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The Human Gene Encoding the Interleukin-1 Receptor Accessory Protein (IL1RAP) Maps to Chromosome 3q28 by Fluorescencein SituHybridization and Radiation Hybrid Mapping
325
BRIEF REPORTS
The Human Gene Encoding the Interleukin-1 Receptor Accessory Protein (IL1RAP) Maps to Chromosome 3q28 by Fluorescence in Situ Hybridization and Radiation Hybrid Mapping Mark Dale, David W. Hammond,* Angela Cox, and Martin J. H. Nicklin1 Section of Molecular Medicine, Department of Molecular and Genetic Medicine, University of Sheffield, Royal Hallamshire Hospital, and *Institute of Cancer Studies, Royal Hallamshire Hospital, Sheffield S10 2JF, United Kingdom Received September 15, 1997; accepted November 4, 1997
Two receptors, Type I and Type II, that have significant affinity for the proinflammatory cytokines interleukin-1 (IL-1)a and IL-1 b have been described. Only the Type I receptor (encoding an 80-kDa protein) has been shown to transduce a signal in response to ligation (13). The role of the Type II receptor seems to be as a competitive binding protein and in this respect may actually antagonize IL-1 signaling (2). The human genes encoding the two receptors (IL1R1 and IL1R2, respectively) have been localized to chromosome 2q12 (3, 11), where they are also closely linked to two genes that encode structural homologs of the Type I receptor protein, namely the T1 gene (14) and the gene encoding the IL-1 receptor-related protein (12). The IL-1 receptor accessory protein (IL-1RAcP) was identified as a subunit of the IL-1 type 1 receptor complex in mouse cells that contributes to the affinity of the receptor for IL1 (4), and it has recently been shown to be required for IL1 signal transduction (9). For simplicity, we propose the gene name IL1RAP for the human homologue of the mouse IL-1RAcP gene. At the time of writing, only the cDNA sequences for mouse and rat IL-1RAcP are available, although many isologous human expressed sequence tags have now been deposited with the GenBank database. We amplified a probe spanning nt 488 – 1106 of the mouse IL-1RAcP (4) from mouse EL4 cDNA. This was used to probe a gridded human PAC (8) library, RPCI1, by filter hybridization (1) at low stringency (washes were performed at 557C in 200 mM sodium phosphate, pH 7.0, 1% NaDodSO 4). Plasmid DNA from the single positive clone (135E11 from the RPCI1 library) was digested with NotI to allow a size estimate of 110 kb for the genomic insert by pulsed-field gel electrophoresis (data not shown). Further digestion with BamHI yielded a 5-kb hybridizing subfragSequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. AF016261. 1 To whom correspondence should be addressed. Telephone: /44 114 271 3261. Fax: /44 114 272 1104. E-mail: m.nicklin@sheffield. ac.uk. GENOMICS 47, 325–326 ARTICLE NO. GE975113
ment that was subcloned as a complete Sau3AI digest. Two hybridizing clones were selected, and their inserts of 374 and 178 bp were entirely sequenced in both directions. They formed a 548-nt contig that has been deposited with GenBank (Accession No. AF016261). The sequence represents an intronic fragment of 250 bp, an exon of 187 nt, and a further intronic fragment of 111 bp. A human EST (GenBank Accession No. T85756) also contains part of the 187-nt exon. Within the predicted coding sequence of the genomic fragments, 87% of nucleotides were identical to nt 485 to 671 of the published mouse cDNA sequence (4). Fifty-two of the sixty-two amino acids encoded by the putative exon are identical to the corresponding mouse polypeptide sequence, and 8 of the 10 changes are functionally conservative. We therefore concluded that clone 135E11 from the RPCI1 human genomic PAC library contains IL1RAP, the human isolog of the mouse IL-1RAcP gene. The entire PAC clone was used as a probe for fluorescence in situ hybridization. The probe was labeled by nicktranslation to incorporate biotin – dUTP conjugate, as described previously (7, 10), and 50 ng was precompeted with 2 m g human Cot1 (Gibco/BRL) DNA for 30 min at 377C to suppress hybridization with repetitive sequences. Biotinylated probe was detected with fluorescein-labeled streptavidin. DNA was counterstained with DAPI. Raw data from the DAPI signal were processed to increase contrast and then further enhanced to accentuate G-banding. Thirteen individual cells were examined. Chromosomes were identified by their size and G-banding patterns. Strong paired signals were seen unequivocally on at least one identifiable copy of chromosome 3 in all cells. An example is given in Fig. 1, showing clear paired labeling of both copies of chromosome 3. Because of the clear banding in the region, the label was mapped to 3q28, which is the most telomeric DAPI-bright band of the chromosome. To confirm the localization, we designed a PCR that was specific to the sequenced genomic fragment described above. The primers corresponded to nucleotides 64 – 90 and 418 – 390 (complement) of the genomic fragment. We screened DNA from the GeneBridge 4 radiation hybrid panel (6) and analyzed the data automatically. The analysis placed the IL1RAP fragment on chromosome 3, 1.8 cR telomeric of D3S1314 (LOD ú 3.0). D3S1314 maps to Ç11 cM from the telomere of chromosome 3q (5) and is thus fully consistent with our in situ hybridization data. We conclude that IL1RAP is not encoded within the interleukin-1 receptor cluster on chromosome 2q12, but is on chromosome 3q28.
ACKNOWLEDGMENTS The human library RPCI1 was created by the group of Pieter de Jong (Roswell Park Cancer Institute, Buffalo, NY) and distributed by the UK Human Genome Project Resource Centre. The GeneBridge 4 panel was made available by the UK Human Genome Project Resource Centre and was analyzed remotely at the European Biological Information Centre, UK. We thank Hazel Holden for automated sequencing and John Mee for the gift of EL4 cDNA. This work was supported by a Programme Grant from the Arthritis and Rheumatism Council, UK (Grant D0528).
(1998)
0888-7543/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
AID
Genom BREP
/
6r58$$$81x
01-05-98 10:55:01
gnmbr
AP: Genomics
326
BRIEF REPORTS
FIG. 1. (A) Signal from fluorescein–avidin detection of biotinylated IL1RAP genomic probe (shown in white), superimposed on an enhanced DAPI image (gray/black). (B) For comparison, the DAPI banding pattern is shown as a negative image.
REFERENCES 1. Church, G. M., and Gilbert, W. (1984). Genomic sequencing. Proc. Natl. Acad. Sci. USA 81: 1991–1995. 2. Colotta, F., Re, F., Muzio, M., Bertini, R., Polentarutti, N., Sironi, M., Giri, J. G., Dower, S. K., Sims, J. E., and Mantovani, A. (1993). Interleukin-1 type II receptor: A decoy target for IL1 that is regulated by IL-4. Science 261: 472–475. 3. Copeland, N. G., Silan, C. M., Kingsley, D. M., Jenkins, N. A., Cannizzaro, L. A., Croce, C. M., Huebner, K., and Sims, J. E. (1991). Chromosomal location of murine and human IL-1 receptor genes. Genomics 9: 44–50. 4. Greenfeder, S. A., Nunes, P., Kwee, L., Labow, M., Chizzonite, R. A., and Ju, G. (1995). Molecular cloning and characterization of a second subunit of the interleukin-1 receptor complex. J. Biol. Chem. 270: 13757–13765. 5. Gyapay, G., Morissette, J., Vignal, A., Dib, C., Fizames, C., Millasseau, P., Marc, S., Bernardi, G., Lathrop, M., and Weissenbach, J. (1994). The 1993–94 Ge´ne´thon human genetic linkage map. Nat. Genet. 7: 246–339. 6. Gyapay, G., Schmitt, K., Fizames, C., Jones, H., Vega-Czarny, N., Spillett, D., Muselet, D., Prud’Homme, J.-F., Dib, C., Auffray, C., Morissette, J., Weissenbach, J., and Goodfellow, P. (1996). A radiation map of the human genome. Hum. Mol. Genet. 3: 339–346. 7. Hammond, D. W., Hancock, B. W., and Goyns, M. H. (1994). Identification of a subclass of double minute chromosomes containing centromere-associated DNA Genes Chromosomes Cancer 10: 139–142.
AID
Genom BREP
/
6r58$$$$$1
01-05-98 10:55:01
8. Ioannou, P. A., and de Jong, P. J. (1996). Construction of bacterial artificial chromosome libraries using a modified P1 (PAC) system. In ‘‘Current Protocols in Human Genetics’’ (Dracopoli et al., Eds.), Unit 5.15, Wiley, New York. 9. Korherr, C., Hofmeister, R., Wesche, H., and Falke, W. (1997). A critical role for IL-1R accessory protein in IL-1 signaling. Eur. J. Immunol. 27: 262–267. 10. Lichter, P., and Cremer, T. (1992). Chromosome analysis by non-isotopic in situ hybridisation. In ‘‘Human Cytogenetics, A Practical Approach’’ (D. E. Rooney, and B. H. Czepukowski, Eds.), pp. 157–192, IRL Press, Oxford. 11. McMahan, C. J., Slack, J. L., Mosley, B., Cosman, D., Lupton, S. D., Brunton, L. L., Grubin, C. E., Wignall, J. M., Jenkins, N. A., Brannan, C. I., Copeland, N. G., Huebner, K., Croce, C. M., Cannizzaro, L. A., Benjamin, D., Dower, S. K., Spriggs, M. K., and Sims, J. E. (1991). A novel IL-1 receptor, cloned from B cells by mammalian expression, is expressed in many cell types. EMBO J. 10: 2821–2832. 12. Parnet, P., Garka, K. E., Bonner, T. P., Dower, S. K., and Sims, J. E. (1996). IL-1Rrp is a novel receptor-like molecule similar to the type I interleukin-1 receptor and its homologs T1/ST2 and IL-1RAcP. J. Biol. Chem. 271: 3967–3970. 13. Sims, J. E., March, C. J., Cosman, D., Widmer, M. B., MacDonald, H. R., McMahan, C. J., Grubin, C. E., Wignall, J. M., Jackson, J. L., Call, S., Friend, D., Alpert, A. R., Gillis, S., Urdal, D. L., and Dower, S. K. (1989). cDNA expression cloning of the IL-1 receptor, a member of the immunoglobulin superfamily. Science 241: 585–589. 14. Sims, J. E., Painter, S. L., and Gow, I. R. (1995). Genomic organization of the type I and type II IL-1 receptors. Cytokine 7: 483–490.
gnmbr
AP: Genomics
BRIEF REPORTS
The Human Gene Encoding the Interleukin-1 Receptor Accessory Protein (IL1RAP) Maps to Chromosome 3q28 by Fluorescence in Situ Hybridization and Radiation Hybrid Mapping Mark Dale, David W. Hammond,* Angela Cox, and Martin J. H. Nicklin1 Section of Molecular Medicine, Department of Molecular and Genetic Medicine, University of Sheffield, Royal Hallamshire Hospital, and *Institute of Cancer Studies, Royal Hallamshire Hospital, Sheffield S10 2JF, United Kingdom Received September 15, 1997; accepted November 4, 1997
Two receptors, Type I and Type II, that have significant affinity for the proinflammatory cytokines interleukin-1 (IL-1)a and IL-1 b have been described. Only the Type I receptor (encoding an 80-kDa protein) has been shown to transduce a signal in response to ligation (13). The role of the Type II receptor seems to be as a competitive binding protein and in this respect may actually antagonize IL-1 signaling (2). The human genes encoding the two receptors (IL1R1 and IL1R2, respectively) have been localized to chromosome 2q12 (3, 11), where they are also closely linked to two genes that encode structural homologs of the Type I receptor protein, namely the T1 gene (14) and the gene encoding the IL-1 receptor-related protein (12). The IL-1 receptor accessory protein (IL-1RAcP) was identified as a subunit of the IL-1 type 1 receptor complex in mouse cells that contributes to the affinity of the receptor for IL1 (4), and it has recently been shown to be required for IL1 signal transduction (9). For simplicity, we propose the gene name IL1RAP for the human homologue of the mouse IL-1RAcP gene. At the time of writing, only the cDNA sequences for mouse and rat IL-1RAcP are available, although many isologous human expressed sequence tags have now been deposited with the GenBank database. We amplified a probe spanning nt 488 – 1106 of the mouse IL-1RAcP (4) from mouse EL4 cDNA. This was used to probe a gridded human PAC (8) library, RPCI1, by filter hybridization (1) at low stringency (washes were performed at 557C in 200 mM sodium phosphate, pH 7.0, 1% NaDodSO 4). Plasmid DNA from the single positive clone (135E11 from the RPCI1 library) was digested with NotI to allow a size estimate of 110 kb for the genomic insert by pulsed-field gel electrophoresis (data not shown). Further digestion with BamHI yielded a 5-kb hybridizing subfragSequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. AF016261. 1 To whom correspondence should be addressed. Telephone: /44 114 271 3261. Fax: /44 114 272 1104. E-mail: m.nicklin@sheffield. ac.uk. GENOMICS 47, 325–326 ARTICLE NO. GE975113
ment that was subcloned as a complete Sau3AI digest. Two hybridizing clones were selected, and their inserts of 374 and 178 bp were entirely sequenced in both directions. They formed a 548-nt contig that has been deposited with GenBank (Accession No. AF016261). The sequence represents an intronic fragment of 250 bp, an exon of 187 nt, and a further intronic fragment of 111 bp. A human EST (GenBank Accession No. T85756) also contains part of the 187-nt exon. Within the predicted coding sequence of the genomic fragments, 87% of nucleotides were identical to nt 485 to 671 of the published mouse cDNA sequence (4). Fifty-two of the sixty-two amino acids encoded by the putative exon are identical to the corresponding mouse polypeptide sequence, and 8 of the 10 changes are functionally conservative. We therefore concluded that clone 135E11 from the RPCI1 human genomic PAC library contains IL1RAP, the human isolog of the mouse IL-1RAcP gene. The entire PAC clone was used as a probe for fluorescence in situ hybridization. The probe was labeled by nicktranslation to incorporate biotin – dUTP conjugate, as described previously (7, 10), and 50 ng was precompeted with 2 m g human Cot1 (Gibco/BRL) DNA for 30 min at 377C to suppress hybridization with repetitive sequences. Biotinylated probe was detected with fluorescein-labeled streptavidin. DNA was counterstained with DAPI. Raw data from the DAPI signal were processed to increase contrast and then further enhanced to accentuate G-banding. Thirteen individual cells were examined. Chromosomes were identified by their size and G-banding patterns. Strong paired signals were seen unequivocally on at least one identifiable copy of chromosome 3 in all cells. An example is given in Fig. 1, showing clear paired labeling of both copies of chromosome 3. Because of the clear banding in the region, the label was mapped to 3q28, which is the most telomeric DAPI-bright band of the chromosome. To confirm the localization, we designed a PCR that was specific to the sequenced genomic fragment described above. The primers corresponded to nucleotides 64 – 90 and 418 – 390 (complement) of the genomic fragment. We screened DNA from the GeneBridge 4 radiation hybrid panel (6) and analyzed the data automatically. The analysis placed the IL1RAP fragment on chromosome 3, 1.8 cR telomeric of D3S1314 (LOD ú 3.0). D3S1314 maps to Ç11 cM from the telomere of chromosome 3q (5) and is thus fully consistent with our in situ hybridization data. We conclude that IL1RAP is not encoded within the interleukin-1 receptor cluster on chromosome 2q12, but is on chromosome 3q28.
ACKNOWLEDGMENTS The human library RPCI1 was created by the group of Pieter de Jong (Roswell Park Cancer Institute, Buffalo, NY) and distributed by the UK Human Genome Project Resource Centre. The GeneBridge 4 panel was made available by the UK Human Genome Project Resource Centre and was analyzed remotely at the European Biological Information Centre, UK. We thank Hazel Holden for automated sequencing and John Mee for the gift of EL4 cDNA. This work was supported by a Programme Grant from the Arthritis and Rheumatism Council, UK (Grant D0528).
(1998)
0888-7543/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
AID
Genom BREP
/
6r58$$$81x
01-05-98 10:55:01
gnmbr
AP: Genomics
326
BRIEF REPORTS
FIG. 1. (A) Signal from fluorescein–avidin detection of biotinylated IL1RAP genomic probe (shown in white), superimposed on an enhanced DAPI image (gray/black). (B) For comparison, the DAPI banding pattern is shown as a negative image.
REFERENCES 1. Church, G. M., and Gilbert, W. (1984). Genomic sequencing. Proc. Natl. Acad. Sci. USA 81: 1991–1995. 2. Colotta, F., Re, F., Muzio, M., Bertini, R., Polentarutti, N., Sironi, M., Giri, J. G., Dower, S. K., Sims, J. E., and Mantovani, A. (1993). Interleukin-1 type II receptor: A decoy target for IL1 that is regulated by IL-4. Science 261: 472–475. 3. Copeland, N. G., Silan, C. M., Kingsley, D. M., Jenkins, N. A., Cannizzaro, L. A., Croce, C. M., Huebner, K., and Sims, J. E. (1991). Chromosomal location of murine and human IL-1 receptor genes. Genomics 9: 44–50. 4. Greenfeder, S. A., Nunes, P., Kwee, L., Labow, M., Chizzonite, R. A., and Ju, G. (1995). Molecular cloning and characterization of a second subunit of the interleukin-1 receptor complex. J. Biol. Chem. 270: 13757–13765. 5. Gyapay, G., Morissette, J., Vignal, A., Dib, C., Fizames, C., Millasseau, P., Marc, S., Bernardi, G., Lathrop, M., and Weissenbach, J. (1994). The 1993–94 Ge´ne´thon human genetic linkage map. Nat. Genet. 7: 246–339. 6. Gyapay, G., Schmitt, K., Fizames, C., Jones, H., Vega-Czarny, N., Spillett, D., Muselet, D., Prud’Homme, J.-F., Dib, C., Auffray, C., Morissette, J., Weissenbach, J., and Goodfellow, P. (1996). A radiation map of the human genome. Hum. Mol. Genet. 3: 339–346. 7. Hammond, D. W., Hancock, B. W., and Goyns, M. H. (1994). Identification of a subclass of double minute chromosomes containing centromere-associated DNA Genes Chromosomes Cancer 10: 139–142.
AID
Genom BREP
/
6r58$$$$$1
01-05-98 10:55:01
8. Ioannou, P. A., and de Jong, P. J. (1996). Construction of bacterial artificial chromosome libraries using a modified P1 (PAC) system. In ‘‘Current Protocols in Human Genetics’’ (Dracopoli et al., Eds.), Unit 5.15, Wiley, New York. 9. Korherr, C., Hofmeister, R., Wesche, H., and Falke, W. (1997). A critical role for IL-1R accessory protein in IL-1 signaling. Eur. J. Immunol. 27: 262–267. 10. Lichter, P., and Cremer, T. (1992). Chromosome analysis by non-isotopic in situ hybridisation. In ‘‘Human Cytogenetics, A Practical Approach’’ (D. E. Rooney, and B. H. Czepukowski, Eds.), pp. 157–192, IRL Press, Oxford. 11. McMahan, C. J., Slack, J. L., Mosley, B., Cosman, D., Lupton, S. D., Brunton, L. L., Grubin, C. E., Wignall, J. M., Jenkins, N. A., Brannan, C. I., Copeland, N. G., Huebner, K., Croce, C. M., Cannizzaro, L. A., Benjamin, D., Dower, S. K., Spriggs, M. K., and Sims, J. E. (1991). A novel IL-1 receptor, cloned from B cells by mammalian expression, is expressed in many cell types. EMBO J. 10: 2821–2832. 12. Parnet, P., Garka, K. E., Bonner, T. P., Dower, S. K., and Sims, J. E. (1996). IL-1Rrp is a novel receptor-like molecule similar to the type I interleukin-1 receptor and its homologs T1/ST2 and IL-1RAcP. J. Biol. Chem. 271: 3967–3970. 13. Sims, J. E., March, C. J., Cosman, D., Widmer, M. B., MacDonald, H. R., McMahan, C. J., Grubin, C. E., Wignall, J. M., Jackson, J. L., Call, S., Friend, D., Alpert, A. R., Gillis, S., Urdal, D. L., and Dower, S. K. (1989). cDNA expression cloning of the IL-1 receptor, a member of the immunoglobulin superfamily. Science 241: 585–589. 14. Sims, J. E., Painter, S. L., and Gow, I. R. (1995). Genomic organization of the type I and type II IL-1 receptors. Cytokine 7: 483–490.
gnmbr
AP: Genomics