- Email: [email protected]
The Human PECAM1 Gene Maps to 17q23
SHORT COMMUNICATION The Human PECAM1 Gene Maps to 17q23 RICHARD J. GUMINA,*,† NANCY E. KIRSCHBAUM,† P. NAGESH RAO,‡ PETER , ,Ø,1 AND PETER J. NEWMAN* †
VANTUINEN,§
*Department of Cellular Biology and Anatomy, §Department of Pathology, and ØDepartment of Pharmacology, Medical College of Wisconsin, Milwaukee, Wisconsin; ‡Molecular Cytogenetics Laboratory, Bowman Gray School of Medicine, Winston-Salem, North Carolina; and †Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53201 Received September 20, 1995; accepted February 20, 1996
We have determined the chromosomal and regional location of the gene encoding PECAM-1 (termed PECAM1 by GBD nomenclature) using a polymerase chain reaction (PCR)-based analysis of somatic cell hybrids. Analysis of a somatic cell hybrid chromosome panel established that the PECAM1 gene is on chromosome 17. Interestingly, several adhesion molecules expressed on platelets and endothelium also localize to chromosome 17: the GP1BA locus (glycoprotein (GP) Iba) has been provisionally mapped to the region 17p12 –pter, the ITGA2B (GPIIb) and the ITGB3 (GPIIIa) loci have been confirmed to the region 17q21.32; and the ICAM2 locus has been provisionally mapped to the region 17q23 – q25. To determine if the PECAM1 locus colocalizes with any of the loci for these adhesion molecules, PCR-based analysis of a regional mapping panel for human chromosome 17 was conducted. We found that the PECAM1 locus is on the long arm of chromosome 17, in the region q23 –qter. To confirm this observation and obtain a more precise localization of the PECAM1 locus, fluorescence in situ hybridization was conducted. Together our data allowed assignment of the PECAM1 locus to the region 17q23. q 1996 Academic Press, Inc.
PECAM-1 (platelet/endothelial cell adhesion molecule-1) is a 130-kDa plasma membrane glycoprotein that is constitutively expressed on the surface of endothelial cells, platelets, and certain leukocyte subtypes (2, 8, 10). PECAM-1 is expressed at high levels on human endothelial cells where it is localized to the cell– cell border at areas of endothelial cell contact, where it plays a role in stabilization of the endothelial cell – cell junction (8) and also appears to function as a mediator of leukocyte transendothelial migration (9, 11). Given what we know concerning the role of PECAM1 as an endothelial cell junctional molecule and as a mediator of leukocyte/endothelial cell–cell interaction, 1 To whom all correspondence should be addressed at Blood Research Institute, The Blood Center of Southeastern Wisconsin, P.O. Box 2178, Milwaukee, WI 53201-2178. Telephone: (414) 937-6237. Fax: (414) 937-6284. E-mail: [email protected].
a knowledge of the structure of the PECAM1 gene and of the regulation of expression of the PECAM1 gene may facilitate our work to understand the cellular interactions mediated by this novel cell adhesion molecule. Work by our laboratory has previously described the cloning of the PECAM-1 cDNA and the characterization of the structure of the PECAM1 gene (6, 10). As a prelude to further analysis of the organization and evolution of the PECAM1 gene, we have mapped the human PECAM1 locus using a polymerase chain reaction (PCR)-based analysis of two panels of somatic cell hybrids and fluorescence in situ hybridization. The first panel consisted of genomic DNA from human –hamster hybrids (BIOS, Inc., New Haven, CT). Seventeen hybrids, which together encompass the full complement of human chromosomes, were screened. The second panel of somatic hybrids, which subdivides human chromosome 17 into 15 regions, has been previously described (16). A subset of these hybrids was utilized in the localization of the human PECAM1 gene. Genomic DNA was isolated from the chromosome 17-specific panel as previously described (7). The loci of the genes for several adhesion molecules expressed on platelets and endothelium have also been previously mapped to chromosome 17. The GP1BA locus (glycoprotein (GP) Iba) has been provisionally mapped to the region 17p12–pter (18), the ITGA2B (GPIIb) and the ITGB3 (GPIIIa) loci have been confirmed to the region 17q21.32 (1, 15), and the ICAM2 locus has been provisionally mapped to the region 17q23–q25 (14). To validate the use of PCR to map gene location with chromosome 17 panel primers routinely used in our lab for the amplification of the GP1A, the ITGA2B and ITGB3 genes were employed to confirm the localization of these genes on chromosome 17. The GP1BA-specific forward primer begins at position 404 of the cDNA with the sequence 5*-TGGACGTCTCCTCAACCGGCTGACA-3 *, and the reverse primer begins at position 1001 in the cDNA with the sequence 5*-GTATGGGCTTTGGTGGGGAACTTGACC-3*. The GP1BA primers yielded a 600-bp product following PCR amplification. The ITGB3-specific forward primer GENOMICS
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begins at position 633 of the cDNA with the sequence 5*-GATATGGAAGACCACCTGCTTGCCCAT-3 *, and the ITGB3-specific reverse primer begins at position 825 of the cDNA with the sequence 5*-GGATGCATCATTCCTCCAGCCAATC-3*. The ITGB3 primers amplify across an intron to yield an 800-bp product. Based upon the ICAM2 genomic structure obtained from the GenBank/EMBL Data Bank (Accession No. M1234), primers specific for exon 2 were designed. The forward primer beginning at nucleotide position 131 in the ICAM2 cDNA sequence with the sequence 5*-TGAGAAGGTATTCGAGGTAC-3* was used in conjunction with a reverse primer beginning at nucleotide position 370 in the ICAM2 cDNA with the sequence 5*-TTCATTGACTCCTGCTTCCC-3*. These primers yielded a 240-bp ICAM2-specific product following PCR amplification. Finally, the PECAM-1 primer set amplifies across intron 14 (6) to generate a 1000-bp product with PCR amplification. The PECAM1-specific forward primer begins at position 2038 of the cDNA with the sequence 5*-ATGCCAGTGGAAATGTCC-3 *, and the reverse primer begins at nucleotide position 2077 with the sequence 5*-TCAGAAGTGGTACTGGTG-3 *. All amplifications were conducted using Taq polymerase at 2.0 U/ml with annealing temperatures between 58 and 607C. GP1BA, ITGB3, and PECAM1 amplification products were electrophoresed through 1% agarose in 11 TAE at 100 V for 1 h. ICAM2 amplification products were electrophoresed through 2% agarose in 11 TAE at 100 V for 2 h. Using two separate PECAM-1-specific primer sets, aliquots of genomic DNA from the somatic cell hybrid chromosome panel were amplified by PCR and indicated that the human PECAM1 locus is on chromosome 17 (data not shown). Interestingly, the genes for several other adhesion molecules expressed on platelets and endothelium have also been previously localized to chromosome 17. To validate our assignment of the PECAM1 gene to chromosome 17 using this PCR-based approach and to determine if the PECAM1 gene colocalizes with any of the genes encoding these adhesion molecules, PCR analysis of a human chromosome 17 regional mapping panel, which subdivides chromosome 17 into 15 regions, was conducted. GPIba is a member of the leucine-rich glycoprotein family of cell adhesion molecules and is a subunit of the vWF receptor expressed on endothelial cells and platelets (3). The GP1BA locus (GPIba) has been provisionally localized to the 17p12–pter region (18). As shown in Fig. 1, using GP1BA-specific primers, only those clones containing the short arm of chromosome 17 were positive for the GP1BA locus. Integrins are a family of cytoadhesive receptors expressed on the surface of cells as Ca2/-depppendent ab heterodimers (5). Glycoprotein IIIa (GPIIIa) is a b subunit of the integrin family that is expressed on endothelial cells and platelets, complexed with different a subunits. On platelets, GPIIIa complexes with glycoprotein IIb (GPIIb) to form the fibrinogen receptor (11). Both the ITGB3 (GPIIIa) and the
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FIG. 1. Sublocalization of the human PECAM1 gene to the 17q23 –qter region. Using a chromosome 17 regional mapping panel, PCR amplification of genomic DNA was conducted with primers for GPIba (GP1BA by GDB nomenclature), GPIIIa (ITGB3), ICAM-2 (ICAM2) and PECAM1 as described in the text. In positive clones, GPIba-specific primers yielded a 600-bp product; GPIIIa-specific primers yielded an 800-bp product; ICAM-2 specific primers yielded a 240-bp product; and PECAM1-specific primers yielded a 1000-bp product.
ITGA2B (GPIIb) loci have been confirmed at the region 17q21.32 (1, 15). Using ITGB3-specific primers, only those clones containing the 17q21.32 region were positive for the ITGB3 locus. The ITGA2B locus also mapped to this same region (data not shown). ICAM2 is an immunoglobulin gene superfamily member that is expressed constitutively on the surface of endothelial cells and cells of myeloid lineage, where it functions as the counterreceptor for the integrin LFA-1 (aLb2 ) found on neutrophils and monocytes (17). The ICAM2 locus has been provisionally localized to the region 17q25–qter (14). Using ICAM2-specific primers, only those clones containing the 17q23–qter region were positive for the ICAM2 locus. Thus, the results of our chromosome 17 regional mapping panel were in agreement with previous mapping of the GP1BA, ITGA2B, ITGB3, and ICAM2 loci on chromosome 17 and validated the use of PCR to map gene location with our chromosome 17 panel. Finally, as shown in Fig. 1, using a PECAM-1-specific primer set, only those clones from the chromosome 17 mapping panel containing the 17q23–qter region were positive for the PECAM1 locus. Thus, the PECAM1 locus lies on the long arm of chromosome 17 in the 17q23–qter region. To verify the assignment of the PECAM1 locus to the long arm of chromosome 17 and to determine more precisely the localization of the PECAM1 locus, we performed fluorescence in situ hybridization (FISH). Briefly, DNA from a previously described P1 genomic clone that contains a portion of the PECAM1 gene (6) was labeled with digoxigenin –dUTP by nick-translation. A biotin-labeled chromosome 17 centromere-spe-
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FIG. 2. Precise localization of the PECAM1 gene to 17q23 by FISH. (A) In situ cohybridization of a PECAM1 P1 genomic clone (6) probe and a chromosome 17 centromere-specific probe (D17Z1) to human metaphase chromosomes. P1 DNA was labeled with digoxigenin –dUTP by nick-translation and hybridized along with a biotin-labeled chromosome 17 centromere-specific probe (D17Z1) to normal metaphase chromosomes. Specific hybridization and detection resulted in labeling the chromosome 17 centromere in red (arrowhead) and localization of the PECAM1 gene in green (arrow). (B) A partial view of a propidium-iodide-stained R-banded metaphase chromosome spread hybridized with the PECAM1 P1 genomic clone. DNA from the P1 clone was labeled by nick-translation with biotin-14 –dATP. Simultaneous detection of in situ hybridization signals and chromosome banding was performed by previously described techniques (4, 12). The chromosomes were observed under a Zeiss Axiophot fluorescence microscope equipped with a filter set simultaneously transmitting FITC-labeled probe sites and PI. True images were captured and stored on a TOMS image analysis system (TOMS, Columbus, GA), and prints were made using a SONY color printer. At least 20 metaphases were analyzed, and consistent signals were observed only on chromosome 17. The arrow denotes PECAM1-specific signals at 17q23.
cific probe (D17Z1) was cohybridized with the P1 probe. Specific hybridization of the PECAM1 probe was detected by incubating the slides in fluoresceinated antidigoxigenin antibodies, while hybridization of D17Z1 was visualized by incubation with Texas red–avidin followed by counterstaining with DAPI. As shown in Fig. 2A, these experiments resulted in the specific labeling of the chromosome 17 centromere in red and localization of the PECAM1 gene on the long arm of chromosome 17 in green. Finally, to determine precise localization of the PECAM1 gene, hybridization of the P1 clone to R-banded metaphase chromosome spreads was conducted. Briefly, purified DNA from the P1 clone was labeled by nick-translation with biotin-14–dATP (Bionick, Gibco-BRL) following the manufacturer’s protocol. Simultaneous detection of in situ hybridization signals and chromosome banding was performed by previously described techniques (4), with some modifications. Standard FISH methods were followed for the hybridization and detection of the probe (13). As shown in Fig. 2B, sublocalization of the PECAM1 locus to chromosomal region 17q23 was unequivocally made on banded metaphase chromosome spreads. Thus, utilizing two independent complementary techniques, PCR analysis of somatic cell hybrids and fluorescence in situ hybridization, we have mapped the human PECAM1 loci to chromosome 17, specifically the region 17q23. ACKNOWLEDGMENTS R.J.G. is the recipient of a Medical Scientist Training Fellowship from the Medical College of Wisconsin and a predoctoral fellowship from the American Heart Association, Wisconsin Affiliate. This work
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was supported by Grant HL40926 (to P.J.N.) and Training Grant HL-07209 from the National Institutes of Health. P.J.N. is an Established Investigator of the American Heart Association.
REFERENCES 1. Bray, P. F., Rosa, J. P., Johnston, G. I., Shiu, D. T., Cook, R. G., Lau, C., Kan, Y. W., McEver, R. P., and Shuman, M. A. (1987). Platelet glycoprotein IIb. Chromosomal localization and tissue expression. J. Clin. Invest. 80: 1812 –1817. 2. Goyert, S. M., Ferrero, E. M., Seremetis, S. V., Winchester, R. J., Silver, J., and Mattison, A. C. (1986). Biochemistry and expression of myelomonocytic antigens. J. Immunol. 137: 3909 – 3914. 3. Handa, R., Titani, K., Holland, L., Roberts, J., and Ruggeri, Z. M. (1986). The von Willebrand factor-binding domain of platelet membrane glycoprotein Ib. J. Biol. Chem. 261: 12579– 12583. 4. Hirai, M., Suto, Y., and Kanoh, M. (1994). A method for simultaneous detection of fluorescent G-bands and in situ hybridization signals. Cytogenet. Cell Genet. 66: 149–151. 5. Hynes, R. O. (1987). Integrins, a family of cell surface receptors. Cell 48: 549–554. 6. Kirschbaum, N., Gumina, R. J., and Newman, P. J. (1994). Organization of the human PECAM-1 gene. Blood 84: 4028 –4037. 7. McFarland, J. G., Aster, R. H., Bussel, J. B., Gianopoulos, J. G., Derbes, R. S., and Newman, P. J. (1991). Prenatal diagnosis of neonatal alloimmune thrombocytopenia using allele-specific oligonucleotide probes. Blood 78: 2276 –2282. 8. Muller, W. A., Ratti, C. M., McDonnell, S. L., and Cohn, Z. A. (1989). A human endothelial cell restricted, externally disposed plasmalemmal protein enriched in intercellular junctions. J. Exp. Med. 170: 399– 414. 9. Muller, W. A., Weigl, S. A., Deng, X., and Phillips, D. M. (1993). PECAM-1 is required for transendothelial migration of leukocytes. J. Exp. Med. 178: 449–460.
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10. Newman, P. J., Berndt, M. C., Gorski, J., White, G. C., Lyman, S., Paddock, P., and Muller, W. A. (1990). PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily. Science 247: 1219 –1222. 11. Ohto, H., Maeda, H., Shibata, Y., Chen, R., Ozaki, Y., Higashihara, M., Takeuchi, A., and Tohyama, H. (1985). A novel leukocyte differentiation antigen: Two monoclonal antibodies TM2 and TM3 define a 120-kd molecule present on neutrophils, monocytes, platelets, and activated lymphoblasts. Blood 66: 873–881. 12. Phillips, D. R., Charo, I. F., and Scarborough, R. M. (1991). GPIIb-IIIa: The responsive integrin. Cell 5: 359–362. 13. Rothschild, C. R., Akots, G., Hayworth, R., Pettenati, M. J., Rao, P. N., Wood, P., Fajans, S. S., and Bowden, D. W. (1993). A genetic map of chromosome 20q12 –q13.1: Multiple highly polymorphic microsatellite and RFLP markers linked to the maturity onset diabetes of the young (MODY) locus. Am. J. Hum. Genet. 52: 110– 123. 14. Sansom, D., Borrow, J., Solomon, E., and Trowsdale, J. (1991).
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15.
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The human ICAM-2 gene maps to 17q23 – 25. Genomics 11: 462– 464. Sosnoski, D. M., Emanuel, B. S., Hawkins, A. L., vanTuinen, P., Ledbetter, D. H., Nussbaum, R. L., Kaos, F., Shwartz, E., Phillips, D., Bennett, J. S., Fitzgerald, L. A., and Poncz, M. (1988). Chromosomal localization of the genes for the vitronectin and fibronectin receptors a subunits and for the platelet glycoprotein IIb and IIIa. J. Clin. Invest. 81: 1993 –1998. vanTuinen, P., Rich, D. C., Summers, K. M., and Ledbetter, D. H. (1987). Regional mapping panel for human chromosome 17: Application to neurofibromatosis Type 1. Genomics 1: 74– 81. Wawryk, S. O., Novotny, J. R., Wicks, I. P., Wilkinson, D., Maher, D., Salvaris, E., Welch, K., Fecondo, J., and Boyd, A. W. (1989). The role of LFA-1/ICAM-1 interaction in human leukocyte homing and adhesion. Immunol. Rev. 108: 135–143. Wenger, R. H., Wicki, A. N., Kieffer, N., Adolph, S., Hameister, H., and Clemtson, K. J. (1989). The 5* flanking region and chromosomal localization of the gene encoding human platelet membrane glycoprotein Iba. Gene 85: 517– 524.
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VANTUINEN,§
*Department of Cellular Biology and Anatomy, §Department of Pathology, and ØDepartment of Pharmacology, Medical College of Wisconsin, Milwaukee, Wisconsin; ‡Molecular Cytogenetics Laboratory, Bowman Gray School of Medicine, Winston-Salem, North Carolina; and †Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53201 Received September 20, 1995; accepted February 20, 1996
We have determined the chromosomal and regional location of the gene encoding PECAM-1 (termed PECAM1 by GBD nomenclature) using a polymerase chain reaction (PCR)-based analysis of somatic cell hybrids. Analysis of a somatic cell hybrid chromosome panel established that the PECAM1 gene is on chromosome 17. Interestingly, several adhesion molecules expressed on platelets and endothelium also localize to chromosome 17: the GP1BA locus (glycoprotein (GP) Iba) has been provisionally mapped to the region 17p12 –pter, the ITGA2B (GPIIb) and the ITGB3 (GPIIIa) loci have been confirmed to the region 17q21.32; and the ICAM2 locus has been provisionally mapped to the region 17q23 – q25. To determine if the PECAM1 locus colocalizes with any of the loci for these adhesion molecules, PCR-based analysis of a regional mapping panel for human chromosome 17 was conducted. We found that the PECAM1 locus is on the long arm of chromosome 17, in the region q23 –qter. To confirm this observation and obtain a more precise localization of the PECAM1 locus, fluorescence in situ hybridization was conducted. Together our data allowed assignment of the PECAM1 locus to the region 17q23. q 1996 Academic Press, Inc.
PECAM-1 (platelet/endothelial cell adhesion molecule-1) is a 130-kDa plasma membrane glycoprotein that is constitutively expressed on the surface of endothelial cells, platelets, and certain leukocyte subtypes (2, 8, 10). PECAM-1 is expressed at high levels on human endothelial cells where it is localized to the cell– cell border at areas of endothelial cell contact, where it plays a role in stabilization of the endothelial cell – cell junction (8) and also appears to function as a mediator of leukocyte transendothelial migration (9, 11). Given what we know concerning the role of PECAM1 as an endothelial cell junctional molecule and as a mediator of leukocyte/endothelial cell–cell interaction, 1 To whom all correspondence should be addressed at Blood Research Institute, The Blood Center of Southeastern Wisconsin, P.O. Box 2178, Milwaukee, WI 53201-2178. Telephone: (414) 937-6237. Fax: (414) 937-6284. E-mail: [email protected].
a knowledge of the structure of the PECAM1 gene and of the regulation of expression of the PECAM1 gene may facilitate our work to understand the cellular interactions mediated by this novel cell adhesion molecule. Work by our laboratory has previously described the cloning of the PECAM-1 cDNA and the characterization of the structure of the PECAM1 gene (6, 10). As a prelude to further analysis of the organization and evolution of the PECAM1 gene, we have mapped the human PECAM1 locus using a polymerase chain reaction (PCR)-based analysis of two panels of somatic cell hybrids and fluorescence in situ hybridization. The first panel consisted of genomic DNA from human –hamster hybrids (BIOS, Inc., New Haven, CT). Seventeen hybrids, which together encompass the full complement of human chromosomes, were screened. The second panel of somatic hybrids, which subdivides human chromosome 17 into 15 regions, has been previously described (16). A subset of these hybrids was utilized in the localization of the human PECAM1 gene. Genomic DNA was isolated from the chromosome 17-specific panel as previously described (7). The loci of the genes for several adhesion molecules expressed on platelets and endothelium have also been previously mapped to chromosome 17. The GP1BA locus (glycoprotein (GP) Iba) has been provisionally mapped to the region 17p12–pter (18), the ITGA2B (GPIIb) and the ITGB3 (GPIIIa) loci have been confirmed to the region 17q21.32 (1, 15), and the ICAM2 locus has been provisionally mapped to the region 17q23–q25 (14). To validate the use of PCR to map gene location with chromosome 17 panel primers routinely used in our lab for the amplification of the GP1A, the ITGA2B and ITGB3 genes were employed to confirm the localization of these genes on chromosome 17. The GP1BA-specific forward primer begins at position 404 of the cDNA with the sequence 5*-TGGACGTCTCCTCAACCGGCTGACA-3 *, and the reverse primer begins at position 1001 in the cDNA with the sequence 5*-GTATGGGCTTTGGTGGGGAACTTGACC-3*. The GP1BA primers yielded a 600-bp product following PCR amplification. The ITGB3-specific forward primer GENOMICS
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0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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begins at position 633 of the cDNA with the sequence 5*-GATATGGAAGACCACCTGCTTGCCCAT-3 *, and the ITGB3-specific reverse primer begins at position 825 of the cDNA with the sequence 5*-GGATGCATCATTCCTCCAGCCAATC-3*. The ITGB3 primers amplify across an intron to yield an 800-bp product. Based upon the ICAM2 genomic structure obtained from the GenBank/EMBL Data Bank (Accession No. M1234), primers specific for exon 2 were designed. The forward primer beginning at nucleotide position 131 in the ICAM2 cDNA sequence with the sequence 5*-TGAGAAGGTATTCGAGGTAC-3* was used in conjunction with a reverse primer beginning at nucleotide position 370 in the ICAM2 cDNA with the sequence 5*-TTCATTGACTCCTGCTTCCC-3*. These primers yielded a 240-bp ICAM2-specific product following PCR amplification. Finally, the PECAM-1 primer set amplifies across intron 14 (6) to generate a 1000-bp product with PCR amplification. The PECAM1-specific forward primer begins at position 2038 of the cDNA with the sequence 5*-ATGCCAGTGGAAATGTCC-3 *, and the reverse primer begins at nucleotide position 2077 with the sequence 5*-TCAGAAGTGGTACTGGTG-3 *. All amplifications were conducted using Taq polymerase at 2.0 U/ml with annealing temperatures between 58 and 607C. GP1BA, ITGB3, and PECAM1 amplification products were electrophoresed through 1% agarose in 11 TAE at 100 V for 1 h. ICAM2 amplification products were electrophoresed through 2% agarose in 11 TAE at 100 V for 2 h. Using two separate PECAM-1-specific primer sets, aliquots of genomic DNA from the somatic cell hybrid chromosome panel were amplified by PCR and indicated that the human PECAM1 locus is on chromosome 17 (data not shown). Interestingly, the genes for several other adhesion molecules expressed on platelets and endothelium have also been previously localized to chromosome 17. To validate our assignment of the PECAM1 gene to chromosome 17 using this PCR-based approach and to determine if the PECAM1 gene colocalizes with any of the genes encoding these adhesion molecules, PCR analysis of a human chromosome 17 regional mapping panel, which subdivides chromosome 17 into 15 regions, was conducted. GPIba is a member of the leucine-rich glycoprotein family of cell adhesion molecules and is a subunit of the vWF receptor expressed on endothelial cells and platelets (3). The GP1BA locus (GPIba) has been provisionally localized to the 17p12–pter region (18). As shown in Fig. 1, using GP1BA-specific primers, only those clones containing the short arm of chromosome 17 were positive for the GP1BA locus. Integrins are a family of cytoadhesive receptors expressed on the surface of cells as Ca2/-depppendent ab heterodimers (5). Glycoprotein IIIa (GPIIIa) is a b subunit of the integrin family that is expressed on endothelial cells and platelets, complexed with different a subunits. On platelets, GPIIIa complexes with glycoprotein IIb (GPIIb) to form the fibrinogen receptor (11). Both the ITGB3 (GPIIIa) and the
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FIG. 1. Sublocalization of the human PECAM1 gene to the 17q23 –qter region. Using a chromosome 17 regional mapping panel, PCR amplification of genomic DNA was conducted with primers for GPIba (GP1BA by GDB nomenclature), GPIIIa (ITGB3), ICAM-2 (ICAM2) and PECAM1 as described in the text. In positive clones, GPIba-specific primers yielded a 600-bp product; GPIIIa-specific primers yielded an 800-bp product; ICAM-2 specific primers yielded a 240-bp product; and PECAM1-specific primers yielded a 1000-bp product.
ITGA2B (GPIIb) loci have been confirmed at the region 17q21.32 (1, 15). Using ITGB3-specific primers, only those clones containing the 17q21.32 region were positive for the ITGB3 locus. The ITGA2B locus also mapped to this same region (data not shown). ICAM2 is an immunoglobulin gene superfamily member that is expressed constitutively on the surface of endothelial cells and cells of myeloid lineage, where it functions as the counterreceptor for the integrin LFA-1 (aLb2 ) found on neutrophils and monocytes (17). The ICAM2 locus has been provisionally localized to the region 17q25–qter (14). Using ICAM2-specific primers, only those clones containing the 17q23–qter region were positive for the ICAM2 locus. Thus, the results of our chromosome 17 regional mapping panel were in agreement with previous mapping of the GP1BA, ITGA2B, ITGB3, and ICAM2 loci on chromosome 17 and validated the use of PCR to map gene location with our chromosome 17 panel. Finally, as shown in Fig. 1, using a PECAM-1-specific primer set, only those clones from the chromosome 17 mapping panel containing the 17q23–qter region were positive for the PECAM1 locus. Thus, the PECAM1 locus lies on the long arm of chromosome 17 in the 17q23–qter region. To verify the assignment of the PECAM1 locus to the long arm of chromosome 17 and to determine more precisely the localization of the PECAM1 locus, we performed fluorescence in situ hybridization (FISH). Briefly, DNA from a previously described P1 genomic clone that contains a portion of the PECAM1 gene (6) was labeled with digoxigenin –dUTP by nick-translation. A biotin-labeled chromosome 17 centromere-spe-
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FIG. 2. Precise localization of the PECAM1 gene to 17q23 by FISH. (A) In situ cohybridization of a PECAM1 P1 genomic clone (6) probe and a chromosome 17 centromere-specific probe (D17Z1) to human metaphase chromosomes. P1 DNA was labeled with digoxigenin –dUTP by nick-translation and hybridized along with a biotin-labeled chromosome 17 centromere-specific probe (D17Z1) to normal metaphase chromosomes. Specific hybridization and detection resulted in labeling the chromosome 17 centromere in red (arrowhead) and localization of the PECAM1 gene in green (arrow). (B) A partial view of a propidium-iodide-stained R-banded metaphase chromosome spread hybridized with the PECAM1 P1 genomic clone. DNA from the P1 clone was labeled by nick-translation with biotin-14 –dATP. Simultaneous detection of in situ hybridization signals and chromosome banding was performed by previously described techniques (4, 12). The chromosomes were observed under a Zeiss Axiophot fluorescence microscope equipped with a filter set simultaneously transmitting FITC-labeled probe sites and PI. True images were captured and stored on a TOMS image analysis system (TOMS, Columbus, GA), and prints were made using a SONY color printer. At least 20 metaphases were analyzed, and consistent signals were observed only on chromosome 17. The arrow denotes PECAM1-specific signals at 17q23.
cific probe (D17Z1) was cohybridized with the P1 probe. Specific hybridization of the PECAM1 probe was detected by incubating the slides in fluoresceinated antidigoxigenin antibodies, while hybridization of D17Z1 was visualized by incubation with Texas red–avidin followed by counterstaining with DAPI. As shown in Fig. 2A, these experiments resulted in the specific labeling of the chromosome 17 centromere in red and localization of the PECAM1 gene on the long arm of chromosome 17 in green. Finally, to determine precise localization of the PECAM1 gene, hybridization of the P1 clone to R-banded metaphase chromosome spreads was conducted. Briefly, purified DNA from the P1 clone was labeled by nick-translation with biotin-14–dATP (Bionick, Gibco-BRL) following the manufacturer’s protocol. Simultaneous detection of in situ hybridization signals and chromosome banding was performed by previously described techniques (4), with some modifications. Standard FISH methods were followed for the hybridization and detection of the probe (13). As shown in Fig. 2B, sublocalization of the PECAM1 locus to chromosomal region 17q23 was unequivocally made on banded metaphase chromosome spreads. Thus, utilizing two independent complementary techniques, PCR analysis of somatic cell hybrids and fluorescence in situ hybridization, we have mapped the human PECAM1 loci to chromosome 17, specifically the region 17q23. ACKNOWLEDGMENTS R.J.G. is the recipient of a Medical Scientist Training Fellowship from the Medical College of Wisconsin and a predoctoral fellowship from the American Heart Association, Wisconsin Affiliate. This work
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was supported by Grant HL40926 (to P.J.N.) and Training Grant HL-07209 from the National Institutes of Health. P.J.N. is an Established Investigator of the American Heart Association.
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