GENOMICS
8,
575-578
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SHORT COMMUNICATION Assignment of the Erythropoietin Receptor (EPOR) Gene to Mouse Chromosome 9 and Human Chromosome 19' M. BUDARF,* K. HuEf3NER,t B. EMANUEL,* C. M. CRocE,t N. G. COPELAND,* N. A. JENKINS,* AND A. D. D’ANDREAS *Division of Human Genetics and Molecular Biology, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19 704; t Fe/s Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19740; *Mammalian Genetics Laboratory, ABL-Basic Research Program, NC/-Frederick Cancer Research Facility, Frederick, Maryland 2 170 1; and §Children’s Hospital, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02 115 Received
April
16, 1990;
revised
June 22, 1990
A 4-kb BumHI fragment of the human EPOR gene, isolated from a human genomic library (Jones et al., 1990), was subcloned into pUC18 (Pharmacia). The 4-kb insert was digested with a variety of restriction enzymes and screened for unique sequences by Southern hybridization using 32P-labeled total human DNA. A 0.6-kb fragment from a PstI digest was relatively repeat free and used in subsequent analyses. The 0.6-kb fragment detected a 5%kb humanspecific band on Southern blots of HindIII-digested DNA samples (Fig. 1, lane 2). The 5%kb Hind111 band is also detected on Southern blots hybridized with 32P-labeled full-length mouse and human EPOR cDNAs. With long exposures, using the 0.6-kb PstI genomic probe, a faint 6.7-kb mouse-specific band and a faint 2.5-kb hamster-specific band can also be detected (data not shown). DNAs from a mouse-human somatic cell hybrid panel (Bauer et al., 1988; Huebner et al., 1988) were used to determine the chromosomal location of the EPOR. The human-specific 5.8-kb Hind111 fragment segregated concordantly with chromosome (Chr) 19 in the hybrid lines, as pictured in Fig. 1 and summarized in Fig. 2. Figures 1 and 2 also illustrate a partial regional localization of the human EPOR gene since hybrid JI4-2 retains 19q12-19qter but is negative for the EPOR gene (Fig. 1, lane 7). Thus, rodent-human hybrid analysis localizes the human EPOR gene to Chr lgpter-19q12. The mouse chromosomal location of the Epor locus was determined by interspecific backcross analysis using progeny derived from matings of ((C57BL/6J X Mus spretus)F, X C57BL/6J) mice. This interspe-
Erythropoietin (EPO), the primary regulator of mammalian erythropoiesis, binds and activates a specific receptor on erythroid progenitors. The human and mouse cDNAs for this receptor (EPOR) have recently been isolated. These cDNAs were used to establish the genomic location of the EPOR gene. By somatic cell hybrid analysis, the locus for the EPOR maps to human chromosome (Chr) lgpter-ql2. By interspecific backcross mapping the locus is tightly linked to the murine Ldlr locus near the centromere of mouse Chr 9. This region of mouse Chr 9 is homologous to a region of human Chr 19p13 carrying the human LDLR and MEL loci, strongly suggesting that the human EPOR gene is at 19~13 near the human LDLR locus. o ISSO Academic Press, inc.
Erythropoietin (EPO), a 34,000-Da glycoprotein hormone, is the primary regulator of mammalian erythropoiesis. EPO is synthesized and released by the kidney and it circulates to the bone marrow, where it stimulates erythroid progenitors via a specific receptor (EPOR). The cDNAs for the murine (D’Andrea et al., 1989a) and human (Jones et al., 1990) EPOR have recently been isolated and characterized. As inferred from the cDNA sequence, the EPOR is a 507-amino-acid polypeptide with a single membrane-spanning domain. More recently, the EPOR has been shown to share extensive amino acid homology with other growth factor receptors, including the receptors for interleukin-2 (D’Andrea et al., 1989b), interleukin-3 (Itoh et al., 1990), interleukin-4 (Idzerda et al., 1990), GM-CSF (Gearing et al., 1989), and prolactin and growth hormone (Bazan, 1989). 575
All
Copyright 0 1990 rights of reproduction
o&3%7543/90 $3.00 by Academic Press, Inc. in any form reserved.
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FIG. 1. The human EPOR gene segregates with Chr 19 in somatic cell hybrids. DNA (-10 pg/lane) was cleaved with restriction enzyme HindIII, electrophoresed in an agarose gel, transferred to nylon filter, and hybridized to the 0.6-kb 32P-labeled EPOR probe. Lane 1, mouse (from LMTKcells); lane 2, human (from K562 cells); lane 3, hybrid EF3 retaining human Chr 7, 14, 16,19,21, and X and segments of Chr 8 and 22; lane 4, hybrid GL3 retaining Chr 4, 6, 7, 14, 15, and 17-20 and a segment of Chr 12; lane 6, hybrid AA2 retaining Chr 3, 6, 10, 11, 13, 14, 19,20, and X as well as 9q34-9qter and 22pter-22qll; lane 6, hybrid GB-8 which retains a portion of the long arm of Chr 19; lane 7, hybrid JI4-2 retaining Chr 3,4,6,7,9-15,17,18,21, and 22 and segments of Chr 1,2,5,8, and 19q; lane 8, hybrid P12 retaining Chr 19 and others; lane 9, hybrid ED8 retaining Chr 14 and 19 and segments of 8 and 22; and lane 10, hybrid BD3 which retains Chr l-8,10-16,18-22, and X. Size in kilobases (kb) of the human-specific EPOR fragment is shown on the right. Chromosomes present in most of the hybrids are depicted diagrammatically in Fig. 2; each hybrid positive for human EPOR sequences (lanes 3-5,8, 9) retains Chr 19; hybrids that do not retain the EPOR gene are GB-8 and JI4-2 (lanes 6,7) which carry partial 19q, as determined by the presence of 19q linked loci and the absence of 19p linked loci (Ref. (19)). Hybrid BD3 is faintly positive (lane 10) but clearly positive on longer exposures (not shown).
Human
cific backcross mapping panel has been typed for over 435 loci distributed among all the mouse autosomes as well as the X chromosome. C57BL/6J and M. spretus DNAs were digested with several restriction enzymes and analyzed by Southern blot hybridization for informative restriction fragment length polymorphisms (RFLPs) using the murine EPOR cDNA probe. The single 7.6-kg M. spretus-specific XbaI fragment was used to follow the segregation of the Epor locus in backcross mice. The mapping results indicated that the Epor locus is located in the proximal region of mouse Chr 9 linked to the low-density lipoprotein receptor (Ldlr), the E26 avian leukemia virus oncogene, the 5’ domain (E&l), and the thymus cell antigen-l (Thy-1) gene (Fig. 3). The ratios of the total number of mice carrying recombinant chromosomes to the total number of mice analyzed for each pair of loci are l/161 for the Ldlr to Epor interval, H/158 for the Epor to Ets-1 interval, and 15/167 for the Ets-1 to Thy-l interval. The recombination distance (in centimorgans) f the standard errors and the most likely gene order are Ldlr-0.6 + 0.6-Epor-11.4 f 2.5-Ets-l10.2 + 2.3-Thy-l. The placement of Ldlr, Ets-1, and Thy-l relative to other Chr 9 markers and a comparison of the interspecific backcross map with the composite intraspecific backcross map have been reported previously (Kingsley et al.,
Chromosomes
Hybrid ci27 N9 cl2 77-31 77-30 s5 EC
M44 G5N PB5- 1 GLSD A83 803 EF3 GL3 AA2 J/4-2 FIG. 2. Presence of the human erythropoietin receptor (EPOR) sequences in a panel of 17 rodent-human hybrids. DNA (10 pg/lane) was digested with HindHI, electrophoresed in an agarose gel, transferred to a nylon filter, and hybridized with a unique 0.6-kb 3ZP-labeled human EPOR genomic probe. Final washes were performed& 65°C in 0.1X SSC and 1% SDS. Presence of the human EPOR gene was scored by the presence or absence of a 5.8-kb Hind111 fragment on Southern blot. A completely stippled box indicates that the hybrid named in the left column contains the chromosome indicated in the upper row; lower-right stippling indicates presence of the long arm (or part of the long arm, indicated by a smaller fraction of stippling) of the chromosome shown above the column; upper left stippling indicates presence of the short arm (or partial short arm) of the chromosome listed above the column; the column for Chr 19 is boldly outlined and stippled to highlight correlation of presence of this chromosome (or chromosome region) with the presence of the EPOR gene. The pattern of retention of the EPOR sequences in the hybrids is shown to the right, where presence of the gene in the hybrids is indicated by a stippled box with a plus sign and absence of the gene is indicated by an open box enclosing a minus sign.
SHORT
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Ets-1
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Thy-l
1 lq22
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COMMUNICATION
10.2
FIG. 3. Position of the Epor locus on mouse Chr 9. The Epor gene was placed on mouse Chr 9 by interspecific backcross analysis. Progeny were generated by mating (C57BL/6J x M. spretm) F, females and C57BL/6J males (4) and a random subset of the 204 backcross progeny were used to map the Epor locus. DNA digestion and Southern hybridization are as previously described (14). The Epor probe was a 0.75kb PstI fragment corresponding to the 3’ end of the mouse cDNA clone 190 (7) and final stringency of washes was 0.5X SSC, 0.1% SDS 65°C. Recombination distances were calculated (10) using the computer program SPRETUS MADNESS (D. Dave, Data Management Services, Inc., and A. M. Buchberg, NCI-FCRDC, ABL-BRP, Frederick, MD). The segregation patterns of Epar and flanking genes in 155 backcross animals that were typed in common for Epor, L&r, Es&I, and Thy-l are shown at the top. For individual pairs of loci more than 155 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J X it4. spretus) F, parent. The shaded boxes represent the presence of a C57BL/6J allele, and white boxes represent the presence of a M. spretlls allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 9 linkage map showing the location of Epor in relation to flanking genes is shown at the bottom. Recombination distances between loci in centimorgans are shown to the left of the chromosome and the positions of loci in human chromosomes are shown to the right.
ley et al., 1988), suggesting that these three loci form conserved groups in the mouse and human genomes. Furthermore, all of the human 19q markers thus far mapped in mouse are localized on mouse Chr 7 (Lalley et aZ., 1988). In contrast, genes that have been localized to human 19p have mapped to three different chromosomes in mouse: 8,9, and 17 (Ceci et al., 1990; Lalley et al., 1988). There are no known disease states suggestive of an EPOR mutation that have been mapped to mouse Chr 9 (Russell, 1979) or human Chr 19 (McKusick, 1988). The mapping of the EPOR to Chr 19p, however, will facilitate linkage studies of diseasesthat may be associated with EPOR defects, such as Diamond-Blackfan anemia (Alter, 1980; Nathan et al., 1978) andpoly&hernia vera (Golde et al., 1981). Further, since the EPOR is a member of a new hematopoietic growth factor receptor superfamily, it will be of interest to determine whether these genes are clustered or dispersed. ACKNOWLEDGMENTS We thank D. A. Swing, D. J. Gilbert, B. Cho, and M. Adams for excellent technical assistance. This research was supported, in part, by the National Cancer Institute, DHHS, under Contract NOl-CO-74101 with ABL and by Grants GM 32592, CA 39926, CA 39860, and KllHL02132-01 from the National Institutes of Health.
REFERENCES 1.
Amer.
J. Pe-
2. BAUER, S., HUEBNER,
K., BUDARF, M., FINAN, J., ERIKSON, J., EMANUEL, B., NOWELL, P. C., CROCE, C. M., AND MELCHERS, F. (1988). The human VpreB gene is located on chromosome 22 near a cluster of V segments. Immunogenetics 28: 328-333.
3. BAZAN, J. F. (1989).
4.
A novel family of growth factor receptors: A common binding domain in the growth hormone, prolactin, erythropoietin, and IL-6 receptors, and the ~75 IL-2 receptor &chain. Biochem. Biophys. Res. Commun. 164: 788-796. BUCHBERG, A. M., BEDIGIAN, H. G., TAYLOR, B. A., BROWNELL, E., IHLE, J. N., NAGATA, S., JENKINS, N. A., AND COPELAND, N. G. (1988). Localization of Evi-2 to chromosome 11: Linkage to other proto-oncogene and growth factor loci using interspecific backcrose mice. Oncogene Res. 2: 149-165.
5. CECI, J. D., JUSTICE, M. J., LOCK, L. F., JENKINS, 6.
The close linkage of Epor to Ldlr on mouse Chr 9 suggeststhat the human EPOR locus maps to human Chr 19p since the human LDLR gene has been mapped to 19p13.2-p13.1 (Le Beau et al., 1989). Also, the MEL oncogene, which maps to human Chr 19p13.2~ten (Le Beau et al., 1989), has been shown to have a homolog on mouse Chr 9 (summarized in Lal-
ALTER, B. P. (1980). Childhood red cell aplasia. diatr. Hematol Oncol. 2: 121-139.
N. COPELAND, N. G. (1990). An interspecific backcross map of mouse chromosome 8. Genomics 6: 72-79. D’ANDREA, A. D., FASMAN, G., AND LODISH, H. F. Erythropoietin receptor and interleukin-2 receptor 6 new receptor family. Cell 58: 1023-1024.
7. D’ANDREA, 8.
A., AND linkage (1989b). chain: A
A. D., LODISH, H. F., AND WONG, G. G. (1989a). Expression cloning of the murine erythropoietin receptor. Cell 67: 277-285. GEARING, D. J., KING, J., GOUGH, N. M., AND NICOLA, N. A. (1989). Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor. EMBO J. 8:
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GOLDE, D. W., HOCKING, W. G., KOEFFLER, H. P., AND ADAMSON, J. W. (1981). Polycythemia: Mechanisms and management. Ann. Intern. Med. 95: 71-84.
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GREEN, E. L. (1981). Linkage, In “Genetics and Probability ments,” pp. 77-113, Macmillan,
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HUEBNER, K., CANNIZARRO, L. A., NAKAMURA, T., HILLOVA, J., MARIAGE-SAMSON, R., HECHT, F., HILL, M., AND CROCE, C. M. (1988). A rearranged transforming gene, tre, is made up of human sequences derived from chromosome regions 5q, 17q, and 18q. Oncogene 3: 449-455.
12.
13.
ITOH, N., YONEHARA, S., SCHREURS, J., GORMAN, D. M., MARUYAMA, K., ISHII, A., YAHARA, I., ARAI, K., AND MIYAJIMA, A. (1990). Cloning of an interleukin-3 receptor gene: A member of a distinct receptor gene family. Science 247: 324327.
14.
JENKINS, N. A., COPELAND, N. G., TAYLOR, B. A., AND LEE, B. K. (1982). Organization, distribution, and stability of endogenous ecotropic murine leukemia virus DNA sequences in chromosomes of Mus musculus. J. Viral. 43: 26-36.
15.
JONES,
A. D., HAINES,
L. L., AND WONG,
erythropoietin characterization.
receptor: Blood
Cloning, ex76: 31-35.
16.
KINGSLEY, D. M., JENKINS, N. A., AND COPELAND, N. G. (1989). A molecular genetic linkage map of mouse chromosome 9 with regional localizations for the Gstu, 2’3g, Ets-1 and Ldlr loci. Genetics 123: 165172.
17.
LALLEY, P. A., DAVISSON, M. T., GRAVES, J. A. M., O’BRIAN, S. J., RODERICK, T. H., DOOLITTLE, D. P., AND HILLARD, A. L. (1988). Report to the committee on comparative mapping. Cytogenet. Cell Genet. 49: 227-235.
18.
LE BEAU, M. M., RYAN, D., AND PERICAK-VANCE, M. A. (1989). Report of the committee on genetic constitution of chromosomes 18 and 19: Human gene mapping 10. Cytogenet. Cell Genet. 51: 338-357.
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MARTINERIE, C., CANNIZZAFXO, L. A., CROCE, C. M., HUEBNER, K., KATZAV, S., AND BARBACID, M. (1990). The human vav protooncogene maps to chromosome region 19p12-19p13.2. Hum. Genet., in press. MCKUSICK, V. A. (1988). “Mendelian Inheritance in Man: Catalogs of Autosomal Dominant, Autosomal Recessive, and X-Linked Phenotypes,” 8th ed. Johns Hopkins Univ. Press, Baltimore, MD.
recombination, and mapping. in Animal Breeding ExperiNew York.
IDZERDA, R. L., MARCH, C. J., MOSLEY, B., LYMAN, S. D., VANDEN Bos, T., GIMPEL, S. D., DIN, W. S., GRABSTEIN, K. H., WIDNER, M. B., PARK, L. S., COSMAN, D., AND BECKMAN, M. P. (1990). Human interleukin 4 receptor confers biological responsiveness and defines a novel receptor superfamily. J. Exp. Med. 171: 861-873.
S. S., D’ANDREA,
G. G. (1990). Human pression, and biological
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NATHAN, D. G., CLARKE, B. J., HILLMAN, D. G., ALTER, B. P., AND HOUSMAN, D. E. (1978). Erythroidprecursors in congenital hypoplastic (Diamond-Blackfan) anemia. J. Clin. Znuest. 61: 489-498. RUSSELL, E. (1979). Hereditary anemias of the mouse: A review for geneticists. Adu. Genet. 20: 373-459.
8,
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(1990)
SHORT COMMUNICATION Assignment of the Erythropoietin Receptor (EPOR) Gene to Mouse Chromosome 9 and Human Chromosome 19' M. BUDARF,* K. HuEf3NER,t B. EMANUEL,* C. M. CRocE,t N. G. COPELAND,* N. A. JENKINS,* AND A. D. D’ANDREAS *Division of Human Genetics and Molecular Biology, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19 704; t Fe/s Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19740; *Mammalian Genetics Laboratory, ABL-Basic Research Program, NC/-Frederick Cancer Research Facility, Frederick, Maryland 2 170 1; and §Children’s Hospital, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02 115 Received
April
16, 1990;
revised
June 22, 1990
A 4-kb BumHI fragment of the human EPOR gene, isolated from a human genomic library (Jones et al., 1990), was subcloned into pUC18 (Pharmacia). The 4-kb insert was digested with a variety of restriction enzymes and screened for unique sequences by Southern hybridization using 32P-labeled total human DNA. A 0.6-kb fragment from a PstI digest was relatively repeat free and used in subsequent analyses. The 0.6-kb fragment detected a 5%kb humanspecific band on Southern blots of HindIII-digested DNA samples (Fig. 1, lane 2). The 5%kb Hind111 band is also detected on Southern blots hybridized with 32P-labeled full-length mouse and human EPOR cDNAs. With long exposures, using the 0.6-kb PstI genomic probe, a faint 6.7-kb mouse-specific band and a faint 2.5-kb hamster-specific band can also be detected (data not shown). DNAs from a mouse-human somatic cell hybrid panel (Bauer et al., 1988; Huebner et al., 1988) were used to determine the chromosomal location of the EPOR. The human-specific 5.8-kb Hind111 fragment segregated concordantly with chromosome (Chr) 19 in the hybrid lines, as pictured in Fig. 1 and summarized in Fig. 2. Figures 1 and 2 also illustrate a partial regional localization of the human EPOR gene since hybrid JI4-2 retains 19q12-19qter but is negative for the EPOR gene (Fig. 1, lane 7). Thus, rodent-human hybrid analysis localizes the human EPOR gene to Chr lgpter-19q12. The mouse chromosomal location of the Epor locus was determined by interspecific backcross analysis using progeny derived from matings of ((C57BL/6J X Mus spretus)F, X C57BL/6J) mice. This interspe-
Erythropoietin (EPO), the primary regulator of mammalian erythropoiesis, binds and activates a specific receptor on erythroid progenitors. The human and mouse cDNAs for this receptor (EPOR) have recently been isolated. These cDNAs were used to establish the genomic location of the EPOR gene. By somatic cell hybrid analysis, the locus for the EPOR maps to human chromosome (Chr) lgpter-ql2. By interspecific backcross mapping the locus is tightly linked to the murine Ldlr locus near the centromere of mouse Chr 9. This region of mouse Chr 9 is homologous to a region of human Chr 19p13 carrying the human LDLR and MEL loci, strongly suggesting that the human EPOR gene is at 19~13 near the human LDLR locus. o ISSO Academic Press, inc.
Erythropoietin (EPO), a 34,000-Da glycoprotein hormone, is the primary regulator of mammalian erythropoiesis. EPO is synthesized and released by the kidney and it circulates to the bone marrow, where it stimulates erythroid progenitors via a specific receptor (EPOR). The cDNAs for the murine (D’Andrea et al., 1989a) and human (Jones et al., 1990) EPOR have recently been isolated and characterized. As inferred from the cDNA sequence, the EPOR is a 507-amino-acid polypeptide with a single membrane-spanning domain. More recently, the EPOR has been shown to share extensive amino acid homology with other growth factor receptors, including the receptors for interleukin-2 (D’Andrea et al., 1989b), interleukin-3 (Itoh et al., 1990), interleukin-4 (Idzerda et al., 1990), GM-CSF (Gearing et al., 1989), and prolactin and growth hormone (Bazan, 1989). 575
All
Copyright 0 1990 rights of reproduction
o&3%7543/90 $3.00 by Academic Press, Inc. in any form reserved.
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FIG. 1. The human EPOR gene segregates with Chr 19 in somatic cell hybrids. DNA (-10 pg/lane) was cleaved with restriction enzyme HindIII, electrophoresed in an agarose gel, transferred to nylon filter, and hybridized to the 0.6-kb 32P-labeled EPOR probe. Lane 1, mouse (from LMTKcells); lane 2, human (from K562 cells); lane 3, hybrid EF3 retaining human Chr 7, 14, 16,19,21, and X and segments of Chr 8 and 22; lane 4, hybrid GL3 retaining Chr 4, 6, 7, 14, 15, and 17-20 and a segment of Chr 12; lane 6, hybrid AA2 retaining Chr 3, 6, 10, 11, 13, 14, 19,20, and X as well as 9q34-9qter and 22pter-22qll; lane 6, hybrid GB-8 which retains a portion of the long arm of Chr 19; lane 7, hybrid JI4-2 retaining Chr 3,4,6,7,9-15,17,18,21, and 22 and segments of Chr 1,2,5,8, and 19q; lane 8, hybrid P12 retaining Chr 19 and others; lane 9, hybrid ED8 retaining Chr 14 and 19 and segments of 8 and 22; and lane 10, hybrid BD3 which retains Chr l-8,10-16,18-22, and X. Size in kilobases (kb) of the human-specific EPOR fragment is shown on the right. Chromosomes present in most of the hybrids are depicted diagrammatically in Fig. 2; each hybrid positive for human EPOR sequences (lanes 3-5,8, 9) retains Chr 19; hybrids that do not retain the EPOR gene are GB-8 and JI4-2 (lanes 6,7) which carry partial 19q, as determined by the presence of 19q linked loci and the absence of 19p linked loci (Ref. (19)). Hybrid BD3 is faintly positive (lane 10) but clearly positive on longer exposures (not shown).
Human
cific backcross mapping panel has been typed for over 435 loci distributed among all the mouse autosomes as well as the X chromosome. C57BL/6J and M. spretus DNAs were digested with several restriction enzymes and analyzed by Southern blot hybridization for informative restriction fragment length polymorphisms (RFLPs) using the murine EPOR cDNA probe. The single 7.6-kg M. spretus-specific XbaI fragment was used to follow the segregation of the Epor locus in backcross mice. The mapping results indicated that the Epor locus is located in the proximal region of mouse Chr 9 linked to the low-density lipoprotein receptor (Ldlr), the E26 avian leukemia virus oncogene, the 5’ domain (E&l), and the thymus cell antigen-l (Thy-1) gene (Fig. 3). The ratios of the total number of mice carrying recombinant chromosomes to the total number of mice analyzed for each pair of loci are l/161 for the Ldlr to Epor interval, H/158 for the Epor to Ets-1 interval, and 15/167 for the Ets-1 to Thy-l interval. The recombination distance (in centimorgans) f the standard errors and the most likely gene order are Ldlr-0.6 + 0.6-Epor-11.4 f 2.5-Ets-l10.2 + 2.3-Thy-l. The placement of Ldlr, Ets-1, and Thy-l relative to other Chr 9 markers and a comparison of the interspecific backcross map with the composite intraspecific backcross map have been reported previously (Kingsley et al.,
Chromosomes
Hybrid ci27 N9 cl2 77-31 77-30 s5 EC
M44 G5N PB5- 1 GLSD A83 803 EF3 GL3 AA2 J/4-2 FIG. 2. Presence of the human erythropoietin receptor (EPOR) sequences in a panel of 17 rodent-human hybrids. DNA (10 pg/lane) was digested with HindHI, electrophoresed in an agarose gel, transferred to a nylon filter, and hybridized with a unique 0.6-kb 3ZP-labeled human EPOR genomic probe. Final washes were performed& 65°C in 0.1X SSC and 1% SDS. Presence of the human EPOR gene was scored by the presence or absence of a 5.8-kb Hind111 fragment on Southern blot. A completely stippled box indicates that the hybrid named in the left column contains the chromosome indicated in the upper row; lower-right stippling indicates presence of the long arm (or part of the long arm, indicated by a smaller fraction of stippling) of the chromosome shown above the column; upper left stippling indicates presence of the short arm (or partial short arm) of the chromosome listed above the column; the column for Chr 19 is boldly outlined and stippled to highlight correlation of presence of this chromosome (or chromosome region) with the presence of the EPOR gene. The pattern of retention of the EPOR sequences in the hybrids is shown to the right, where presence of the gene in the hybrids is indicated by a stippled box with a plus sign and absence of the gene is indicated by an open box enclosing a minus sign.
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Thy-l
1 lq22
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FIG. 3. Position of the Epor locus on mouse Chr 9. The Epor gene was placed on mouse Chr 9 by interspecific backcross analysis. Progeny were generated by mating (C57BL/6J x M. spretm) F, females and C57BL/6J males (4) and a random subset of the 204 backcross progeny were used to map the Epor locus. DNA digestion and Southern hybridization are as previously described (14). The Epor probe was a 0.75kb PstI fragment corresponding to the 3’ end of the mouse cDNA clone 190 (7) and final stringency of washes was 0.5X SSC, 0.1% SDS 65°C. Recombination distances were calculated (10) using the computer program SPRETUS MADNESS (D. Dave, Data Management Services, Inc., and A. M. Buchberg, NCI-FCRDC, ABL-BRP, Frederick, MD). The segregation patterns of Epar and flanking genes in 155 backcross animals that were typed in common for Epor, L&r, Es&I, and Thy-l are shown at the top. For individual pairs of loci more than 155 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J X it4. spretus) F, parent. The shaded boxes represent the presence of a C57BL/6J allele, and white boxes represent the presence of a M. spretlls allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 9 linkage map showing the location of Epor in relation to flanking genes is shown at the bottom. Recombination distances between loci in centimorgans are shown to the left of the chromosome and the positions of loci in human chromosomes are shown to the right.
ley et al., 1988), suggesting that these three loci form conserved groups in the mouse and human genomes. Furthermore, all of the human 19q markers thus far mapped in mouse are localized on mouse Chr 7 (Lalley et aZ., 1988). In contrast, genes that have been localized to human 19p have mapped to three different chromosomes in mouse: 8,9, and 17 (Ceci et al., 1990; Lalley et al., 1988). There are no known disease states suggestive of an EPOR mutation that have been mapped to mouse Chr 9 (Russell, 1979) or human Chr 19 (McKusick, 1988). The mapping of the EPOR to Chr 19p, however, will facilitate linkage studies of diseasesthat may be associated with EPOR defects, such as Diamond-Blackfan anemia (Alter, 1980; Nathan et al., 1978) andpoly&hernia vera (Golde et al., 1981). Further, since the EPOR is a member of a new hematopoietic growth factor receptor superfamily, it will be of interest to determine whether these genes are clustered or dispersed. ACKNOWLEDGMENTS We thank D. A. Swing, D. J. Gilbert, B. Cho, and M. Adams for excellent technical assistance. This research was supported, in part, by the National Cancer Institute, DHHS, under Contract NOl-CO-74101 with ABL and by Grants GM 32592, CA 39926, CA 39860, and KllHL02132-01 from the National Institutes of Health.
REFERENCES 1.
Amer.
J. Pe-
2. BAUER, S., HUEBNER,
K., BUDARF, M., FINAN, J., ERIKSON, J., EMANUEL, B., NOWELL, P. C., CROCE, C. M., AND MELCHERS, F. (1988). The human VpreB gene is located on chromosome 22 near a cluster of V segments. Immunogenetics 28: 328-333.
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