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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 O&N, University of Leuven, B-3000, Belgium

Received September 30, 1991; revised January 23, 1992

The assignment of the gene encoding the cY,-macroglobulin receptor (ASMR), which was first described as the low-den- sity lipoprotein receptor-related protein, was confirmed by nonisotopic and isotopic in situ hybridizations on normal hu- man 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 Robert- sonian 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 12q. Q 1992 Academic Press, Inc.

a,-Macroglobulin (A2M) is a plasma glycoprotein that binds and inhibits proteinases from all subclasses (14,17). The resultant A2M-proteinase complex is then internalized in fibroblasts and macrophages by recep- tor-mediated endocytosis (16). Hertz et at. (7) described a cDNA encoding a .500-kDa liver membrane protein that is closely related to the LDL receptor, with the complete sequence coding for a protein of 4544 amino acids. They proposed that this LDL receptor-related protein (LRP) is a recycling lipoprotein receptor with possible growth-modulating effects. The gene for the LRP receptor was localized by in situ hybridization to chromosome 12q13-q14 (13), a chromosome different from that containing the LDL receptor gene (19p13.2- ~13.1) (5). Evidence supporting the fact that LRP is ac- tually the A2MR has been presented by two different groups (10, 15), both of which demonstrated that the cDNA sequence of LRP is homologous with the partial protein sequence of the human ABMR. The cDNA clone LRP-9 (7) and the probe pBR-12 (l), specific for the pericentric region of chromosome 12, were labeled with biotin-11-dUTP by nick-translation. The hybridization and subsequent detection were performed according to the protocol of Lawrence et al. (11). The l.l-kb probe used for the isotopic in situ hybridization was generated by the polymerase chain reaction, essentially as de- scribed previously (8), to produce a nonrepetitive region for which a high degree of homology existed between the

GENOMICS 13,472-474 (1992)

human A2MR and the mouse A2mr (95%, Van Leuven, unpublished). The isotopic labeling and in situ hybridiza- tion 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 chromo- some 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 hybridiza- tion using the entire LRP-9 cDNA clone combined with the pericentric probe for chromosome 12 supported the localization of ASMR to 12q13-q14 (results not shown).

For the mouse, 80 metaphases containing the Robert- sonian translocation chromosome (6;15) were scored for a total of 196 grains showing significant hybridization to chromosome 15 (45/196 grains or 23%) (Fig. 1). The hy- bridization signal was predominantly localized to the re- gion 15B2-Dl (30/45 grains or 66%).

Previous studies concluded that LRP is an apolipo- protein E-binding receptor (2) that is dependent on Ca2+ ions for binding the Apo E complex. This feature, which is important for receptor confirmation and ligand recog- nition, is also found in the human ASMR (12). We re- cently reported that the A2M gene is among a homolo- gous group of genes present on human chromosome 12p and mouse chromosome 6 (9). A different conserved group of genes located on human chromosome 12q was located on mouse chromosome 15: ELA 1 (Ela-1), PFKX (Pfk-4), GPD 1 (Gdc-1), Hox 3 (Hox-3), WNT 1 (Wnt- l), and KRT 4 (Krt-2) (4). The localization of A2mr to mouse chromosome 15 adds additional support to the linkage group with human chromosome 12q as discussed above. Moreover, this localization is different from that of the classical LDL receptor gene, located on mouse chromosome 9 (18), and also from any other known re- ceptor containing the type of repeats found in LDL-R and LRP (6). We believe, therefore, to have located the authentic ABMR gene. Further studies are being under- taken to clarify the possible dual recognition function of this receptor and to understand the link, if any, between lipoprotein and proteinase inhibitor metabolism.

08&x-7543/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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FIG. 1. Complete histogram of Rb(6;15)1Ald translocation chromosomes hybridized with the PCR-generated probe labeled with tritium dCTP, for the ASMR gene.

ACKNOWLEDGMENTS 6.

The authors thank Dr. Paola Dal Cin for reviewing the human chro- mosomes, Lou &as for technical support, and Karel Rondou for the 7. photographic artwork. Probe LRP-9 was a gift form Dr. J. Hertz, and probe pBR12 was obtained from Dr. M. Rocchi.

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