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of mouse mr-s cDNA\nWe used the bioinformatics method Digital Differential Display (NCBI, UniGene) to screen novel mouse genes expressed preferentially in the retina. Some of the clusters in the UniGene database were mainly from mouse retinal cDNAs. One clone in these clusters (#Mm. 246385) has a weak homology with the polyhomeotic family genes. A 735-bp cDNA fragment of this clone, encoding amino acids 140–384 was amplified by RT-PCR from mouse P0 retinal cDNA. This fragment was used as the probe for library screening, in situ hybridization and Northern hybridization. A mouse P0-P3 retinal cDNA library was screened using this mouse cDNA fragment. Positive bacteriophage clones were isolated and the full-length mr-s fragment was inserted into pBluescriptII (Stratagene). DNA sequencing was performed on both strands by the cycle sequencing method. The nucleotide sequence for mr-s gene has been deposited in the GenBank database under GenBank Accession Number #AY458844.\n\nIn situ hybridization\nThe 735-bp cDNA fragment of mr-s amplified by RT-PCR was used as a probe for in situ hybridization. In situ hybridization was performed as described previously [49].\n\nCell culture and transfection\nHEK293T cells were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Sigma), 100 IU/ml penicillin and 100 μg/ml streptomycin. Transient transfection of HEK293T cells was carried out using calcium phosphate method or Fugene6 transfection reagent (Roche). Y79 retinoblastoma cells were maintained in Iscove's modified Dulbecco's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 20% FBS. Transient transfection was carried out using TransIt LT1 (Mirus).\n\nNorthern-blot analysis\nRNA was extracted from the tissues of adult mice using Trizol (Invitrogen). A 5 μg of total RNA was electrophoresed in a 1.0% agarose-formaldehyde gel and transferred to a nylon membrane (Zeta-Probe GT, Bio-Rad). The 735-bp cDNA fragment encoding the SAM domain of mr-s was used as a probe for hybridization. Hybridization was performed according to the manufacturer's protocol. Washes with increasing stringency were performed, the last being at 50°C in 0.1× standard saline citrate/0.1% sodium dodecyl sulfate (SDS).\n\nRT-PCR analysis\nTotal RNA was isolated from each tissue using Trizol. A 1 μg of total RNA was reverse transcribed using SuperscriptII (Invitrogen). RT-PCR primers, which span introns, for detection of mr-s cDNA were 5'-TGTCCAGCCCAGCCAACCCAAGGAGACGACA-3' and 5'-TGTGGTCTCCTCATCAGTGAAGA-3'. Product size was 292 bp (positions 965–1256 of Genbank Accession Number #AY458844). Primer pairs for mouse G3PDH were 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA- 3' which amplified a 452-bp product (positions 587–1038 of Genbank Accession Number #BC85275).\n\nYeast two-hybrid screening and GAL4 assay\nWe carried out yeast two-hybrid experiments using the MATCHMAKER GAL4 two-hybrid system 3 (BD Bioscience) as recommended by the manufacturer. We cloned the full-length mr-s into the pGBKT7 vector and used it to screen a library of mouse P0-P3 retinal cDNAs in the pGADT7 vector. Transformants that conferred growth were picked, isolated and re-introduced with the bait into AH109 to confirm interaction. Plasmid DNA was isolated from yeast using RPM Yeast Plasmid Isolation Kit (Qbiogene). In order to confirm the protein interactions, single colonies were picked and grown individually in synthetic complete media lacking leucine, tryptophan and containing X-gal. After 4–5 days, X-gal positive clones were selected and analyzed. To test self-interaction of mr-s, we subcloned the full-length, N-terminus (amino acids 1–400) and C-terminus (amino acids 391–542) of mr-s into pGBKT7 and pGADT7 vectors. We assessed interactions by scoring blue color on plates of medium containing X-gal.\n\nImmunoprecipitation assay\nFor immunoprecipitation assay, 4× hemagglutinin (HA) or 3× Flag tagged cDNA fragment encoding full-length mr-s (full-HA and Flag-mrs), a cDNA fragment encoding amino acids 1–400 (ΔSAM-HA and Flag-ΔSAM), and a cDNA fragment encoding amino acids 400–542 (Flag-SAM) were subcloned into the pcDNA3 (Invitrogen) expression vector. We also constructed two site-directed mutants, Flag-W404A and Flag-G453A, using PCR. Each of these mutations was also introduced into Flag-mr-s. We transfected HEK293T cells with 5 μg of plasmid DNA per 6 cm dish by calcium phosphate method. Approximately 48 hr after transfection, cells were harvested in immunoprecipitation buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 2 mM MgCl2, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride [Wako], 1% Nonidet P-40, 10% glycerol) in the presence of protease inhibitor cocktail tablets (Roche). For each reaction, 1 mg of cell lysate was mixed with 0.5 μg of anti-Flag antibody (SIGMA, F3165) and 15 μl of protein G-Sepharose (Amersham) on a rotating wheel at 4°C for 2 hrs. Protein concentration was determined by the BCA protein assay system (Pierce). The beads were then washed three times with immunoprecipitation buffer and followed by three times with wash buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 20 mM MgCl2). Proteins were then boiled for 5 min in SDS sample buffer (1% SDS, 1 mM Tris [pH 6.8], 40 mM DTT, 4% glycerol, 0.01% pyronine Y). The supernatants were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the nitrocellulose membrane (Trans-Blot Transfer Medium, Bio-Rad). Western blotting was performed with rabbit polyclonal anti-HA antibody (Santa Cruz, #sc-805). Signals were detected with horseradish peroxidase- conjugated goat anti-rabbit IgG and ECL plus Western Blotting Detection System (Amersham).\n\nSubcellular localization analysis\nWe transfected the plasmid encoding HA-tagged full-length mr-s into HEK293T cells, seeded cells on coverslips coated with collagen 48 hr after transfection, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 20 min, and washed with PBS. Then we permeabilized cells in PBS containing 0.1% Triton X-100 for 15 min, washed again with PBS, and incubated in PBS containing 0.1% bovine serum albumin (BSA) for 30 min at room temperature. We incubated cells with anti-HA antibody overnight at 4°C (1:200 in PBS containing 0.2% BSA). The following day, we washed cells three times with PBS and incubated with a Cy3-conjugated goat IgG against rabbit IgG (1:400 in PBS, Jackson ImmunoReseach Laboratories) for 30 min. We rinsed cells three times with PBS, followed by observation using a confocal microscope FV300 (Olympus) equipped with a 60× objective lens.\n\nLuciferase assay\nFor transcriptional analysis of the 1.2-kb promoter region of mr-s, we constructed a reporter plasmid (pro1.2k) by subcloning a 1.2-kb upstream genomic fragment of mr-s gene into a pGL3 luciferase reporter plasmid (Promega). The expression vectors were constructed by subcloning full-length Crx, Otx2, Nrl genes into a pMIK expression vector (a gift from Dr. K. Maruyama), respectively. For the \"mut1259\" vector, the Crx binding site at the -1259 bp position was mutated by replacing GGATTA with AGATCT. For the \"mut198\" vector, the Crx binding site at the -198 bp position was mutated by replacing TAATCC with GAATTC. For the \"mut72\" vector, the Crx binding site at the -72 bp position was mutated by replacing GGATTA with GAATTC. For the \"mut all\" vector, all of three Crx binding sites were mutated. 5xGAL4-pGL3 Control reporter plasmid, which contains five copies of the GAL4 DNA recognition sequence positioned immediately upstream of SV40 minimal promoter, was kindly gifted from Dr. T. Noguchi and used for analysis of the transcriptional activity of mr-s [50]. To generate effector plasmids, various deletion fragments were produced by PCR reaction and subcloned into pGBKT7 vector, respectively. These fragments were digested with the sequence, which encodes GAL4 DNA binding domain, and inserted into pcDNA3. We transfected 0.1 μg of reporter plasmid DNA and 2 μg of the expression vector DNA per 6 cm dish into HEK293T cells using Fugene6 transfection reagent. We analyzed luciferase activity 48 hr after transfection."}

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of mouse mr-s cDNA\nWe used the bioinformatics method Digital Differential Display (NCBI, UniGene) to screen novel mouse genes expressed preferentially in the retina. Some of the clusters in the UniGene database were mainly from mouse retinal cDNAs. One clone in these clusters (#Mm. 246385) has a weak homology with the polyhomeotic family genes. A 735-bp cDNA fragment of this clone, encoding amino acids 140–384 was amplified by RT-PCR from mouse P0 retinal cDNA. This fragment was used as the probe for library screening, in situ hybridization and Northern hybridization. A mouse P0-P3 retinal cDNA library was screened using this mouse cDNA fragment. Positive bacteriophage clones were isolated and the full-length mr-s fragment was inserted into pBluescriptII (Stratagene). DNA sequencing was performed on both strands by the cycle sequencing method. The nucleotide sequence for mr-s gene has been deposited in the GenBank database under GenBank Accession Number #AY458844.\n\nIn situ hybridization\nThe 735-bp cDNA fragment of mr-s amplified by RT-PCR was used as a probe for in situ hybridization. In situ hybridization was performed as described previously [49].\n\nCell culture and transfection\nHEK293T cells were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Sigma), 100 IU/ml penicillin and 100 μg/ml streptomycin. Transient transfection of HEK293T cells was carried out using calcium phosphate method or Fugene6 transfection reagent (Roche). Y79 retinoblastoma cells were maintained in Iscove's modified Dulbecco's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 20% FBS. Transient transfection was carried out using TransIt LT1 (Mirus).\n\nNorthern-blot analysis\nRNA was extracted from the tissues of adult mice using Trizol (Invitrogen). A 5 μg of total RNA was electrophoresed in a 1.0% agarose-formaldehyde gel and transferred to a nylon membrane (Zeta-Probe GT, Bio-Rad). The 735-bp cDNA fragment encoding the SAM domain of mr-s was used as a probe for hybridization. Hybridization was performed according to the manufacturer's protocol. Washes with increasing stringency were performed, the last being at 50°C in 0.1× standard saline citrate/0.1% sodium dodecyl sulfate (SDS).\n\nRT-PCR analysis\nTotal RNA was isolated from each tissue using Trizol. A 1 μg of total RNA was reverse transcribed using SuperscriptII (Invitrogen). RT-PCR primers, which span introns, for detection of mr-s cDNA were 5'-TGTCCAGCCCAGCCAACCCAAGGAGACGACA-3' and 5'-TGTGGTCTCCTCATCAGTGAAGA-3'. Product size was 292 bp (positions 965–1256 of Genbank Accession Number #AY458844). Primer pairs for mouse G3PDH were 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA- 3' which amplified a 452-bp product (positions 587–1038 of Genbank Accession Number #BC85275).\n\nYeast two-hybrid screening and GAL4 assay\nWe carried out yeast two-hybrid experiments using the MATCHMAKER GAL4 two-hybrid system 3 (BD Bioscience) as recommended by the manufacturer. We cloned the full-length mr-s into the pGBKT7 vector and used it to screen a library of mouse P0-P3 retinal cDNAs in the pGADT7 vector. Transformants that conferred growth were picked, isolated and re-introduced with the bait into AH109 to confirm interaction. Plasmid DNA was isolated from yeast using RPM Yeast Plasmid Isolation Kit (Qbiogene). In order to confirm the protein interactions, single colonies were picked and grown individually in synthetic complete media lacking leucine, tryptophan and containing X-gal. After 4–5 days, X-gal positive clones were selected and analyzed. To test self-interaction of mr-s, we subcloned the full-length, N-terminus (amino acids 1–400) and C-terminus (amino acids 391–542) of mr-s into pGBKT7 and pGADT7 vectors. We assessed interactions by scoring blue color on plates of medium containing X-gal.\n\nImmunoprecipitation assay\nFor immunoprecipitation assay, 4× hemagglutinin (HA) or 3× Flag tagged cDNA fragment encoding full-length mr-s (full-HA and Flag-mrs), a cDNA fragment encoding amino acids 1–400 (ΔSAM-HA and Flag-ΔSAM), and a cDNA fragment encoding amino acids 400–542 (Flag-SAM) were subcloned into the pcDNA3 (Invitrogen) expression vector. We also constructed two site-directed mutants, Flag-W404A and Flag-G453A, using PCR. Each of these mutations was also introduced into Flag-mr-s. We transfected HEK293T cells with 5 μg of plasmid DNA per 6 cm dish by calcium phosphate method. Approximately 48 hr after transfection, cells were harvested in immunoprecipitation buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 2 mM MgCl2, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride [Wako], 1% Nonidet P-40, 10% glycerol) in the presence of protease inhibitor cocktail tablets (Roche). For each reaction, 1 mg of cell lysate was mixed with 0.5 μg of anti-Flag antibody (SIGMA, F3165) and 15 μl of protein G-Sepharose (Amersham) on a rotating wheel at 4°C for 2 hrs. Protein concentration was determined by the BCA protein assay system (Pierce). The beads were then washed three times with immunoprecipitation buffer and followed by three times with wash buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 20 mM MgCl2). Proteins were then boiled for 5 min in SDS sample buffer (1% SDS, 1 mM Tris [pH 6.8], 40 mM DTT, 4% glycerol, 0.01% pyronine Y). The supernatants were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the nitrocellulose membrane (Trans-Blot Transfer Medium, Bio-Rad). Western blotting was performed with rabbit polyclonal anti-HA antibody (Santa Cruz, #sc-805). Signals were detected with horseradish peroxidase- conjugated goat anti-rabbit IgG and ECL plus Western Blotting Detection System (Amersham).\n\nSubcellular localization analysis\nWe transfected the plasmid encoding HA-tagged full-length mr-s into HEK293T cells, seeded cells on coverslips coated with collagen 48 hr after transfection, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 20 min, and washed with PBS. Then we permeabilized cells in PBS containing 0.1% Triton X-100 for 15 min, washed again with PBS, and incubated in PBS containing 0.1% bovine serum albumin (BSA) for 30 min at room temperature. We incubated cells with anti-HA antibody overnight at 4°C (1:200 in PBS containing 0.2% BSA). The following day, we washed cells three times with PBS and incubated with a Cy3-conjugated goat IgG against rabbit IgG (1:400 in PBS, Jackson ImmunoReseach Laboratories) for 30 min. We rinsed cells three times with PBS, followed by observation using a confocal microscope FV300 (Olympus) equipped with a 60× objective lens.\n\nLuciferase assay\nFor transcriptional analysis of the 1.2-kb promoter region of mr-s, we constructed a reporter plasmid (pro1.2k) by subcloning a 1.2-kb upstream genomic fragment of mr-s gene into a pGL3 luciferase reporter plasmid (Promega). The expression vectors were constructed by subcloning full-length Crx, Otx2, Nrl genes into a pMIK expression vector (a gift from Dr. K. Maruyama), respectively. For the \"mut1259\" vector, the Crx binding site at the -1259 bp position was mutated by replacing GGATTA with AGATCT. For the \"mut198\" vector, the Crx binding site at the -198 bp position was mutated by replacing TAATCC with GAATTC. For the \"mut72\" vector, the Crx binding site at the -72 bp position was mutated by replacing GGATTA with GAATTC. For the \"mut all\" vector, all of three Crx binding sites were mutated. 5xGAL4-pGL3 Control reporter plasmid, which contains five copies of the GAL4 DNA recognition sequence positioned immediately upstream of SV40 minimal promoter, was kindly gifted from Dr. T. Noguchi and used for analysis of the transcriptional activity of mr-s [50]. To generate effector plasmids, various deletion fragments were produced by PCR reaction and subcloned into pGBKT7 vector, respectively. These fragments were digested with the sequence, which encodes GAL4 DNA binding domain, and inserted into pcDNA3. We transfected 0.1 μg of reporter plasmid DNA and 2 μg of the expression vector DNA per 6 cm dish into HEK293T cells using Fugene6 transfection reagent. We analyzed luciferase activity 48 hr after transfection."}

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of mouse mr-s cDNA\nWe used the bioinformatics method Digital Differential Display (NCBI, UniGene) to screen novel mouse genes expressed preferentially in the retina. Some of the clusters in the UniGene database were mainly from mouse retinal cDNAs. One clone in these clusters (#Mm. 246385) has a weak homology with the polyhomeotic family genes. A 735-bp cDNA fragment of this clone, encoding amino acids 140–384 was amplified by RT-PCR from mouse P0 retinal cDNA. This fragment was used as the probe for library screening, in situ hybridization and Northern hybridization. A mouse P0-P3 retinal cDNA library was screened using this mouse cDNA fragment. Positive bacteriophage clones were isolated and the full-length mr-s fragment was inserted into pBluescriptII (Stratagene). DNA sequencing was performed on both strands by the cycle sequencing method. The nucleotide sequence for mr-s gene has been deposited in the GenBank database under GenBank Accession Number #AY458844.\n\nIn situ hybridization\nThe 735-bp cDNA fragment of mr-s amplified by RT-PCR was used as a probe for in situ hybridization. In situ hybridization was performed as described previously [49].\n\nCell culture and transfection\nHEK293T cells were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Sigma), 100 IU/ml penicillin and 100 μg/ml streptomycin. Transient transfection of HEK293T cells was carried out using calcium phosphate method or Fugene6 transfection reagent (Roche). Y79 retinoblastoma cells were maintained in Iscove's modified Dulbecco's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 20% FBS. Transient transfection was carried out using TransIt LT1 (Mirus).\n\nNorthern-blot analysis\nRNA was extracted from the tissues of adult mice using Trizol (Invitrogen). A 5 μg of total RNA was electrophoresed in a 1.0% agarose-formaldehyde gel and transferred to a nylon membrane (Zeta-Probe GT, Bio-Rad). The 735-bp cDNA fragment encoding the SAM domain of mr-s was used as a probe for hybridization. Hybridization was performed according to the manufacturer's protocol. Washes with increasing stringency were performed, the last being at 50°C in 0.1× standard saline citrate/0.1% sodium dodecyl sulfate (SDS).\n\nRT-PCR analysis\nTotal RNA was isolated from each tissue using Trizol. A 1 μg of total RNA was reverse transcribed using SuperscriptII (Invitrogen). RT-PCR primers, which span introns, for detection of mr-s cDNA were 5'-TGTCCAGCCCAGCCAACCCAAGGAGACGACA-3' and 5'-TGTGGTCTCCTCATCAGTGAAGA-3'. Product size was 292 bp (positions 965–1256 of Genbank Accession Number #AY458844). Primer pairs for mouse G3PDH were 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA- 3' which amplified a 452-bp product (positions 587–1038 of Genbank Accession Number #BC85275).\n\nYeast two-hybrid screening and GAL4 assay\nWe carried out yeast two-hybrid experiments using the MATCHMAKER GAL4 two-hybrid system 3 (BD Bioscience) as recommended by the manufacturer. We cloned the full-length mr-s into the pGBKT7 vector and used it to screen a library of mouse P0-P3 retinal cDNAs in the pGADT7 vector. Transformants that conferred growth were picked, isolated and re-introduced with the bait into AH109 to confirm interaction. Plasmid DNA was isolated from yeast using RPM Yeast Plasmid Isolation Kit (Qbiogene). In order to confirm the protein interactions, single colonies were picked and grown individually in synthetic complete media lacking leucine, tryptophan and containing X-gal. After 4–5 days, X-gal positive clones were selected and analyzed. To test self-interaction of mr-s, we subcloned the full-length, N-terminus (amino acids 1–400) and C-terminus (amino acids 391–542) of mr-s into pGBKT7 and pGADT7 vectors. We assessed interactions by scoring blue color on plates of medium containing X-gal.\n\nImmunoprecipitation assay\nFor immunoprecipitation assay, 4× hemagglutinin (HA) or 3× Flag tagged cDNA fragment encoding full-length mr-s (full-HA and Flag-mrs), a cDNA fragment encoding amino acids 1–400 (ΔSAM-HA and Flag-ΔSAM), and a cDNA fragment encoding amino acids 400–542 (Flag-SAM) were subcloned into the pcDNA3 (Invitrogen) expression vector. We also constructed two site-directed mutants, Flag-W404A and Flag-G453A, using PCR. Each of these mutations was also introduced into Flag-mr-s. We transfected HEK293T cells with 5 μg of plasmid DNA per 6 cm dish by calcium phosphate method. Approximately 48 hr after transfection, cells were harvested in immunoprecipitation buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 2 mM MgCl2, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride [Wako], 1% Nonidet P-40, 10% glycerol) in the presence of protease inhibitor cocktail tablets (Roche). For each reaction, 1 mg of cell lysate was mixed with 0.5 μg of anti-Flag antibody (SIGMA, F3165) and 15 μl of protein G-Sepharose (Amersham) on a rotating wheel at 4°C for 2 hrs. Protein concentration was determined by the BCA protein assay system (Pierce). The beads were then washed three times with immunoprecipitation buffer and followed by three times with wash buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 20 mM MgCl2). Proteins were then boiled for 5 min in SDS sample buffer (1% SDS, 1 mM Tris [pH 6.8], 40 mM DTT, 4% glycerol, 0.01% pyronine Y). The supernatants were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the nitrocellulose membrane (Trans-Blot Transfer Medium, Bio-Rad). Western blotting was performed with rabbit polyclonal anti-HA antibody (Santa Cruz, #sc-805). Signals were detected with horseradish peroxidase- conjugated goat anti-rabbit IgG and ECL plus Western Blotting Detection System (Amersham).\n\nSubcellular localization analysis\nWe transfected the plasmid encoding HA-tagged full-length mr-s into HEK293T cells, seeded cells on coverslips coated with collagen 48 hr after transfection, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 20 min, and washed with PBS. Then we permeabilized cells in PBS containing 0.1% Triton X-100 for 15 min, washed again with PBS, and incubated in PBS containing 0.1% bovine serum albumin (BSA) for 30 min at room temperature. We incubated cells with anti-HA antibody overnight at 4°C (1:200 in PBS containing 0.2% BSA). The following day, we washed cells three times with PBS and incubated with a Cy3-conjugated goat IgG against rabbit IgG (1:400 in PBS, Jackson ImmunoReseach Laboratories) for 30 min. We rinsed cells three times with PBS, followed by observation using a confocal microscope FV300 (Olympus) equipped with a 60× objective lens.\n\nLuciferase assay\nFor transcriptional analysis of the 1.2-kb promoter region of mr-s, we constructed a reporter plasmid (pro1.2k) by subcloning a 1.2-kb upstream genomic fragment of mr-s gene into a pGL3 luciferase reporter plasmid (Promega). The expression vectors were constructed by subcloning full-length Crx, Otx2, Nrl genes into a pMIK expression vector (a gift from Dr. K. Maruyama), respectively. For the \"mut1259\" vector, the Crx binding site at the -1259 bp position was mutated by replacing GGATTA with AGATCT. For the \"mut198\" vector, the Crx binding site at the -198 bp position was mutated by replacing TAATCC with GAATTC. For the \"mut72\" vector, the Crx binding site at the -72 bp position was mutated by replacing GGATTA with GAATTC. For the \"mut all\" vector, all of three Crx binding sites were mutated. 5xGAL4-pGL3 Control reporter plasmid, which contains five copies of the GAL4 DNA recognition sequence positioned immediately upstream of SV40 minimal promoter, was kindly gifted from Dr. T. Noguchi and used for analysis of the transcriptional activity of mr-s [50]. To generate effector plasmids, various deletion fragments were produced by PCR reaction and subcloned into pGBKT7 vector, respectively. These fragments were digested with the sequence, which encodes GAL4 DNA binding domain, and inserted into pcDNA3. We transfected 0.1 μg of reporter plasmid DNA and 2 μg of the expression vector DNA per 6 cm dish into HEK293T cells using Fugene6 transfection reagent. We analyzed luciferase activity 48 hr after transfection."}