.

The bovine alpha and murine beta subunits of rod-photoreceptor cGMP-phosphodiesterase (PDE alpha and PDE beta) were expressed in adenovirus-transformed 293 human embryonic kidney cells. RNA blots from transfected cells showed transcripts of 3.0 and 2.