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Volumn 279, Issue 5348, 1998, Pages 237-242

Functional expression of a mammalian odorant receptor

Author keywords

[No Author keywords available]

Indexed keywords

ADENOVIRUS; ANIMAL CELL; ARTICLE; ELECTROOCULOGRAM; MAMMAL; NONHUMAN; ODOR; OLFACTORY EPITHELIUM; PRIORITY JOURNAL; SIGNAL TRANSDUCTION;

EID: 0032498150     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5348.237     Document Type: Article
Times cited : (530)

References (43)
  • 1
    • 0028373297 scopus 로고
    • H. Breer, Semin. Cell Biol. 5, 25 (1994); G. M. Shepherd, Neuron 13, 771 (1994).
    • (1994) Semin. Cell Biol. , vol.5 , pp. 25
    • Breer, H.1
  • 2
    • 0027945342 scopus 로고
    • H. Breer, Semin. Cell Biol. 5, 25 (1994); G. M. Shepherd, Neuron 13, 771 (1994).
    • (1994) Neuron , vol.13 , pp. 771
    • Shepherd, G.M.1
  • 5
    • 0027154580 scopus 로고
    • We assume here from previous in situ hybridization studies that each of the approximately 1000 receptors is expressed in roughly 0.1% of the total olfactory neurons [K. J. Ressler, S. L. Sullivan, L. B. Buck, Cell 73, 597 (1993); R. Vassar, J. Ngai, R. Axel, ibid. 74, 309 (1993)], so that if expression of one particular receptor could be induced in as few as 1 to 10% of the neurons, the additional response attributable to activation of that receptor by its particular ligands would be relatively easy to detect.
    • (1993) Cell , vol.73 , pp. 597
    • Ressler, K.J.1    Sullivan, S.L.2    Buck, L.B.3
  • 6
    • 0027328684 scopus 로고
    • We assume here from previous in situ hybridization studies that each of the approximately 1000 receptors is expressed in roughly 0.1% of the total olfactory neurons [K. J. Ressler, S. L. Sullivan, L. B. Buck, Cell 73, 597 (1993); R. Vassar, J. Ngai, R. Axel, ibid. 74, 309 (1993)], so that if expression of one particular receptor could be induced in as few as 1 to 10% of the neurons, the additional response attributable to activation of that receptor by its particular ligands would be relatively easy to detect.
    • (1993) Cell , vol.74 , pp. 309
    • Vassar, R.1    Ngai, J.2    Axel, R.3
  • 7
    • 0002256791 scopus 로고
    • D. Ottoson, Acta Physiol. Scand. 35, 1 (1956); Handbook of Sensory Physiology (Olfaction) (Springer-Verlag, Berlin, 1971), vol. 4, pp. 95-131; A. Mackay-Sim and S. Kesteven, J. Neurophysiol. 71, 150 (1994).
    • (1956) Acta Physiol. Scand. , vol.35 , pp. 1
    • Ottoson, D.1
  • 8
    • 0002289611 scopus 로고
    • Springer-Verlag, Berlin
    • D. Ottoson, Acta Physiol. Scand. 35, 1 (1956); Handbook of Sensory Physiology (Olfaction) (Springer-Verlag, Berlin, 1971), vol. 4, pp. 95-131; A. Mackay-Sim and S. Kesteven, J. Neurophysiol. 71, 150 (1994).
    • (1971) Handbook of Sensory Physiology (Olfaction) , vol.4 , pp. 95-131
  • 9
    • 0027983004 scopus 로고
    • D. Ottoson, Acta Physiol. Scand. 35, 1 (1956); Handbook of Sensory Physiology (Olfaction) (Springer-Verlag, Berlin, 1971), vol. 4, pp. 95-131; A. Mackay-Sim and S. Kesteven, J. Neurophysiol. 71, 150 (1994).
    • (1994) J. Neurophysiol. , vol.71 , pp. 150
    • Mackay-Sim, A.1    Kesteven, S.2
  • 10
    • 0002025785 scopus 로고
    • F. L. Graham and L. Prevec, Methods Mol. Biol. 7, 109 (1991); T. C. Becker et al., in Protein Expression in Animal Cells, M. G. Roth, Ed. (Academic Press, San Diego, CA, 1994), pp. 162-189; M. A. Rosenfeld et al., Cell 68, 143 (1992); J. Zabner et al., ibid. 75, 207 (1993); G. Le Gal Le Salle et al., Science 259, 988 (1993); K. Moriyoshi, L. J. Richards, C. Akazawa, D. D. M. O'Leary, S. Nakanishi, Neuron 16, 255 (1996); A. J. G. D. Holtmaat et al., Mol. Brain Res. 41, 148 (1996).
    • (1991) Methods Mol. Biol. , vol.7 , pp. 109
    • Graham, F.L.1    Prevec, L.2
  • 11
    • 0001095874 scopus 로고
    • M. G. Roth, Ed. Academic Press, San Diego, CA
    • F. L. Graham and L. Prevec, Methods Mol. Biol. 7, 109 (1991); T. C. Becker et al., in Protein Expression in Animal Cells, M. G. Roth, Ed. (Academic Press, San Diego, CA, 1994), pp. 162-189; M. A. Rosenfeld et al., Cell 68, 143 (1992); J. Zabner et al., ibid. 75, 207 (1993); G. Le Gal Le Salle et al., Science 259, 988 (1993); K. Moriyoshi, L. J. Richards, C. Akazawa, D. D. M. O'Leary, S. Nakanishi, Neuron 16, 255 (1996); A. J. G. D. Holtmaat et al., Mol. Brain Res. 41, 148 (1996).
    • (1994) Protein Expression in Animal Cells , pp. 162-189
    • Becker, T.C.1
  • 12
    • 0026558496 scopus 로고
    • F. L. Graham and L. Prevec, Methods Mol. Biol. 7, 109 (1991); T. C. Becker et al., in Protein Expression in Animal Cells, M. G. Roth, Ed. (Academic Press, San Diego, CA, 1994), pp. 162-189; M. A. Rosenfeld et al., Cell 68, 143 (1992); J. Zabner et al., ibid. 75, 207 (1993); G. Le Gal Le Salle et al., Science 259, 988 (1993); K. Moriyoshi, L. J. Richards, C. Akazawa, D. D. M. O'Leary, S. Nakanishi, Neuron 16, 255 (1996); A. J. G. D. Holtmaat et al., Mol. Brain Res. 41, 148 (1996).
    • (1992) Cell , vol.68 , pp. 143
    • Rosenfeld, M.A.1
  • 13
    • 0027377523 scopus 로고
    • F. L. Graham and L. Prevec, Methods Mol. Biol. 7, 109 (1991); T. C. Becker et al., in Protein Expression in Animal Cells, M. G. Roth, Ed. (Academic Press, San Diego, CA, 1994), pp. 162-189; M. A. Rosenfeld et al., Cell 68, 143 (1992); J. Zabner et al., ibid. 75, 207 (1993); G. Le Gal Le Salle et al., Science 259, 988 (1993); K. Moriyoshi, L. J. Richards, C. Akazawa, D. D. M. O'Leary, S. Nakanishi, Neuron 16, 255 (1996); A. J. G. D. Holtmaat et al., Mol. Brain Res. 41, 148 (1996).
    • (1993) Cell , vol.75 , pp. 207
    • Zabner, J.1
  • 14
    • 0027457561 scopus 로고
    • F. L. Graham and L. Prevec, Methods Mol. Biol. 7, 109 (1991); T. C. Becker et al., in Protein Expression in Animal Cells, M. G. Roth, Ed. (Academic Press, San Diego, CA, 1994), pp. 162-189; M. A. Rosenfeld et al., Cell 68, 143 (1992); J. Zabner et al., ibid. 75, 207 (1993); G. Le Gal Le Salle et al., Science 259, 988 (1993); K. Moriyoshi, L. J. Richards, C. Akazawa, D. D. M. O'Leary, S. Nakanishi, Neuron 16, 255 (1996); A. J. G. D. Holtmaat et al., Mol. Brain Res. 41, 148 (1996).
    • (1993) Science , vol.259 , pp. 988
    • Le Gal Le Salle, G.1
  • 15
    • 0030048198 scopus 로고    scopus 로고
    • F. L. Graham and L. Prevec, Methods Mol. Biol. 7, 109 (1991); T. C. Becker et al., in Protein Expression in Animal Cells, M. G. Roth, Ed. (Academic Press, San Diego, CA, 1994), pp. 162-189; M. A. Rosenfeld et al., Cell 68, 143 (1992); J. Zabner et al., ibid. 75, 207 (1993); G. Le Gal Le Salle et al., Science 259, 988 (1993); K. Moriyoshi, L. J. Richards, C. Akazawa, D. D. M. O'Leary, S. Nakanishi, Neuron 16, 255 (1996); A. J. G. D. Holtmaat et al., Mol. Brain Res. 41, 148 (1996).
    • (1996) Neuron , vol.16 , pp. 255
    • Moriyoshi, K.1    Richards, L.J.2    Akazawa, C.3    O'Leary, D.D.M.4    Nakanishi, S.5
  • 16
    • 0030571042 scopus 로고    scopus 로고
    • F. L. Graham and L. Prevec, Methods Mol. Biol. 7, 109 (1991); T. C. Becker et al., in Protein Expression in Animal Cells, M. G. Roth, Ed. (Academic Press, San Diego, CA, 1994), pp. 162-189; M. A. Rosenfeld et al., Cell 68, 143 (1992); J. Zabner et al., ibid. 75, 207 (1993); G. Le Gal Le Salle et al., Science 259, 988 (1993); K. Moriyoshi, L. J. Richards, C. Akazawa, D. D. M. O'Leary, S. Nakanishi, Neuron 16, 255 (1996); A. J. G. D. Holtmaat et al., Mol. Brain Res. 41, 148 (1996).
    • (1996) Mol. Brain Res. , vol.41 , pp. 148
    • Holtmaat, A.J.G.D.1
  • 17
    • 0028799961 scopus 로고
    • The adenoviral vector AdexCAG-I7-IRES-GFP (Ad-I7) was constructed in the following manner. Adenoviruses lacking the E1 region of their genome are replication-incompetent and are grown in the complementary human embryonic kidney (HEK) 293 cell line, which provides the E1 genes in trans. The entire coding sequence for the rat odorant receptor I7 (2) was amplified by the polymerase chain reaction (PCR) using Pfu DNA polymerase (Stratagene) from the I7 clone plasmid with the upstream primer 5′-CCCTCGAGTATGGAGCGAAGGAACCAC-3′ and the downstream primer 5′-GCTCTAGACTAACCAATTTTGCTGCCT-3′. The 0.6-kb IRES (9) fragment was cut with Eco RI and Bam HI from plasmid p1162. The fragments of I7, IRES, and the S65T mutant of GFP were first conjugated in the multicloning sites of the expression vector pCA4 (Microbix, Ontario, Canada) and tested by Northern blot with a I7 probe for transcription of mRNA, and by green fluorescence for IRES-driven GFP expression in HEK 293 cells (ATCC, CRL-1573). The I7-IRES-GFP sequence was then subcloned into the Swa I site of the cosmid vector pAdex1pCAw (26) [Y. Kanegae et al., Nucleic Acids Res. 23, 3816 (1995)] to create the cosmid vector pAdexI7-IRES-GFP. The pAdex1pCAw cosmid was created from the human adenovirus type 5 genome from which the E1a, E1b, and E3 regions were deleted and replaced with an expression unit containing the CAG promoter, composed of the cytomegalovirus enhancer plus the chicken β-actin promoter [H. Niwa, K. Yamamura, J. Miyazaki, Gene 108, 193 (1991)], a Swa I site, and the rabbit β-globin polyadenylation signal. The I7 sequence was confirmed by sequencing. Nucleotide 104 in the I7 sequence from GenBank (accession number M64386) is undefined. This nucleotide is deoxycytidine according to our sequence analysis; therefore, amino acid 35 in the deduced protein sequence is alanine. The cosmid vector pAdexI7-IRES-GFP and the Eco T22I-digested DNA-terminal protein complex (DNA-TPC) of Ad5-dIX, which is a human type 5 adenovirus lacking the E3 region, were cotransfected into HEK293 cells by calcium phosphate precipitation. The recombinant adenovirus AdexCAG-I7-IRES-GFP was then generated by homologous recombination in the HEK 293 cells. The DNA-TPC method has been described in detail (26) [S. Miyake et al., Proc. Natl. Acad. Sci. U.S.A. 93, 1320 (1996)]. Bam HI and Xba I digestion of the genomic DNA of AdexI7-IRES-GFP produced the appropriate band pattern, and positive PCR amplification of I7 also verified the construct. Because recombinant viruses do not include the E1a genes, PCR amplification of the E1a region was performed with the primers 5′-ATTACCGAAGAAATGGCCGC-3′ and 5′-CCCATTTAACACACGCCATGCA-3′, as a control for contamination by wild-type adenovirus (Ad5-dIX). Negative PCR amplification of the E1a gene was observed in every stock of recombinant adenovirus. The recombinant adenovirus was propagated in HEK 293 cells and purified by cesium gradient centrifugation [Y. Kanegae, M. Makimura, I. Saito, Jpn. J. Med. Sci. Biol. 47, 157 (1994)]. The viral titer was determined by plaque-forming assay on HEK 293 cells.
    • (1995) Nucleic Acids Res. , vol.23 , pp. 3816
    • Kanegae, Y.1
  • 18
    • 0025884056 scopus 로고
    • The adenoviral vector AdexCAG-I7-IRES-GFP (Ad-I7) was constructed in the following manner. Adenoviruses lacking the E1 region of their genome are replication-incompetent and are grown in the complementary human embryonic kidney (HEK) 293 cell line, which provides the E1 genes in trans. The entire coding sequence for the rat odorant receptor I7 (2) was amplified by the polymerase chain reaction (PCR) using Pfu DNA polymerase (Stratagene) from the I7 clone plasmid with the upstream primer 5′-CCCTCGAGTATGGAGCGAAGGAACCAC-3′ and the downstream primer 5′-GCTCTAGACTAACCAATTTTGCTGCCT-3′. The 0.6-kb IRES (9) fragment was cut with Eco RI and Bam HI from plasmid p1162. The fragments of I7, IRES, and the S65T mutant of GFP were first conjugated in the multicloning sites of the expression vector pCA4 (Microbix, Ontario, Canada) and tested by Northern blot with a I7 probe for transcription of mRNA, and by green fluorescence for IRES-driven GFP expression in HEK 293 cells (ATCC, CRL-1573). The I7-IRES-GFP sequence was then subcloned into the Swa I site of the cosmid vector pAdex1pCAw (26) [Y. Kanegae et al., Nucleic Acids Res. 23, 3816 (1995)] to create the cosmid vector pAdexI7-IRES-GFP. The pAdex1pCAw cosmid was created from the human adenovirus type 5 genome from which the E1a, E1b, and E3 regions were deleted and replaced with an expression unit containing the CAG promoter, composed of the cytomegalovirus enhancer plus the chicken β-actin promoter [H. Niwa, K. Yamamura, J. Miyazaki, Gene 108, 193 (1991)], a Swa I site, and the rabbit β-globin polyadenylation signal. The I7 sequence was confirmed by sequencing. Nucleotide 104 in the I7 sequence from GenBank (accession number M64386) is undefined. This nucleotide is deoxycytidine according to our sequence analysis; therefore, amino acid 35 in the deduced protein sequence is alanine. The cosmid vector pAdexI7-IRES-GFP and the Eco T22I-digested DNA-terminal protein complex (DNA-TPC) of Ad5-dIX, which is a human type 5 adenovirus lacking the E3 region, were cotransfected into HEK293 cells by calcium phosphate precipitation. The recombinant adenovirus AdexCAG-I7-IRES-GFP was then generated by homologous recombination in the HEK 293 cells. The DNA-TPC method has been described in detail (26) [S. Miyake et al., Proc. Natl. Acad. Sci. U.S.A. 93, 1320 (1996)]. Bam HI and Xba I digestion of the genomic DNA of AdexI7-IRES-GFP produced the appropriate band pattern, and positive PCR amplification of I7 also verified the construct. Because recombinant viruses do not include the E1a genes, PCR amplification of the E1a region was performed with the primers 5′-ATTACCGAAGAAATGGCCGC-3′ and 5′-CCCATTTAACACACGCCATGCA-3′, as a control for contamination by wild-type adenovirus (Ad5-dIX). Negative PCR amplification of the E1a gene was observed in every stock of recombinant adenovirus. The recombinant adenovirus was propagated in HEK 293 cells and purified by cesium gradient centrifugation [Y. Kanegae, M. Makimura, I. Saito, Jpn. J. Med. Sci. Biol. 47, 157 (1994)]. The viral titer was determined by plaque-forming assay on HEK 293 cells.
    • (1991) Gene , vol.108 , pp. 193
    • Niwa, H.1    Yamamura, K.2    Miyazaki, J.3
  • 19
    • 0030070724 scopus 로고    scopus 로고
    • The adenoviral vector AdexCAG-I7-IRES-GFP (Ad-I7) was constructed in the following manner. Adenoviruses lacking the E1 region of their genome are replication-incompetent and are grown in the complementary human embryonic kidney (HEK) 293 cell line, which provides the E1 genes in trans. The entire coding sequence for the rat odorant receptor I7 (2) was amplified by the polymerase chain reaction (PCR) using Pfu DNA polymerase (Stratagene) from the I7 clone plasmid with the upstream primer 5′-CCCTCGAGTATGGAGCGAAGGAACCAC-3′ and the downstream primer 5′-GCTCTAGACTAACCAATTTTGCTGCCT-3′. The 0.6-kb IRES (9) fragment was cut with Eco RI and Bam HI from plasmid p1162. The fragments of I7, IRES, and the S65T mutant of GFP were first conjugated in the multicloning sites of the expression vector pCA4 (Microbix, Ontario, Canada) and tested by Northern blot with a I7 probe for transcription of mRNA, and by green fluorescence for IRES-driven GFP expression in HEK 293 cells (ATCC, CRL-1573). The I7-IRES-GFP sequence was then subcloned into the Swa I site of the cosmid vector pAdex1pCAw (26) [Y. Kanegae et al., Nucleic Acids Res. 23, 3816 (1995)] to create the cosmid vector pAdexI7-IRES-GFP. The pAdex1pCAw cosmid was created from the human adenovirus type 5 genome from which the E1a, E1b, and E3 regions were deleted and replaced with an expression unit containing the CAG promoter, composed of the cytomegalovirus enhancer plus the chicken β-actin promoter [H. Niwa, K. Yamamura, J. Miyazaki, Gene 108, 193 (1991)], a Swa I site, and the rabbit β-globin polyadenylation signal. The I7 sequence was confirmed by sequencing. Nucleotide 104 in the I7 sequence from GenBank (accession number M64386) is undefined. This nucleotide is deoxycytidine according to our sequence analysis; therefore, amino acid 35 in the deduced protein sequence is alanine. The cosmid vector pAdexI7-IRES-GFP and the Eco T22I-digested DNA-terminal protein complex (DNA-TPC) of Ad5-dIX, which is a human type 5 adenovirus lacking the E3 region, were cotransfected into HEK293 cells by calcium phosphate precipitation. The recombinant adenovirus AdexCAG-I7-IRES-GFP was then generated by homologous recombination in the HEK 293 cells. The DNA-TPC method has been described in detail (26) [S. Miyake et al., Proc. Natl. Acad. Sci. U.S.A. 93, 1320 (1996)]. Bam HI and Xba I digestion of the genomic DNA of AdexI7-IRES-GFP produced the appropriate band pattern, and positive PCR amplification of I7 also verified the construct. Because recombinant viruses do not include the E1a genes, PCR amplification of the E1a region was performed with the primers 5′-ATTACCGAAGAAATGGCCGC-3′ and 5′-CCCATTTAACACACGCCATGCA-3′, as a control for contamination by wild-type adenovirus (Ad5-dIX). Negative PCR amplification of the E1a gene was observed in every stock of recombinant adenovirus. The recombinant adenovirus was propagated in HEK 293 cells and purified by cesium gradient centrifugation [Y. Kanegae, M. Makimura, I. Saito, Jpn. J. Med. Sci. Biol. 47, 157 (1994)]. The viral titer was determined by plaque-forming assay on HEK 293 cells.
    • (1996) Proc. Natl. Acad. Sci. U.S.A. , vol.93 , pp. 1320
    • Miyake, S.1
  • 20
    • 0028037530 scopus 로고
    • The adenoviral vector AdexCAG-I7-IRES-GFP (Ad-I7) was constructed in the following manner. Adenoviruses lacking the E1 region of their genome are replication-incompetent and are grown in the complementary human embryonic kidney (HEK) 293 cell line, which provides the E1 genes in trans. The entire coding sequence for the rat odorant receptor I7 (2) was amplified by the polymerase chain reaction (PCR) using Pfu DNA polymerase (Stratagene) from the I7 clone plasmid with the upstream primer 5′-CCCTCGAGTATGGAGCGAAGGAACCAC-3′ and the downstream primer 5′-GCTCTAGACTAACCAATTTTGCTGCCT-3′. The 0.6-kb IRES (9) fragment was cut with Eco RI and Bam HI from plasmid p1162. The fragments of I7, IRES, and the S65T mutant of GFP were first conjugated in the multicloning sites of the expression vector pCA4 (Microbix, Ontario, Canada) and tested by Northern blot with a I7 probe for transcription of mRNA, and by green fluorescence for IRES-driven GFP expression in HEK 293 cells (ATCC, CRL-1573). The I7-IRES-GFP sequence was then subcloned into the Swa I site of the cosmid vector pAdex1pCAw (26) [Y. Kanegae et al., Nucleic Acids Res. 23, 3816 (1995)] to create the cosmid vector pAdexI7-IRES-GFP. The pAdex1pCAw cosmid was created from the human adenovirus type 5 genome from which the E1a, E1b, and E3 regions were deleted and replaced with an expression unit containing the CAG promoter, composed of the cytomegalovirus enhancer plus the chicken β-actin promoter [H. Niwa, K. Yamamura, J. Miyazaki, Gene 108, 193 (1991)], a Swa I site, and the rabbit β-globin polyadenylation signal. The I7 sequence was confirmed by sequencing. Nucleotide 104 in the I7 sequence from GenBank (accession number M64386) is undefined. This nucleotide is deoxycytidine according to our sequence analysis; therefore, amino acid 35 in the deduced protein sequence is alanine. The cosmid vector pAdexI7-IRES-GFP and the Eco T22I-digested DNA-terminal protein complex (DNA-TPC) of Ad5-dIX, which is a human type 5 adenovirus lacking the E3 region, were cotransfected into HEK293 cells by calcium phosphate precipitation. The recombinant adenovirus AdexCAG-I7-IRES-GFP was then generated by homologous recombination in the HEK 293 cells. The DNA-TPC method has been described in detail (26) [S. Miyake et al., Proc. Natl. Acad. Sci. U.S.A. 93, 1320 (1996)]. Bam HI and Xba I digestion of the genomic DNA of AdexI7-IRES-GFP produced the appropriate band pattern, and positive PCR amplification of I7 also verified the construct. Because recombinant viruses do not include the E1a genes, PCR amplification of the E1a region was performed with the primers 5′-ATTACCGAAGAAATGGCCGC-3′ and 5′-CCCATTTAACACACGCCATGCA-3′, as a control for contamination by wild-type adenovirus (Ad5-dIX). Negative PCR amplification of the E1a gene was observed in every stock of recombinant adenovirus. The recombinant adenovirus was propagated in HEK 293 cells and purified by cesium gradient centrifugation [Y. Kanegae, M. Makimura, I. Saito, Jpn. J. Med. Sci. Biol. 47, 157 (1994)]. The viral titer was determined by plaque-forming assay on HEK 293 cells.
    • (1994) Jpn. J. Med. Sci. Biol. , vol.47 , pp. 157
    • Kanegae, Y.1    Makimura, M.2    Saito, I.3
  • 21
    • 0000557447 scopus 로고    scopus 로고
    • B. N. Fields et al., Eds. Lippincott-Raven, Philadelphia
    • The production of other viral proteins is also hindered because the E1 genes provide an essential function as transcriptional activators [T. Shenk, in Fields Virology, B. N. Fields et al., Eds. (Lippincott-Raven, Philadelphia, 1996), vol. 2, pp. 2111-2138]. In an earlier study using an adenovirus vector containing the lacZ marker gene, we observed strong but heterogeneous viral infection and protein expression in rat nasal epithelium, and no virally induced cell loss out to 21 days after infection [H. Zhao, J. M. Otaki, S. Firestein, J. Neurobiol. 30, 521 (1996)].
    • (1996) Fields Virology , vol.2 , pp. 2111-2138
    • Shenk, T.1
  • 22
    • 0030017352 scopus 로고    scopus 로고
    • The production of other viral proteins is also hindered because the E1 genes provide an essential function as transcriptional activators [T. Shenk, in Fields Virology, B. N. Fields et al., Eds. (Lippincott-Raven, Philadelphia, 1996), vol. 2, pp. 2111-2138]. In an earlier study using an adenovirus vector containing the lacZ marker gene, we observed strong but heterogeneous viral infection and protein expression in rat nasal epithelium, and no virally induced cell loss out to 21 days after infection [H. Zhao, J. M. Otaki, S. Firestein, J. Neurobiol. 30, 521 (1996)].
    • (1996) J. Neurobiol. , vol.30 , pp. 521
    • Zhao, H.1    Otaki, J.M.2    Firestein, S.3
  • 24
    • 6844227448 scopus 로고    scopus 로고
    • note
    • 9 pfu/ml and 0.3% fast green dye was slowly injected through the nostril into the right side of the nasal cavity with a thin plastic tubing. The solution was allowed to remain in the nasal cavity. After recovery, the animals were maintained at room temperature with no other treatment until they were killed.
  • 25
    • 0004136246 scopus 로고
    • Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
    • Northern blot for detection of I7 mRNA was performed using a standard procedure [J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)]. Total RNAs were extracted from tissues with TRlozol reagent (Gibco-BRL); 20 μg of total RNA was loaded on each lane of the gel. The I7 probe was synthesized by PCR with primers that covered the entire I7 coding sequence and was labeled with digoxigenin (DIG-11-dUTP, Boehringer Mannheim) according to the manufacturer's protocol. After hybridization, the probe was detected with the DIG Nucleic Acid Detection Kit (Boehringer Mannheim).
    • (1989) Molecular Cloning: A Laboratory Manual
    • Sambrook, J.1    Fritsch, E.F.2    Maniatis, T.3
  • 27
    • 6844238223 scopus 로고    scopus 로고
    • note
    • The olfactory turbinates were dissected out, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 2 hours, and cryoprotected in 20% sucrose. Cryostat sections (15 μm) were cut and incubated with polyclonal antibody to GFP (Clontech). Specific staining was then visualized with the Vectastain Elite ABC kit (Vector Lab).
  • 28
    • 6844230226 scopus 로고    scopus 로고
    • note
    • A panel of 74 odors were screened. They included odors from several classes and groups: Aromatics Alcohols Cinnamyl alcohol, eugenol, guaiacol Aldehydes para-Anisaldehyde, (-)-carvone, cinnamaldehyde, salicylaldehyde, lilial Esters Cinnamylformate, geranyl acetate, isoamyl salicylate, linalyl formate Ethers Anisole, cineole, 2-methylanisole, 4-methylanisole, isoeugenol, methyl eugenol Heterocycles 2-Isobutyl-3-methoxypyrazin Hydrocarbons 2-Ethyltoluene, 3-ethyltoluene, 1,2-diethylbenzene, limonen Ketones Acetophenone, 2-decalone Aliphatics Alcohols n-Propyl alcohol, n-butyl alcohol, n-pentyl alcohol, n-hexyl alcohol, n-heptyl alcohol, n-octyl alcohol, n-nonyl alcohol, n-decyl alcohol, 2-ethylfenchol, geraniol, β-citronellol, linalool Aldehydes Propion aldehyde, isobutyraldehyde, n-valeraldehyde, n-hexaldehyde, n-heptaldehyde, n-octyl aldehyde, n-nonyl aldehyde, n-decyl aldehyde, undecylic aldehyde, dodecyl aldehyde, frans-2-octenal, 2-octynal, trans-2-tridecanal, citral, lyral Acids Propionic acid, n-valeric acid, n-octanoic acid, n-nonanoic acid Alkanes n-Octane, n-nonane, n-decane Amines Isopentylamine, phenethylamine Esters Amyl acetate, ethyl butyrate, ethyl hexanoate, isoamyl acetate, octyl butyrate, octyl isovalerate Ethers Citral diethyl acetal, citral dimethyl acetal Ketones 2,3-Butanedione, 1-fenchone, 2-nonanone Other Heptyl cyanide, 1,1,3,3-tetramethylbutyl isocyanide
  • 29
    • 6844230225 scopus 로고    scopus 로고
    • note
    • 3 airspace was allowed to equilibrate for more than 1 hour. All solutions were used within 8 hours. Two 18-gauge needles provided the input and output ports for the odorant-containing vapor above the solution. For stimulation, a 100-ms pulse of the odorant vapor at 9 psi was injected into the continuous stream of humidified air. The pulse was controlled by a Picospritzer solenoid-controlled valve (General Valve). The odorant stimulus pathway was cleaned by air between each stimulus presentation. The minimum interval between two adjacent stimuli was 1 min.
  • 30
    • 0023378353 scopus 로고
    • The animal was overdosed with anesthetics (ketamine and xylazine) and decapitated. The head was cut open sagitally and the septum was removed to expose the medial surface of the olfactory turbinates [S. G. Shirley, E. H. Polak, D. A. Edwards, M. A. Wood, G. H. Dodd, Biochem. J. 245, 185 (1987)]. The right half of the head was mounted in a wax dish filled with rat Ringer solution. The medial surface of turbinates was face up and exposed to the air. A continuous stream of humidified clean air was gently blown on the turbinates through tubing to prevent tissue from drying. The opening of the tubing was 8 mm in diameter and was placed about 10 mm from the turbinate surface. The EOG recording electrode was an Ag-AgCl wire in a capillary glass pipette filled with rat Ringer solution containing 0.6% agarose. The electrode resistance was 0.5 to 1 megohm. The recording pipette was placed on the surface of the olfactory epithelium and connected to a differential amplifier (DP-301, Warner Instruments). Placement of the electrode was determined by visualizing GFP fluorescence with a modified stereomicroscope (Kramer Scientific). The EOG potential was observed on a chart recorder, recorded with a digital audio tape recorder, and later transferred to computer. For most experiments, two electrodes and two amplifiers were used to record EOGs from two different sites of epithelium simultaneously. All experiments were performed at room temperature (22° to 25°C).
    • (1987) Biochem. J. , vol.245 , pp. 185
    • Shirley, S.G.1    Polak, E.H.2    Edwards, D.A.3    Wood, M.A.4    Dodd, G.H.5
  • 31
    • 6844219597 scopus 로고    scopus 로고
    • note
    • -3 M were used alternately in each experiment.
  • 33
    • 6844234185 scopus 로고    scopus 로고
    • note
    • Five animals were infected with virus containing only the lacZ gene. In each animal the EOG electrode was positioned in several areas over the epithelium. After recording, the epithelia were reacted with X-gal and 18 electrode positions were determined to have been within areas of high infection. Responses to octanal at those positions were not different from those of uninfected animals.
  • 35
    • 0028922651 scopus 로고
    • T. Sato, J. Hirono, M. Tonioke, M. Takebayashi, J. Neurophysiol. 72, 2980 (1994). K. Mori and Y. Yoshihara, Prog. Neurobiol. 45, 585 (1995).
    • (1995) Prog. Neurobiol. , vol.45 , pp. 585
    • Mori, K.1    Yoshihara, Y.2
  • 36
    • 0027475469 scopus 로고
    • K. Raming et al., Nature 361, 353 (1993).
    • (1993) Nature , vol.361 , pp. 353
    • Raming, K.1
  • 37
    • 0029887597 scopus 로고    scopus 로고
    • P. Sengupta, J. H. Chou, C. I. Bargmann, Cell 84, 899 (1996); Y. Zhang, J. H. Chou, J. Bradley, C. I. Bargmann, K. Zinn, Proc. Natl. Acad. Sci. U.S.A. 94, 12162 (1997).
    • (1996) Cell , vol.84 , pp. 899
    • Sengupta, P.1    Chou, J.H.2    Bargmann, C.I.3
  • 43
    • 6844264251 scopus 로고    scopus 로고
    • note
    • We thank K. Mikoshiba, E. Falck-Pedersen, M. Chao, and in particular S. O. Yoon for expert advice and assistance; L. Buck and H. Breer for receptor clones; T. Lufkin for the IRES construct; L. Richards and K. Moriyoshi for GFP adenovirus; IFF and Harmon & Reimer Inc. for lyral and lilial; Y. Huang for technical assistance; and R. Axel, D. Kelley, and P. Mombaerts for thoughtful comments. J.M.O. would like to dedicate this paper to the memory of Yoko Sakaki. This work was supported by the Whitehall and McKnight Foundations and the National Institute on Deafness and Other Communication Disorders.


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