SHORT COMMUNICATION Assignment of the Gene Coding for the a,-Macroglobulin Receptor to Mouse Chromosome 15 and to Human Chromosome 12ql3-q14 by Isotopic and Nonisotopic in Situ Hybridization CARL HILLIKER,
FRED VAN LEUVEN, AND HERMAN VAN DEN BERGHE
Center for Human Genetics, Campus Gasthuisberg Received
September
30, 1991;
O&N, University of Leuven, B-3000, Belgium revised
January
23, 1992
human A2MR and the mouse A2mr (95%, Van Leuven, unpublished). The isotopic labeling and in situ hybridization were performed according to the procedure of Harper and Saunders (6) with the staining procedure set forth by Cannizzarro and Emanuel (3). Analysis of 75 metaphases from normal human lymphocytes yielded 170 grains showing significant hybridization on chromosome 12 (26/170 or 15.5%; results not shown), with the highest concentration of grains occurring in the region 12q13-q14 (16/26 or 62%). Nonisotopic insitu hybridization using the entire LRP-9 cDNA clone combined with the pericentric probe for chromosome 12 supported the 12q. Q 1992 Academic Press, Inc. localization of ASMR to 12q13-q14 (results not shown). For the mouse, 80 metaphases containing the Roberta,-Macroglobulin (A2M) is a plasma glycoprotein sonian translocation chromosome (6;15) were scored for that binds and inhibits proteinases from all subclasses a total of 196 grains showing significant hybridization to (14,17). The resultant A2M-proteinase complex is then chromosome 15 (45/196 grains or 23%) (Fig. 1). The hyinternalized in fibroblasts and macrophages by recep- bridization signal was predominantly localized to the retor-mediated endocytosis (16). Hertz et at. (7) described gion 15B2-Dl (30/45 grains or 66%). Previous studies concluded that LRP is an apolipoa cDNA encoding a .500-kDa liver membrane protein that is closely related to the LDL receptor, with the protein E-binding receptor (2) that is dependent on Ca2+ ions for binding the Apo E complex. This feature, which complete sequence coding for a protein of 4544 amino is important for receptor confirmation and ligand recogacids. They proposed that this LDL receptor-related nition, is also found in the human ASMR (12). We reprotein (LRP) is a recycling lipoprotein receptor with possible growth-modulating effects. The gene for the cently reported that the A2M gene is among a homoloLRP receptor was localized by in situ hybridization to gous group of genes present on human chromosome 12p and mouse chromosome 6 (9). A different conserved chromosome 12q13-q14 (13), a chromosome different group of genes located on human chromosome 12q was from that containing the LDL receptor gene (19p13.2~13.1) (5). Evidence supporting the fact that LRP is ac- located on mouse chromosome 15: ELA 1 (Ela-1), PFKX (Pfk-4), GPD 1 (Gdc-1), Hox 3 (Hox-3), WNT 1 (Wnttually the A2MR has been presented by two different l), and KRT 4 (Krt-2) (4). The localization of A2mr to groups (10, 15), both of which demonstrated that the mouse chromosome 15 adds additional support to the cDNA sequence of LRP is homologous with the partial protein sequence of the human ABMR. The cDNA clone linkage group with human chromosome 12q as discussed LRP-9 (7) and the probe pBR-12 (l), specific for the above. Moreover, this localization is different from that of the classical LDL receptor gene, located on mouse pericentric region of chromosome 12, were labeled with chromosome 9 (18), and also from any other known rebiotin-11-dUTP by nick-translation. The hybridization and subsequent detection were performed according to ceptor containing the type of repeats found in LDL-R the protocol of Lawrence et al. (11). The l.l-kb probe and LRP (6). We believe, therefore, to have located the authentic ABMR gene. Further studies are being underused for the isotopic in situ hybridization was generated by the polymerase chain reaction, essentially as de- taken to clarify the possible dual recognition function of scribed previously (8), to produce a nonrepetitive region this receptor and to understand the link, if any, between for which a high degree of homology existed between the lipoprotein and proteinase inhibitor metabolism. The assignment of the gene encoding the cY,-macroglobulin receptor (ASMR), which was first described as the low-density lipoprotein receptor-related protein, was confirmed by nonisotopic and isotopic in situ hybridizations on normal human metaphases to the region 12q13-q14. The same human cDNA, which has 95% sequence identity with the mouse A2mr, was hybridized to metaphases containing the Robertsonian translocation Rb(6;15)1Ald. The mouse A2mr gene was assigned to chromosome 15 in the region B2-Dl. This locus and other loci on mouse chromosome 15 have been shown to be homologous with loci on human chromosome
GENOMICS 13,472-474 (1992) 08&x-7543/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Inc. reserved.
472
SHORT
FIG. dCTP,
1. Complete for the ASMR
histogram gene.
of Rb(6;15)1Ald
translocation
COMMUNICATION
chromosomes
ACKNOWLEDGMENTS
M., Archidiacono, N., A human alpha satellite 12. Am. J. Hum. Genet.
4.
probe
labeled
with
tritium
7.
Hertz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H., and Stanley, K. K. (1988). Surface location and high affinity for calcium of a 500-kDa liver membrane protein closely related to the LDL-receptor suggests a physiological role as a lipoprotein receptor. EMBO J. 7: 4119-4127.
8.
Hilliker, C. E., Darville, M. I., Aly, M. S., Chirki, M., Szpirer, C., Marynen, P. A., Rousseau, G. G., and Cassiman, J. J. (1991). Human and rat chromosomal localization of two genes for 6phosphofructo-2-kinase:fructose 2,6-bisphosphatase by analysis of somatic cell hybrids and in situ hybridization. Genomics 10:
Miller, 0.
J., and DNA subset spe46: 784-788.
2. Beisiegel, 3.
the PCR-generated
M. E., and Saunders, G. F. (1981). Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization. Chromosoma 83: 431-439.
REFERENCES Baldini, A., Rocchi, Miller, D. A. (1990). cific for chromosome
with
6. Harper,
The authors thank Dr. Paola Dal Cin for reviewing the human chromosomes, Lou &as for technical support, and Karel Rondou for the photographic artwork. Probe LRP-9 was a gift form Dr. J. Hertz, and probe pBR12 was obtained from Dr. M. Rocchi.
1.
hybridized
U., Weber, W., Gudrum, I., Hertz, J., and Stanley, K. K. (1989). The LDL-receptor-related protein, LRP, is an apolipoprotein E-binding protein. Nature 341: 162-164. Cannizzarro, L., and Emanuel, B. S. (1984). An improved method for G-banding chromosomes after in situ hybridization. Cytogenet. Cell Genet. 38: 308-309. Davisson, M. T., Lalley, P. A., Peters, J., Doolittle, D. P., Hillyard, A. L., and Searle, A. G. (1990). Report of the comparative subcommittee for human and mouse homologies: Update to the Tenth International Workshop on Human Gene Mapping (Oxford Congress). Cytogenet. Cell Genet. 55: 434-456.
867-873. 9.
Hilliker, C., Overbergh, L., Van Leuven, F., and Van den Berghe, H. (1992). The gene coding for mouse a-2-macroglobulin is localized to chromosome 6F3-G3 by in situ hybridization. Submitted for publication.
10.
Kristensen, T., Moestrup, S., Gliemann, J., Bendtsen, L., Sandi, O., and Sottrup-Jensen, L. (1991). Evidence that the newly cloned low-density-lipoprotein receptor related protein (LRP) is the o-2-macroglobulin receptor. FEBS L&t. 276: 151-155.
ll.
Lawrence, J. B., Villnave, C. A., and Singer, R. H. (1988). Sensitive/high resolution chromatin and chromosome mapping in situ: Presence and orientation of two closely integrated copies of EBV in a lymphoma cell line. Cell 52: 51-61.
12.
Moestrup, S., Kaltoft, K., Sottrup-Jensen, L., and Gliemann, J. (1990). The human a-2-macroglobulin receptor contains high af-
5. Francke,
U., Brown, M. S., and Goldstein, J. L. (1984). Assignment of the human gene for the low density lipoprotein receptor to chromosome 19: Synteny of a receptor, a ligand, and a genetic disease. Proc. Natl. Acad. Sci. USA 81: 2826-2830.
474
SHORT fmity calcium binding and ligand recognition.
13.
14.
15.
sites important J. Biol. Chem.
COMMUNICATION
for receptor confirmation 256: 12,623-12,628.
Myklebost, O., Arheden, K., Rogne, S., Geurts van Kessel, A., Mandahl, N., Hertz, J., Stanley, K. K., Heim, S., and Mitelman, F. (1989). The gene coding for the putative apoE receptor is on chromosome 12 in the segment q13-q14. Genomics 5: 65-69. Sottrup-Jensen, L. (1989). Alpha macroglobulins: Structure, shape, and mechanism of proteinase complex formation. J. Biol. Chem. 264: 11,539-11,542. Strickland, D. K., Ashcom, J. D., Williams, S., Burgess, W. H., Migliorini, M., and Argraves, W. S. (1990). Sequence identity between the a-2-macroglobulin receptor low density lipoprotein
receptor functional 16.
17. 18.
related protein suggests that this molecule receptor. J. Biol. Chem. 265: 17,401-17,404.
is a multi-
Van Leuven, F., Cassiman, J. J., and Van den Berghe, H. (1979). Demonstration of an a-2-macroglobulin receptor in human fibroblasts, absent in tumor-derived cell lines. J. Biol. Chem. 254: 5155-5160. Van Leuven, F. (1982). Human a-2-macroglobulin: Structure and function. Trends Biochem. Sci. 7: 185-187. Wang, L. M., Killary, A. M., Fong, X-E., Parriott, S. K., Lalley, P. A., Bell, G. I., and Sakaguchi, A. Y. (1988). Chromosome assignment of mouse insulin, colony stimulating factor I, and lowdensity lipoprotein receptor. Genomics 3: 172-175.
FRED VAN LEUVEN, AND HERMAN VAN DEN BERGHE
Center for Human Genetics, Campus Gasthuisberg Received
September
30, 1991;
O&N, University of Leuven, B-3000, Belgium revised
January
23, 1992
human A2MR and the mouse A2mr (95%, Van Leuven, unpublished). The isotopic labeling and in situ hybridization were performed according to the procedure of Harper and Saunders (6) with the staining procedure set forth by Cannizzarro and Emanuel (3). Analysis of 75 metaphases from normal human lymphocytes yielded 170 grains showing significant hybridization on chromosome 12 (26/170 or 15.5%; results not shown), with the highest concentration of grains occurring in the region 12q13-q14 (16/26 or 62%). Nonisotopic insitu hybridization using the entire LRP-9 cDNA clone combined with the pericentric probe for chromosome 12 supported the 12q. Q 1992 Academic Press, Inc. localization of ASMR to 12q13-q14 (results not shown). For the mouse, 80 metaphases containing the Roberta,-Macroglobulin (A2M) is a plasma glycoprotein sonian translocation chromosome (6;15) were scored for that binds and inhibits proteinases from all subclasses a total of 196 grains showing significant hybridization to (14,17). The resultant A2M-proteinase complex is then chromosome 15 (45/196 grains or 23%) (Fig. 1). The hyinternalized in fibroblasts and macrophages by recep- bridization signal was predominantly localized to the retor-mediated endocytosis (16). Hertz et at. (7) described gion 15B2-Dl (30/45 grains or 66%). Previous studies concluded that LRP is an apolipoa cDNA encoding a .500-kDa liver membrane protein that is closely related to the LDL receptor, with the protein E-binding receptor (2) that is dependent on Ca2+ ions for binding the Apo E complex. This feature, which complete sequence coding for a protein of 4544 amino is important for receptor confirmation and ligand recogacids. They proposed that this LDL receptor-related nition, is also found in the human ASMR (12). We reprotein (LRP) is a recycling lipoprotein receptor with possible growth-modulating effects. The gene for the cently reported that the A2M gene is among a homoloLRP receptor was localized by in situ hybridization to gous group of genes present on human chromosome 12p and mouse chromosome 6 (9). A different conserved chromosome 12q13-q14 (13), a chromosome different group of genes located on human chromosome 12q was from that containing the LDL receptor gene (19p13.2~13.1) (5). Evidence supporting the fact that LRP is ac- located on mouse chromosome 15: ELA 1 (Ela-1), PFKX (Pfk-4), GPD 1 (Gdc-1), Hox 3 (Hox-3), WNT 1 (Wnttually the A2MR has been presented by two different l), and KRT 4 (Krt-2) (4). The localization of A2mr to groups (10, 15), both of which demonstrated that the mouse chromosome 15 adds additional support to the cDNA sequence of LRP is homologous with the partial protein sequence of the human ABMR. The cDNA clone linkage group with human chromosome 12q as discussed LRP-9 (7) and the probe pBR-12 (l), specific for the above. Moreover, this localization is different from that of the classical LDL receptor gene, located on mouse pericentric region of chromosome 12, were labeled with chromosome 9 (18), and also from any other known rebiotin-11-dUTP by nick-translation. The hybridization and subsequent detection were performed according to ceptor containing the type of repeats found in LDL-R the protocol of Lawrence et al. (11). The l.l-kb probe and LRP (6). We believe, therefore, to have located the authentic ABMR gene. Further studies are being underused for the isotopic in situ hybridization was generated by the polymerase chain reaction, essentially as de- taken to clarify the possible dual recognition function of scribed previously (8), to produce a nonrepetitive region this receptor and to understand the link, if any, between for which a high degree of homology existed between the lipoprotein and proteinase inhibitor metabolism. The assignment of the gene encoding the cY,-macroglobulin receptor (ASMR), which was first described as the low-density lipoprotein receptor-related protein, was confirmed by nonisotopic and isotopic in situ hybridizations on normal human metaphases to the region 12q13-q14. The same human cDNA, which has 95% sequence identity with the mouse A2mr, was hybridized to metaphases containing the Robertsonian translocation Rb(6;15)1Ald. The mouse A2mr gene was assigned to chromosome 15 in the region B2-Dl. This locus and other loci on mouse chromosome 15 have been shown to be homologous with loci on human chromosome
GENOMICS 13,472-474 (1992) 08&x-7543/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Inc. reserved.
472
SHORT
FIG. dCTP,
1. Complete for the ASMR
histogram gene.
of Rb(6;15)1Ald
translocation
COMMUNICATION
chromosomes
ACKNOWLEDGMENTS
M., Archidiacono, N., A human alpha satellite 12. Am. J. Hum. Genet.
4.
probe
labeled
with
tritium
7.
Hertz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H., and Stanley, K. K. (1988). Surface location and high affinity for calcium of a 500-kDa liver membrane protein closely related to the LDL-receptor suggests a physiological role as a lipoprotein receptor. EMBO J. 7: 4119-4127.
8.
Hilliker, C. E., Darville, M. I., Aly, M. S., Chirki, M., Szpirer, C., Marynen, P. A., Rousseau, G. G., and Cassiman, J. J. (1991). Human and rat chromosomal localization of two genes for 6phosphofructo-2-kinase:fructose 2,6-bisphosphatase by analysis of somatic cell hybrids and in situ hybridization. Genomics 10:
Miller, 0.
J., and DNA subset spe46: 784-788.
2. Beisiegel, 3.
the PCR-generated
M. E., and Saunders, G. F. (1981). Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization. Chromosoma 83: 431-439.
REFERENCES Baldini, A., Rocchi, Miller, D. A. (1990). cific for chromosome
with
6. Harper,
The authors thank Dr. Paola Dal Cin for reviewing the human chromosomes, Lou &as for technical support, and Karel Rondou for the photographic artwork. Probe LRP-9 was a gift form Dr. J. Hertz, and probe pBR12 was obtained from Dr. M. Rocchi.
1.
hybridized
U., Weber, W., Gudrum, I., Hertz, J., and Stanley, K. K. (1989). The LDL-receptor-related protein, LRP, is an apolipoprotein E-binding protein. Nature 341: 162-164. Cannizzarro, L., and Emanuel, B. S. (1984). An improved method for G-banding chromosomes after in situ hybridization. Cytogenet. Cell Genet. 38: 308-309. Davisson, M. T., Lalley, P. A., Peters, J., Doolittle, D. P., Hillyard, A. L., and Searle, A. G. (1990). Report of the comparative subcommittee for human and mouse homologies: Update to the Tenth International Workshop on Human Gene Mapping (Oxford Congress). Cytogenet. Cell Genet. 55: 434-456.
867-873. 9.
Hilliker, C., Overbergh, L., Van Leuven, F., and Van den Berghe, H. (1992). The gene coding for mouse a-2-macroglobulin is localized to chromosome 6F3-G3 by in situ hybridization. Submitted for publication.
10.
Kristensen, T., Moestrup, S., Gliemann, J., Bendtsen, L., Sandi, O., and Sottrup-Jensen, L. (1991). Evidence that the newly cloned low-density-lipoprotein receptor related protein (LRP) is the o-2-macroglobulin receptor. FEBS L&t. 276: 151-155.
ll.
Lawrence, J. B., Villnave, C. A., and Singer, R. H. (1988). Sensitive/high resolution chromatin and chromosome mapping in situ: Presence and orientation of two closely integrated copies of EBV in a lymphoma cell line. Cell 52: 51-61.
12.
Moestrup, S., Kaltoft, K., Sottrup-Jensen, L., and Gliemann, J. (1990). The human a-2-macroglobulin receptor contains high af-
5. Francke,
U., Brown, M. S., and Goldstein, J. L. (1984). Assignment of the human gene for the low density lipoprotein receptor to chromosome 19: Synteny of a receptor, a ligand, and a genetic disease. Proc. Natl. Acad. Sci. USA 81: 2826-2830.
474
SHORT fmity calcium binding and ligand recognition.
13.
14.
15.
sites important J. Biol. Chem.
COMMUNICATION
for receptor confirmation 256: 12,623-12,628.
Myklebost, O., Arheden, K., Rogne, S., Geurts van Kessel, A., Mandahl, N., Hertz, J., Stanley, K. K., Heim, S., and Mitelman, F. (1989). The gene coding for the putative apoE receptor is on chromosome 12 in the segment q13-q14. Genomics 5: 65-69. Sottrup-Jensen, L. (1989). Alpha macroglobulins: Structure, shape, and mechanism of proteinase complex formation. J. Biol. Chem. 264: 11,539-11,542. Strickland, D. K., Ashcom, J. D., Williams, S., Burgess, W. H., Migliorini, M., and Argraves, W. S. (1990). Sequence identity between the a-2-macroglobulin receptor low density lipoprotein
receptor functional 16.
17. 18.
related protein suggests that this molecule receptor. J. Biol. Chem. 265: 17,401-17,404.
is a multi-
Van Leuven, F., Cassiman, J. J., and Van den Berghe, H. (1979). Demonstration of an a-2-macroglobulin receptor in human fibroblasts, absent in tumor-derived cell lines. J. Biol. Chem. 254: 5155-5160. Van Leuven, F. (1982). Human a-2-macroglobulin: Structure and function. Trends Biochem. Sci. 7: 185-187. Wang, L. M., Killary, A. M., Fong, X-E., Parriott, S. K., Lalley, P. A., Bell, G. I., and Sakaguchi, A. Y. (1988). Chromosome assignment of mouse insulin, colony stimulating factor I, and lowdensity lipoprotein receptor. Genomics 3: 172-175.
