Canine Cone -y 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 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 findingssugges t 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)

are signal-transducing guanine nucleotide- transduction cascade. Hydrolysis of GTP to GDP by the binding regulatory proteins that couple activated intrinsic guanosine (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 at positions 36 or 37 in the y subunit interact with of downstream components of the corresponding signal the 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 (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

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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 . 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. As control for the immunoreactions, the retinal sections were incubated without primary antibodies in the Immunocytochemistry

corresponding solutions. These sections confirmed the ab- In the normal dog retina, intense Tyc immunoreactivity was sence of reaction product. Biotinylated secondary antibodies limited to cones that showed uniform labeling of the entire (Zymed, San Francisco, CA) and diaminobenzidine solution outer segments (Fig- 1A). Cone inner segments, rod photore- (Kirkegaard and Perry, Gaithersburg, MD) as chromogen were ceptors, and all other retinal neurons remained unlabeled. In used to visualize the immunoreaction. Immunoreactivity in the areas where the retina and the retinal pigment epithelium cd retinas was judged in comparison with normal control (RPE) had artifactually separated, some of the cone outer seg- samples, which were run concurrently. By using 2-ju,m sections ments had been severed at the thin connecting cilium and of diethylene glycol distearate- embedded tissues, it was pos- remained attached to the RPE by the extensive villous network sible to visualize individual rod and cone photoreceptors and of the RPE cone sheath. These cones also showed intense Ty 24 26 27 c evaluate the immunolabeling pattern. ' ' immunoreactivity (Fig. IB). In striking contrast, cone outer All procedures with animals conformed to the ARVO State- segments in the cd retina were not labeled with the Tyc ment for the Use of Animals in Ophthalmic and Vision Re- antibody. This was apparent in areas where the retina and RPE search and to the guidelines of the committees on animal were attached or artifactually separated (Figs. 1C, ID). research at our respective institutions. The immunolabeling studies in the cd retina were per- formed in 2.2-month-old dogs. At this age at which structurally RNA Isolation and Northern Blot Analysis intact cones, including the outer segments, are visible by light Total RNA from normal (7.5-week-old) and cd-affected (6-week- microscopy.23 To confirm further the presence of cones, we old) canine retinas was isolated using the TRIzol reagent- used the COS-1 antibody, which labels the cone outer seg-

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m> i

B

FIGURE 1. Sections from normal (A, B) and cone-degeneration (cd) (C, D) retinas showing the immunoreaction to the yc- transducin (Tyc) antibody. In the normal retina, there is discrete labeling of the cone outer segments {thin arrows). This immimoreactivity is visualized in areas where the retina and retina! pigment epithelium (*) are attached (A) or separated (B). The section with the artifactual detachment shows that fragments of labeled cone outer segments remain adherent to the retinal pigment epithelial apical cone sheath (thin arrow pairs). In the cd retina (C, D), there is absence of cone outer segment labeling with the Tyc antibody. Horizontal thick arrows (A, B, C, D) identify the external limiting membrane. Magnification, X800.

ments in a pattern indistinguishable from that produced by the cone-specific Ty cDNA. Five positive clones were identified, Tyc antibody in normal cones. The COS-1 antibody showed no two from the normal and three from affected retinal cDNA dog difference in cone outer segment immunoreactivity between libraries. These clones contained inserts varying in size be- cd and normal control retinas (data not shown). The results tween 1.6 kb and 1.9 kb. Sequence analysis of the cDNA with the COS-1 antibody were identical with our previous fragments showed that the fiveclone s encoded Tyc. One clone 2 1 findings. ' from each library contained the full-length Tyc cDNA. The complete nucleotide and deduced amino acid sequences of the Cloning and Characterization of Canine T-yc cDNA normal canine Tyc are shown in Figure 2. The cDNA includes Approximately 100,000 plaques from each normal and cd- 215 bp of 5'-untranslated sequence, and the open reading affected dog retinal cDNA library were screened with a bovine frame of 207 bp encodes a 69 -amino acid protein. The trans-

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MAQELSEKELLKMEVEQL 19

360 KKEVKNPRALISKTGKEIKDYVEAEAGNDP 49

450 LLKGIPEDKNPFKEKGGCMIS *** 69

540 630 720 ATGGCAATCCTTAGAGAC|ATTTA|CTCCTCCCCGAAGCTGGGCCATTCTTTCCCTCTGCACTCTTCCTAGGACCCAGTCTCTTATTCAGAA 810 900 ACCCCAGAGGGCCTGAGGCAAAAGAGTCTATAAAGTCACAATGTCATGATTG|ATTTA|ATATTATGGAGTGTCTGTTCCTTGCTGGGTACT 990 1080 1170 1260 1350 1440 VrTTAfTAGAG 1530 TTTACAGTCCTGCAAGACAAAATGTCTTAGTGlATTTACCAGGGGAAAAATAACCCTTCTGGAAGTTAGGTTATTGATAACCTAACTTCCA 1620 1710 1800 CCCCCTGAAGTTGGGCACCTGGCAGCTCTTTCTGCCTACGGGTCCTGTCCCCGTGTTTGi ^.CCATTTTGCACCAAAAAAAAAAAA 1890 AAAAA 1895

FIGURE 2. Nucleotide sequence of canine transducin-yc cDNA and predicted amino acid sequence. The stop codon is indicated by asterisks. ATTTA pentanucleotides are boxed. A potential polyadenylation signal (AATAAA) is underlined.

lation initiation codon was chosen based on the comparison of the levels of expression of the Tyc mRNAs in the normal and the dog, bovine, and human predicted protein sequences. affected dog retinas are very similar. Canine Tyc cDNA shares 94% and 87% homology in the coding region with the corresponding regions of the bovine and hu- Genomic Organization of the Tyc Gene man Tyc cDNA, respectively. The Tyc transcript has a long To determine the exon-intron structure, as well as the 3' and 5' 3'-untranslated region (UTR) (1470 bp) that includes 4 pen- splicing sequences of the canine Tyc gene (GNGT2), we used tanucleotides 5'-ATTTA-3'. These pentanucleotides can func- specific primers from the Tyc cDNA sequence to amplify dog tion as a target site for RNase, and could be implicated in genomic DNA by polymerase chain reaction (PCR). The PCR mRNA stability.30'31 Comparison of the open reading frame fragments were subcloned into the Bluescript (SK+) vector for and the 5'- and 3'-UTR sequences showed no differences be- direct sequencing. The genomic organization of canine GNGT2 tween cDNA of the normal and cd dogs. is shown in Figure 4. It consists of three exons and two introns, the first exon located in the 5'-UTR of the Tyc mRNA. Part of Northern Blot Analysis the 5'-UTR (38 nucleotides) and the 84 nucleotides encoding Northern blot hybridizations were performed to compare the the first N-terminal 28 amino acid residues are in exon 2. Exon transcript size and level of expression of the Tyc mRNAs in 3 is the longest and contains sequence encoding the remainder normal and cd dog retinas. Total retinal RNA samples were of the protein and the entire 3'-UTR. The structures of the hybridized with the full length Tyc cDNA, and with a 441-bp canine and human GNGT2 genes are similar: both consist of 32 fragment from the 3'-UTR of the dog Tyc cDNA (1059-1500 three exons and two introns. The size of the introns, how- bp; Fig. 2). As shown on Figure 3, the full size cDNA probe ever, is different; moreover, the firstintro n 3' splice site in the revealed two transcripts of approximately 0.7 kb and 1.8 kb canine gene is located 38 nucleotides upstream from the ATG (lanes 1 and 2). The 0.7-kb band is probably the transcript initiation codon, whereas in the human GNGT2 it is found at corresponding to the rod-specific Ty, mRNA, inasmuch as rod- 22 nucleotides from the translation start site. and cone-specific Ty subunits are 66% identical in their coding The human ortholog for canine GNGT2 maps to human regions. The 44l-bp cDNA probe hybridized only to the 1.8-kb 17q21, the locus at which RP17, a form of auto- transcript (lanes 3 and 4), suggesting that this mRNA corre- somal dominant retinitis pigmentosa, has been mapped. Thus, sponds to Tyc Furthermore, these experiments showed that GNGT2 is a candidate gene for this disease, although the

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1 2 kb 3 4

2.37

1.35

0.24

FIGURE 3. Northern blot analysis of canine total retinal RNA from normal (lanes 1 and 3) and cone-degeneration (c

phenotype does not involve cone dystrophy as expected of a The remaining proteins of this family (y5, y7, yM, and yl0) have cone-specific gene. much less homology (~30%). All members of the Gy family contain in the C terminus a CAAX motif (C, cysteine; A, ali- Comparison of G-Protein y Subunits phatic; and X, any amino acid), which is the protein isopreny- Canine Tyc is almost identical to bovine Tyc (98.5% identity) lation site. Two types of prenyl groups can be involved in this with only one amino acid difference. Although still similar to modification: C,g, all-frww-farnesyl or C20, all-/r«ws-gera- human Tyc, the identity between the proteins of dog and nylgeranyl. The amino acid at the X position plays a major role human retinas is 85.5% (Fig. 5A). Alignment of the amino acid in determining whether the cysteine is geranylgeranylated or sequence of proteins from the Gy family (Fig. 5B) shows that farnesylated. Proteins with polar amino acids such as serine, the members of the y,, yc, and y,, group share approximately alanine, or methionine at the X position are farnesylated; if X is 60% amino acid identity, whereas the components of the y2l a hydrophobic amino acid, especially leucine, proteins are 33 y3, and yA group share approximately 68% amino acid identity. modified by addition of a geranylgeranyl group. The three

AGgtaggt atttgtcataccagC TGgtgagc ttgttttctcatagA

215bp 210bp 1470bp

FIGURE 4. Genomic organization of the canine cone transducin-y subunit gene (GNGT2). The sizes of the exons (gray boxes) and introns (lines) are shown. The nucleotide sequences represent the 5'- and 3'- splice sites of each exon-intron. The bottom portion of the diagram illustrates the structure of the transducin-yc cDNA (open boxes correspond to the 5'- and 3'-UTR, respectively, and the solid box indicates the coding region).

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I III

A. MAQDLSEKDLLKMEVEQLKKEVKNTRIPISKAGKEIKEYVEAQAGNDPFLKGIPEDKNPFKE-KGGCLIS HYc MAQELSEKELLKMEVEQLKKEVKNPRALISKTGKEIKDYVEAEAGNDPLLKGIPEDKNPFKE-KGGCMIS MAQELSEKELLKMEVEQLKKEVKNPRALISKTGKEIKDYVEAEAGNDPLLKGIPEDKNPFKE-KGGCIIS Bye

B. MAQELSEKELLKMEVEQLKKEVKNPRALISKTGKEIKDYVEAEAGNDPLLKGIPEDKNPFKE-KGGCMIS MPVINIEDLTEKDKLKMEVDQLKKEVTLERMLVSKCCEEVRDYVEERSGEDPLVKGIPEDKNPFKELKGGCVIS MPALHIEDLPEKEKLKMEVEQLRKEVKLQRQQVSKCSEEIKNYIEERSGEDPLVKGIPEDKNPFKE-KGSCVIS

MASNNTASIAQARKLVEQLKMEANIDRIKVSKAAADLMAYCEAHAKEDPLLTPVPASENPFREKKFFCAIL Y2 MKGETPVNSTMSIGQARKMVEQLKIEASLCRIKVSKAAADLMTYCDAHACEDPLITPVPTSENPFREKKFFCALL MKEGMSNNSTTSISQARKAVEQLKMEACMDRVKVSQAAADLLAYCEAHVREDPLIIPVPASENPFREKKFFCTIL

MSGSSSVAAMKKWQQLRLEAGLNRVKVSQAAADLKQFCLQNAQHDPLLTGVSSSTNPFRPQKV-CSFL MSATNNIAQARKLVEQLRIEAGIERIKVSKAASDLMSYCEQHARNDPLLVGVPASENPFKDKKP-CIIL Y7 MSNNMAKIAEARKTVEQLKLEVNIDRMKVSQAAAELLAFCETHAKDDPLVTPVPAAENPFRDKRLFCTLL MSSGASASALQRLVEQLKLEAGVERIKVSQAAAELQQYCMQNACKDALLVGVPAGSNPFREPRS-CALL

FIGURE 5. Amino acid sequence comparison of G-protein 7 subunits. (A) Comparison of human, canine, and bovine transducin-yc (T7C) proteins. (B) Comparison between G-protein y-subunits. Identical amino acid residues are boxed. I and II are the a-y and /3-y interaction regions, respectively14; region III shows the CAAX motif (C, cysteine; A, aliphatic; and X, any amino acid).

members of the first group of Gy proteins, y,, yc and y,,, are reasonable to suggest that the absence of T/33 and Tyc is caused farnesylated. All other Gy proteins are geranylgeranylated. The by defective posttranslational modification of the Ty subunit X amino acid in Tyc of the three species that have been studied (isoprenylation or carboxymethylation of the COOH-terminal). (bovine, human, and canine) is serine. Protein isoprenylation is needed not only to facilitate mem- In this study we found no difference between the primary brane anchoring, but also to mediate protein-protein inter- structure of the Tyc cDNA from normal and affected cd dog actions. Deficiency in the activity of component A of retinas or in their transcription levels. However, we have geranylgeranyl is the cause of in 35 shown the absence of Tyc immunoreactivity in cone outer humans, a type of X-linked retinal degeneration. It will be segments of dogs affected with the cd mutation. A similar loss interesting to analyze the activity of geranylgeranyl transferase of immunoreactivity was observed with Tj33, another cone- in the cd dog retina, to isolate the genes encoding the compo- specific transducin subuntt.24 These abnormalities occur even nents of this and to determine whether defective though cone visual pigments and other proteins associated isoprenylation is implicated in the etiology of the cd dog cone with the rod and cone photoreceptors have normal localization photoreceptor degeneration. and levels of expression determined by immunocytochemis- try.24 Furthermore, we found normal expression of transcripts from middle-wave-sensitive cones in the cd retina. The ab- Acknowledgments

sence of Tj33 and Tyc immunoreactivity in cone outer segments The authors thank Bernard K.-K. Fung for providing the Tyc antibody of the cd retina suggests a potential role for these proteins in and Clyde Yamashita for valuable discussions. the disease process. Since there are no differences in the T/33 and Tyc cDNA sequences from normal and mutant dogs, and the transcripts are normal in abundance and size, a primary References abnormality of either protein can be ruled out as causally 1. Gilman AG. G proteins: transducers of receptor-generated signals. associated with the disease. Expression studies have shown Annu Rev Biochem. 1987;56:6l5-649. that formation of the TjSy dimer is essential for stability and 2. Simon MI, Strathmann MP, Gautam N. Diversity of G proteins in posttranslational modification of the Ty subunit and for mem- . Science, 1991;252:802-808. 34 3. Neer EJ. Heterotrimeric G proteins: organizers of transmembrane brane translocation of the complex. The absence of cytolog- signals. Cell. 1995;80:249-257. ically detectable Tyc immunoreactivity in the cd dog retina 4. Hurley JB, Fong HKW, Teplow DB, Dreyer wj, Simon MI. Isolation leads us to propose that the rapid degradation of Tyc is the and characterization of a cDNA clone for the y subunit of bovine result of the lack of proper cellular localization of Tj33. It is also retinal transducin. Proc Natl Acad Set USA. 1984;81:6948-6952.

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