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Canine Cone Transducin-Y Gene and Cone Degeneration in the Cd Dog

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Canine Cone Transducin-Y Gene and Cone Degeneration in the Cd Dog Canine Cone Transducin-y Gene and Cone Degeneration in the cd Dog Novrouz B. Akhmedov,1 Natik I. Piriev,1 Sue Pearce-Kelling,5 Gregory M. Acland,5 Gustavo D. Aguirre,5 and Debora B. Farber1'2 PURPOSE. TO characterize the cDNA and the organization of the gene encoding the cone-specific y subunit of transducin (Tyc) and to examine this gene as a candidate for the recessively inherited cone photoreceptor degeneration in the cd dog. METHODS. Canine Tyc cDNA was cloned and sequenced. Polymerase chain reaction (PCR) was used to define the Tyc gene structure, northern blot analysis to examine the level of expression of Tyc mRNA in control and cd-affected retinas, and immunocytochemistry to determine the presence and localization of Tyc in normal and cd retinas. RESULTS. Immunocytochemical results showed Tyc localized to cone photoreceptor outer segments in the normal retina, whereas no Tyc immunoreactivity was observed in the cd retinas. However, the level of transcription and the primary structure of the cloned cDNA coding for the 69 -amino acid protein were identical in retinas from wild-type and affected dogs. CONCLUSIONS. Although Tyc immunoreactivity was specifically absent in the cd dog retina, no differences were detected between normal and cd retinas in the nucleotide sequence of Tyc mRNA or in its synthesis. These results indicate that a mutation in the Tyc gene may not be causally associated with the cd dog disease. These findings suggest that possible abnormalities in posttrans- lational modification of Tyc or defective assembly of the transducin a/3y complex could lead to rapid degradation of Tyc. (Invest Ophthalmol Vis Sci. 1998;39:1775-1781) proteins are signal-transducing guanine nucleotide- transduction cascade. Hydrolysis of GTP to GDP by the binding regulatory proteins that couple activated intrinsic guanosine triphosphatase (GTPase) activity of Ga cell surface receptors and intercellular effectors.1 terminates the cycle. G 2 3 4 14 Most members of this family of proteins are structurally and Seventeen forms of Ga, five of Gj3, and ten of Gy " functionally homologous heterotrimers consisting of a, j3, have been identified by molecular cloning from different mam- and y subunits. In response to a variety of signals, the malian sources. The molecular details of interactions between surface receptors stimulate the exchange of bound G-protein subunits have been extensively studied; however, many questions remain. It has been shown that the N-terminus guanosine diphosphate (GDP) for guanosine triphosphate l5 (GTP) on the G-protein a subunit (Ga). In the inactive state, of G/3 is an essential domain for Gj3y assembly, and that the Ga is bound to GDP (GaGDP) and forms a complex with the 14 -amino acid region (amino acids 36 - 49) of the yl subunit is sufficient for yl to discriminate between the j3 subunits.16 In G/3y dimer; and in the active state, GaGTP dissociates from cross-linking experiments, Bubis and Khorana17 showed that G/3y. GaGTP and Gj3y are capable of regulating the activity cysteines at positions 36 or 37 in the y subunit interact with of downstream components of the corresponding signal the cysteine at position 25 in /3l. Furthermore, with the use of /32//31 chimeras and site-directed mutagenesis, it has been demonstrated that the 56 amino acids from the NH2-terminus From the 'Jules Stein Eye Institute, University of California Los 2 of /32 do not correspond to the portion of the protein neces- Angeles School of Medicine; Molecular Biology Institute, University of 18 California Los Angeles; and 3The James A. Baker Institute for Animal sary for selective interaction with the y subunit and that Health, College of Veterinary Medicine, Cornell University, Ithaca, cysteine residues in j31, y2, and y3 are not needed for /3y 19 New York. dimerization. Studies based on the crystal structure of the The nucleotide sequence data reported in this study have been heterotrimeric Ta/GDP.T/3y20 have shown that interaction be- submitted to GenBank with the accession number AF038862. tween Ta and Tj3y occurs at two distinct interfaces between Supported by Grants EY08285 (DBF) and EY06855 (GMA, GDA) from the National Institutes of Health, Bethesda, Maryland; and by Ta and the amino terminus of T/3. Direct interaction between grants from the George Gund Foundation and The Foundation Fighting Ta and Ty was not observed. However, other studies indicate Blindness, Hunt Valley, Maryland; The Morris Animal Foundation, that a 15 amino acid stretch of the N-terminus of Ty can Englewood, Colorado; The Seeing Eye, Morristown, New Jersey; and directly interact with Ta.2122 In spite of the importance and the American Kennel Club-Canine Health Foundation, Aurora, Ohio. complexity of the G-protein interactions involved in photo- DBF is the recipient of a Research to Prevent Blindness Senior Scientific transduction, only one disease (Nougaret's nyctalopia) has Investigator Award. 23 Submitted for publication January 7, 1998; revised April 3, 1998; been associated with a mutation in a retinal G protein (Ta). accepted April 29, 1998. Proprietary interest category: N. We have recently shown specific absence of immunore- Reprint requests: Debora B. Farber, Jules Stein Eye Institute, UCLA activity against antibodies to cone photoreceptor Tj33 in the cd School of Medicine, 100 Stein Plaza, Los Angeles, CA 90095. dog retina. The cd dog has an inherited cone-specific photore- Investigative Ophthalmology & Visual Science, September 1998, Vol. 39, No. 10 Copyright © Association for Research in Vision and Ophthalmology 1775 Downloaded from iovs.arvojournals.org on 09/27/2021 1776 Akhmedov et al. IOVS, September 1998, Vol. 39, No. 10 ceptor degeneration and is the only animal model for congen- chloroform extraction procedure (TRIzol; Life Technologies, ital achromatopsia in man.24 Based on this observation, we Grand Island, NY) or by guanidinium thiocyanate extraction examined the transcription level of the cone Tj33 gene in and cesium trifluoroacetate separation (CsTFA; Pharmacia, Pis- retinas from normal and affected dogs and cloned the corre- cataway, NJ). (There are no morphologic or electroretinogram sponding cDNA and genes. No differences in T/33 nucleotide differences between the 7.5-week-old normal and 6-week-old sequences and retinal mRNA levels were found between nor- cd dog retinas.) The RNA samples were fractionated on a 1.2% mal and affected dogs.25 Additionally, linkage studies of a agarose gel containing 2.2 M formaldehyde and were blotted Tsp45 I restriction fragment length polymorphism and the overnight onto nylon membranes (Hybond-N+; Amersham, Ar- disease were used to exclude the T/33 gene as a candidate for lington Heights, IL). The membranes were prehybridized and the cd mutation. One of the possible explanations for the lack hybridized in 0.5 M phosphate buffer (pH 7.2), 1 mM EDTA, 7% of agreement between the immunocytochemical studies and sodium dodecyl sulfate, and 1% bovine serum albumin. cDNA the mRNA concentration and nucleotide sequence analyses is probes were labeled by multiprime DNA labeling systems (Am- that there may be problems in the formation of the transducin ersham) in the presence of [a-32P]deoxycytidine triphosphate heterotrimer because of an abnormal transducin-yc (Tyc) sub- (3000 Ci/mmol; Amersham), and oligonucleotides were end unit. To this end, we have now cloned the canine cone-specific labeled with [y-32P]adenosine triphosphate (6000 Ci/mmol; Tyc cDNA and examined its possible involvement in this cone- Amersham) using T4 polynucleotide kinase (Promega, Madi- selective inherited photoreceptor degeneration. son, WI). The blots were washed at a final stringency of 0.2 X SSC and 0.2% sodium dodecyl sulfate and exposed to x-ray film (Hyperfilm-MP; Amersham) at — 80°C, using intensifying MATERIALS AND METHODS screens. The temperatures of hybridization and washing of the Immunocytochemistry blots were calculated based on the nature of the probe. The eyes from light-adapted control and crf-affected dogs were enucleated between 12 PM and 2 PM after intravenous anes- Construction and Screening of cDNA Libraries thesia with sodium pentobarbital, and the dogs were then and Sequence Analysis euthanatized with an overdose of the barbiturate. The retinas Oligo(dT)-primed unidirectional (Notl-Saft) cDNA retinal librar- were fixed in 4% paraformaldehyde in 0.1 M phosphate-buff- ies from normal and crf-affected dogs were constructed in ered saline (PBS) and embedded in diethylene glycol distearate Agt22A using the Super Script Lambda System (Life Technolo- (Polysciences, Warrington, PA), as previously described.2627 gies) and Gigapack III Gold Packaging Extract (Stratagene, La Two-micrometer sections were cut using glass knives, and the Jolla, CA). The libraries were screened using bovine cone- sections were mounted on silane-coated slides. The sections specific Ty cDNA labeled by random priming. Positive plaques were dewaxed with toluene, rehydrated, and immunoreacted were isolated, and the inserts were excised and subcloned with a polyclonal antibody raised in rabbit against an N-termi- into pBluescript II SK (Stratagene). Nucleotide sequence nal peptide (MAQELSEKELLKME) corresponding to residues 1 determinations were performed by the dideoxynucleotide through 14 of bovine Tyc (1:2,500 dilution). As a control for chain termination method vising Sequenase (USB, Cleveland, the presence of cones, the monoclonal antibody COS-1 was OH), [a-35S]deoxyadenosine triphosphate (1000 Ci/mmol; Am- used (1:10,000 dilution). This antibody identifies middle-wave- ersham), or with a Taq DyeDeoxy Terminator Cycle sequenc- sensitive cone opsins. Because this is the predominant class of ing kit (ABI, Foster City, CA). Fragments needed to cover gaps cones in the retina of most mammalian species, including dogs, in the cDNA sequence were obtained using specific internal this antibody was used to determine the presence and integrity sequencing primers. of cones.24'28'29 The immunoreactions were performed using 2.5% bovine serum albumin as a blocking agent and incorpo- rating 0.25% Triton X-100 in all washes and antibody-contain- RESULTS AND DISCUSSION ing solutions.
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