- Email: [email protected]
The hemopexin gene maps to the same location as the β-globin gene cluster on human chromosome 11
GENOMICS
3,48-52
(1988)
The Hemopexin Gene Maps to the Same Location Gene Cluster on Human Chromosome MARTHA
LIAO LAw,*,t
GUANG-YUN
as the @-Globin 11
CAI,* JUDITH A. HARTZ,* CAROL JONES,*‘*
AND FA-TEN KAO*,$
*Eleanor Roosevelt Institute for Cancer Research, 1899 Gaylord Street, Denver, Colorado 80206, and Departments of tpediatrics and *Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver, Colorado80262 Received
November
16, 1987;
May 23, 1988
structure of hemopexin deduced from a cDNA sequence was examined by Altruda et al. (1985). The characterization and physiological function of hemopexin have been widely investigated since it was discovered in 1958 (Neale et al., 1958). Independently, we used mouse monoclonal antibody SBV22 against hemopexin (Cai et al., 1983) to screen a human Xgtll cDNA library and isolated five positive clones. Using these cDNA clones, we assigned the human gene encoding hemopexin to chromosome 11 by Southern blot analysis of somatic cell hybrids (Cai and Law, 1986). This assignment was confirmed by Naylor et al. (1987), who further regionally mapped the gene to the short arm of the chromosome. In this report, we present a more refined localization of the gene to llp15.4-~15.5, the same location as the @-globin gene cluster, by in situ chromosome hybridization. The organization of the hemopexin gene in various species was also analyzed.
Using human hemopexin cDNA clones isolated from Xgtll cDNA library as probes, we have carried out Southern blot analysis of a series of human-Chinese hamster somatic cell hybrids containing different combinations of human chromosomes. Synteny analysis revealed 100% concordance between the hemopexin gene and human chromosome 11. In situ hybridization of ‘H-labeled hemopexin cDNA to metaphase chromosomes prepared from human lymphocytes further localized the gene to the region p16.4-~15.6, the same location as the @-globin gene cluster. 8lS8SAcademicF'ma,Inc.
Hemopexin is a @-glycoprotein found in various mammalian sera (Bremmer, 1964; Witz and Gross, 1965; Muller-Eberhard and English, 1967; Thorbecke et al., 1973; Cai et al., 1983; Strati1 et al., 1984). It binds to heme with high affinity (Hrkal et al., 1974; Morgan et al., 1976) and acts as a transport protein mediating the transfer of circulating heme to liver parenchymal cells (Muller-Eberhard et al., 1970; Smith and Morgan, 1978, 1979). The heme-hemopexin complex interacts with the liver cell surface, and the heme is internalized by a specific receptor-mediated process whereby the apoprotein is released to the plasma (Smith and Morgan, 1979,198l). A variety of cultured nonliver cells can also ingest hemopexin from the ambient media, and this might serve in part as a source for intracellular iron (Davies et al., 1979; Cai et al., 1983; Taketani et al., 1986, 1987). Human hemopexin (M, 63,000) consists of a single polypeptide chain of 439 amino acid residues and six oligosaccharides linked to different amino acids (Takahashi et al., 1985). Internal repeating homology of the primary
MATERIALS Hempexin
AND METHODS
cDNA Probes
Five human hemopexin cDNA clones including XHx-1 and XHx-5 were isolated from a Xgtll-cDNA library with monoclonal antibody SBV22 against hemopexin (Cai et al., 1983). Nucleotide sequencing revealed that XHx-1 contains a l.O-kb partial cDNA fragment of hemopexin, and XHx-5 contains a 1.6-kb cDNA fragment encoding the entire mature hemopexin and part of the signal peptide (Cai et al., manuscript in preparation). The cDNA fragments were released from the Xgtll phage by EcoRI digestion and subcloned into the M13mp19 phage vector for sequence analysis. The nucleotide sequence confirms the previously reported amino acid sequence of human hemopexin (Takahashi et al., 1985). One of the subclones, designated MHx-Sa, containing the 1.6-kb insert was used for in situ chromosome hybridization.
Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. 503048. cw3-7543&3$3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
revised
48
HEMOPEXIN
Somatic Cell HybricEs and DNA Preparation A series of human-rodent cell hybrids used in the present study was derived from several fusions involving various Chinese hamster ovary (CHO) auxotrophic mutants and human fibroblasts or lymphocytes. The human chromosome content in these hybrids and the methods of analyses have been described (Law et al., 1986; Morse et al., 1982). Human cell line HT1080 (Croce, 1976) and the CHO auxotrophic mutant Gly-A (Law and Kao, 1978) were used as genomic DNA controls for human and Chinese hamster, respectively. The probe used in Southern blot studies of cell hybrids was XHx-1. The procedures for DNA preparation from the cultured cells and the cell hybrids have been described (Gusella et al., 1979). In Situ Chromosome Hybridization The procedures used for in situ hybridization to metaphase chromosomes were similar to those described by Harper and Saunders (1981). Briefly, human chromosomes were prepared from phytohemagglutinin-stimulated peripheral blood lymphocytes, synchronized with BrdU and thymidine, and incubated with colcemid (0.1 fig/ml) for 10 min prior to harvest. The chromosome spreads were aged for 5 days, treated with RNase, and denatured with formamide. The hemopexin cDNA clone MHx-5a was labeled with [3H]dCTP, [3H]TTP, and [3H]dATP to a specific activity of 1.1 X 1Oa cpmlrg DNA by the random-primer labeling technique of Feinberg and Vogelstein (1983,1984). Hybridization was carried out at 37°C for 18 h in the presence of formamide and dextran sulfate. After hybridization and extensive washing, the slides were coated with Kodak NTB2 nuclear track emulsion and stored at 4°C for 14 or 21 days before developing. The slides were then stained with Hoechst H33258 for 15 min, placed under uv light for 75 min, restained with Giemsa, and examined under the microscope. Southern Blot Analysis of Genomic DNAs from Different Species Genomic DNAs were prepared from lymphocytes of human, bovine, sheep, rabbit, guinea pig, mouse, dog, pig, chicken, monkey, and rat. Blood (10 ml) from each species was centrifuged at 3000 rpm for 20 min. The supernatant was removed and the cell pellet was washed and centrifuged twice with PBS. Five and a half packed cell volumes of hemolytic buffer (0.131 it4 ammonium chloride, 0.91 m&f ammonium carbonate) was added to each sample. After incubation in an ice bath for 20 min, the supernatant, which contained erythrocyte lysate, was removed by centrifugation. The pellet was resuspended in 10 ml of STE (10 mM Tris-Cl, pH 7.6, 1 mM EDTA, 0.1 M NaCl), treated
GENE
49
MAPPING
with 0.25 ml of proteinase K (2 mg/ml) and 0.25 ml of SDS (20%) at 37°C overnight, extracted with phenolchloroform, precipitated with ethanol, and redissolved in TE buffer. EcoRI or HindIII restriction endonuclease was used to digest the DNAs to completion. A Southern blot of these digested DNAs electrophoresed on 0.8% agarose gel was hybridized to the 32P-labeled 1.6-kb hemopexin cDNA fragment released from XHx-5 by the method of Wahl et al. (1979). RESULTS
Assignment of the Hemopexin Chromosome llp15.4-~15.5
Gene to Human
Upon HindIII digestion, human genomic DNA exhibited an 11-kb band that hybridized to the human hemopexin cDNA clone XHx-1 (Fig. 1). HindIII-digested Chinese hamster genomic DNA produced two hybridizing bands at different positions, 6.0 and 2.8 kb (Fig. 1, lane 12), indicating homology between the hamster and the human hemopexin gene sequences. Table 1 presents hybridization results from 10 cell hybrids. Synteny analysis based on the human chromosome content in the hybrids localized the hemopexin gene to human chromosome 11. Definitive evidence came from the positive hybridization of hybrid Jl (Fig. 1, lane ll), which contains human chromosome 11 as its only human genomic material (Kao et al., 1976). In situ hybridization of 3H-labeled MHx-5a to metaphase chromosomes prepared from human lympho1 2 3 4 5 6 7 8 9 101112
Kb
-
7.1 6.1
-
5.1
-
4.1
-
3.1
-
2.0
DNA8 from FIG. 1. Southern blot analysis of HindHI-digested human, hamster, and cell hybrids using 32P-labeled XHx-1 a8 probe: Lane 1, human (HT1030); lane 2, CP3-1; lane 3, CP5-1; lane 4, CP12-1; lane 5, CP14-1; lane 6, CP15-1; lane 7, CP17-1; lane 8, CPU%1; lane 9, CP20-1; lane 10, CP%-1; lane 11, Jl; lane 12, hamster (Gly-A). The 1-kb DNA ladder (BRL) is shown on the far right.
50
LAW
Assignment
of the Human Hemopexin
ET
AL.
TABLE
1
Gene to Chromosome Human
Hybrid
1
CP3-1 CP5-1
(i Presence
3
4
5
6
7
8
9
11
10
20
60
40
(+) or absence
(-)
40
and Synteny Analysis
12
13
14
15
16
17
18
19
20
21
22
X
Human Hx gene + + + _ +
-----
-
-+++---
+
2644344445 (%)
Hybridization
chromosome
---++-----++-+-++++++-+ +---++-+++-++++++++++++-+---+++++++-------++ ---+++----------++--+++ ---+++----+++-++++----+--++------+----+-----+------+--+--++-+++----+--++--+-++----++--++--+ +--++--++-----------++-----------
CP12-1 CP14-1 CP15-1 CP17-1 CPM-1 CP20-1 CP2f3-1 Jl Concordant hybrids Concordant frequency
2
11 by Molecular
30
40
40
of the hybridizing
40
40
human
50
bands
10
5
5
5
5
6
5
5
4
4
4
3
5
100
50
50
50
50
60
50
50
40
40
40
30
60
using
cytes further localized the hemopexin gene to the distal portion of the short arm of chromosome 11. In 52 random spreads, 21 had grains on chromosome 11. Among 216 grains in the 45 spreads that were analyzed in detail, 65 grains (30%) were on chromosome 11, 37 grains (57%) of which were clustered in the region p15.4-~15.5 (Fig. 2).
the human
hemopexin
probe.
Southern Blot Analysis Figure 3 shows that hemopexin cDNA cross-hybridizes to DNAs extracted from lymphocytes of human, bovine, sheep, rabbit, guinea pig, mouse, dog, pig, monkey, and rat (lanes l-8,10 and ll), but not from chicken lymphocytes (lane 9). The human and mon-
11 FIG. 2. Grain distribution on human a probe. Grains are mainly in the region
chromosome llp16.4-~15.5.
11 after
in situ hybridization
to metaphase
chromosomes
using
‘H-labeled
MHx-5a
as
HEMOPEXIN
123456789lOll
51
GENE MAPPING
1234567891011
*-
6.1 5.1 4.1
-
20
-
1.6
-
1.0
FIG. 8. Southern blot analysis of restriction endonuclease-digested DNAs from various animal species. Lane 1, human; lane 2, bovine; lane 3, sheep; lane 4, rabbit; lane S, guinea pig; lane 6, mouse; lane ‘7, dog, lane 8, pig; lane 9, chicken; lane 10, monkey; lane 11, rat. Restriction enzymes used: (A) EcoRI; (B) HindHI. DNAs from all species cross-hybridized with the hemopexin cDNA except that from chicken. The 1-kb DNA ladder (BRL) is shown on the far right.
key hemopexin genomic sequences have HindI restriction fragments of approximately 11 kb which cross-hybridized to the cDNA probe. DISCUSSION
Previously we have reported preliminary results on the localization of hemopexin gene to human chromosome 11 (Cai and Law, 1986). Naylor et al. (1987) regionally mapped the gene to the short arm of the chromosome. Further assignment of hemopexin gene to the llp15.4-~15.5 region as shown in the present study implies close linkage between the hemopexin gene and the b-globin gene cluster, which was also localized to the same region (Lin et al., 1985). Hemopexin and p-globin have the common feature of being heme carriers, but they are expressed in different tissues. The assignment of the hemopexin gene to the same region as the @-globin gene complex may provide a basis for studying their physical proximity using pulsed-field gel electrophoresis (Schwartz and Cantor, 1984) and for further analysis of the structurefunction relationship among these related genes during development and differentiation in mammals. Although DNA sequences undergo changes during evolution, the hemopexin gene appears to contain a very conserved region that is present in the genomes of various mammalian species as shown by their cross-hybridization to the cDNA probe in Southern blot analysis (Fig. 3). The relatively simple hybridization pattern using either EcoRI or Hi&II implies that there is probably a single gene encoding hemo-
pexin in the mammalian haploid genome and that no pseudogenes are present. To date, no genetic disease is known to result from mutations of the hemopexin gene. However, there are disorders such as hemolytic anemias in which the level of hemopexin is reduced because of increased hemopexin catabolism (Wochner et al., 1974), and other disorders such as chronic neuromuscular diseases and acute intermittent porphyria in which the level of hemopexin is elevated due to increased rates of synthesis (Adornato et al., 1978; Foidart et aZ., 1983). Elevated levels of hemopexin were also found in mice with certain solid tumors (Ishiguro et aZ., 1984). Interestingly, CAMP can dramatically increase the rate of hemopexin synthesis in cultured hepatoma cells (McCracken et al., 1984). The physical map of the hemopexin gene in the human genome provides a genetic basis which will facilitate the studies on the mechanisms regulating hemopexin synthesis in these pathways, as well as the gene structure and its developmental biology in mammals. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health (HD17449, HD02080, GM33903) and the National Science Foundation (PCM-8306832) and by a grant from R. J. Reynolds Industries. REFERENCES 1.
ADORNATO,
B. T., ENGEL,
W. K., AND FOIDART-DESALLE,
(1978). Elevations of hemopexin levels in neuromuscular ease. Arch. Neural. 35: 577-530.
M.
dis-
LAW ET AL.
52
2. ALTRUDA, F., POLI, V., RESTAGNO, G., ARGOS, P., CORTESE, R., AND SILENGO, L. (1985). The primary structure of human hemopexin deduced from cDNA sequence: Evidence for internal, repeating homology. Nucleic Acids Res. 13: 3841-3859. 3. BREMNER, K. C. (1964). Studies on hapotoglobin and haemopexin in the plasma of cattle. Au& J. Exp. Med. Sci. 42: 643-656. 4. CAI, G.-Y., AND LAW, M. L. (1986). Cloning and characterization of a human gene coding for hemopexin. Amer. J. Hum. Genet. 39: A191. 5. CAI, G.-Y., SUHAN, J., BLOSE, G. A., AND BLOSE, S. H. (1983). Detection of hemopexin (a heme-binding plasma fl-glycoprotein) in the lysosomes of cultured cells using a monoclonai antibody against hemopexin. In “Cold Spring Harbor Laboratory Annual Report 1983,” pp. 25-27, Cold Spring Harbor, NY. 6. CROCE, C. M. (1976). Loss of chromosomes in somatic cell hybrids between HT-1080 human fibrosarcoma cells and mouse peritoneal macrophages. Proc. Natl. Acad. Sci. USA 73: 3248-3252. 7. DAVIES, G. A., SMITH, A., AND MULLER-EBERHARD, U. (1979). Hepatic subcellular metabolism of heme from hemehemopexin: Incorporation of iron into ferritin. Biochem. Biophys. Res. Commun. 91: 1504-1511. 8. FEINBERG, A. P., AND VOGELSTEIN, B. (1983). A technique for radiolabeiing DNA restriction endonuclease fragments to high specific activity. AnaL B&hem. 132: 6-13. 9. FEINBERG, A. P., AND VOGELSTEIN, B. (1984). Addendum: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137: 266-267. 10. FOIDART, M., LIEM, H. H., ADORNATO, B. T., ENGEL, W. K., AND MULLER-EBERHARD, U. (1983). Hemopexin metabolism in patients with altered serum levels. J. Lab. Clin. Med. 102: 838-846. 11. GUSELLA, J., VARSANYI-BREINER, A., KAO, F. T., JONES, C., PUCK, T. T., KEYS, C., ORKIN, S., AND HOUSEMAN, D. (1979). Precise localization of human j3-globin gene complex on chromosome 11. Proc. NatL Acad. Sci. USA 76: 5239-5243. 12. HARPER, M. E., AND SAUNDERS, G. F. (1981). Localization of single copy DNA sequence on G-banded human chromosomes by in situ hybridization. Chromosoma 83: 431-439. 13. HRKAL, Z., VODRAZKA, Z., AND KALOUSEK, I. (1974). Transfer of heme from ferrihemoglobin and ferrihemoglobin isolated chains to hemopexin. Eur. J. Biochem. 43: 73-78. 14. ISHIGURO, T., IMANISHI, K., AND SUZUKI, I. (1984). Hemopexin levels in mice. Znt. J. Zmmunopharmacol. 6: 241-244. 15. KAO, F. T., JONES, C., AND PUCK, T. T. (1976). Genetics of somatic mammalian cells: Genetic, immunologic, and biochemical analysis with Chinese hamster cell hybrids containing selected human chromosomes. Pmt. Natl. Acad. Sci. USA 73: 193-197. 16. LAW, M. L., CAI, G.-Y., LIN, F-K., WEI, Q., HUANG, S. Z., HARTZ, J. H., MORSE, H., LIN, C-H., JONES, C., AND KAO, F.-T. (1986). Chromosomal assignment of the erythropoietin gene and its DNA polymorphism. Proc. NatL Acad. Sci. USA 83: 6920-6924. 17. LAW, M. L., AND KAO, F.-T. (1978). Induced segregation of human syntenic genes by 5-bromodeoxyuridine + near-visible light. Somat. Cell Genet. 4: 465-476. 18. LIN, C. C., DRAPER, P. N., AND DE BRAEKELEER, M. (1985). High-resolution chromosomal localization of the &gene of the human @-globin gene complex by in situ hybridization. Cytogenet.
Cell Genet.
39:
269-274.
19. 20. 21. 22. 23.
24. 25.
26. 27. 28. 29. 30.
31.
32.
33.
MCCRACKEN, A. A., EMMETT, M., CROWLE, A. J., AND BROWN, J. L. (1984). Studies on the secretion of serum proteins from rat hepatoma cells. Hepatology 4: 715-721. MORGAN, W. T., LIEM, H. H., SUTOR, R. P., AND MULLEREBERHARD, U. (1976). Transfer of heme from heme-albumin to hemopexin. Bicchem. Biophys. Acta 444: 435-445. MORSE, H. G., PATTERSON, P., AND JONES, C. (1982). Giemsa-11 technique: Applications in basic research. Mamm. Chromosomes News 23: 127-133. MULLER-EBERHARD, U., AND ENGLISH, E. C. (1967). Purification and partial characterization of human hemopexin. J. Lab. Clin. Med. 70: 619-626. MULLER-EBERHARD, U., BOSMAN, C., AND LIEM, H. H. (1970). Tissue localization of the heme-hemopexin complex in the rabbit and the rat as studied by light microscope with use of radioisotopes. J. Lab. Clin. Med. 76: 426-431. NAYLOR, S. L., ALTRUDA, F., MARSHALL, A., SILENGO, L., AND BOWMAN, B. H. (1987). Hemopexin is localized to human chromosome 11. Somat. Cell Genet. 13: 355-358. NEALE, F. C., ABER, G. M., AND NORTHAM, B. E. (1958). The demonstration of intravascular haemolysis by means of serum paper electrophoresis and modification of Schumm’s reaction. J. Clin. Pathol. 11: 206-219. SCHWARTZ, D. C., AND CANTOR, C. R. (1984). Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37: 67-75. SMITH, A., AND MORGAN, W. T. (1978). Transport of heme by hemopexin to the liver: Evidence for receptor-mediated uptake. Biochem. Biophys. Res. Commun. 04: 151-157. SMITH, A., AND MORGAN, W. T. (1979). Haem transport to the liver by haemopexin, receptor-mediated uptake with recycling of the protein. B&hem. J. 162: 47-54. SMITH, A., AND MORGAN, W. T. (1981). Hemopexin-mediated transport of heme into isolated rat hepatocytes. J. BioL Chem. 256: 10902-10909. STRATIL, A., GLASNAK, V., TOMASEK, V., WILLIAMS, J., AND CLAMP, J. R. (1984). Haemopexin in sheep, mouflon and goat: Genetic polymorphism, heterogeneity and partial characterization. Anim. Blood Groups B&hem. Genet. 15: 285-297. TAKAHASHI, N., TAKAHASHI, Y., AND PUTNAM, F. W. (1985). Complete amino acid sequence of human hemopexin, the heme-binding protein of serum. Proc. NatL Acad. Sci. USA 02: 73-77. TAKETANI, S., KOHNO, H., AND TOKUNAGA, R. (1986). Receptor-mediated heme uptake from hemopexin by human erythroleukemia K562 cells. Biochem. Znt. 13: 307-312. TAKETANI, S., KONNO, H., AND TOKUNAGA, R. 0987). Cell surface receptor for hemopexin in human leukemia HL60 cells, specific binding, affinity labeling, and fate of heme. J. Biol.
Chem.
262:
4639-4643.
34. THORBECKE, G. J., LIEM, H. H., KNIGHT, S., Cox, K., AND MULLER-EBERHARD, U. (1973). Sites of formation of the serum proteins transfer& and hemopexin. J. Clin. Invest. 52: 725-731. 35. WAHL, G. M., STERN, M., AND STARK, G. R. (1979). Efficient transfer of large DNA fragments from agarose gels to diaxobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc. Natl. Acad. Sci. USA 76: 3683-3687. 36. WITZ, I., AND GROSS, J. (1965). Purification and partial characterization of mouse hemopexin (Beta S-111 globulin). Proc. Sot. Ezp. Biol. Med. 121: 111-116. 37. WOCHNER, R. D., SPILBERG, I., 110, A., LIEM, H. H., AND MULLER-EBERHARD, U. (1974). Hemopexin metabolism in sickle-cell disease, porphyrias and control subjects-Effects of heme injection. N. EngL J. Med. 290: 822-826.
3,48-52
(1988)
The Hemopexin Gene Maps to the Same Location Gene Cluster on Human Chromosome MARTHA
LIAO LAw,*,t
GUANG-YUN
as the @-Globin 11
CAI,* JUDITH A. HARTZ,* CAROL JONES,*‘*
AND FA-TEN KAO*,$
*Eleanor Roosevelt Institute for Cancer Research, 1899 Gaylord Street, Denver, Colorado 80206, and Departments of tpediatrics and *Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver, Colorado80262 Received
November
16, 1987;
May 23, 1988
structure of hemopexin deduced from a cDNA sequence was examined by Altruda et al. (1985). The characterization and physiological function of hemopexin have been widely investigated since it was discovered in 1958 (Neale et al., 1958). Independently, we used mouse monoclonal antibody SBV22 against hemopexin (Cai et al., 1983) to screen a human Xgtll cDNA library and isolated five positive clones. Using these cDNA clones, we assigned the human gene encoding hemopexin to chromosome 11 by Southern blot analysis of somatic cell hybrids (Cai and Law, 1986). This assignment was confirmed by Naylor et al. (1987), who further regionally mapped the gene to the short arm of the chromosome. In this report, we present a more refined localization of the gene to llp15.4-~15.5, the same location as the @-globin gene cluster, by in situ chromosome hybridization. The organization of the hemopexin gene in various species was also analyzed.
Using human hemopexin cDNA clones isolated from Xgtll cDNA library as probes, we have carried out Southern blot analysis of a series of human-Chinese hamster somatic cell hybrids containing different combinations of human chromosomes. Synteny analysis revealed 100% concordance between the hemopexin gene and human chromosome 11. In situ hybridization of ‘H-labeled hemopexin cDNA to metaphase chromosomes prepared from human lymphocytes further localized the gene to the region p16.4-~15.6, the same location as the @-globin gene cluster. 8lS8SAcademicF'ma,Inc.
Hemopexin is a @-glycoprotein found in various mammalian sera (Bremmer, 1964; Witz and Gross, 1965; Muller-Eberhard and English, 1967; Thorbecke et al., 1973; Cai et al., 1983; Strati1 et al., 1984). It binds to heme with high affinity (Hrkal et al., 1974; Morgan et al., 1976) and acts as a transport protein mediating the transfer of circulating heme to liver parenchymal cells (Muller-Eberhard et al., 1970; Smith and Morgan, 1978, 1979). The heme-hemopexin complex interacts with the liver cell surface, and the heme is internalized by a specific receptor-mediated process whereby the apoprotein is released to the plasma (Smith and Morgan, 1979,198l). A variety of cultured nonliver cells can also ingest hemopexin from the ambient media, and this might serve in part as a source for intracellular iron (Davies et al., 1979; Cai et al., 1983; Taketani et al., 1986, 1987). Human hemopexin (M, 63,000) consists of a single polypeptide chain of 439 amino acid residues and six oligosaccharides linked to different amino acids (Takahashi et al., 1985). Internal repeating homology of the primary
MATERIALS Hempexin
AND METHODS
cDNA Probes
Five human hemopexin cDNA clones including XHx-1 and XHx-5 were isolated from a Xgtll-cDNA library with monoclonal antibody SBV22 against hemopexin (Cai et al., 1983). Nucleotide sequencing revealed that XHx-1 contains a l.O-kb partial cDNA fragment of hemopexin, and XHx-5 contains a 1.6-kb cDNA fragment encoding the entire mature hemopexin and part of the signal peptide (Cai et al., manuscript in preparation). The cDNA fragments were released from the Xgtll phage by EcoRI digestion and subcloned into the M13mp19 phage vector for sequence analysis. The nucleotide sequence confirms the previously reported amino acid sequence of human hemopexin (Takahashi et al., 1985). One of the subclones, designated MHx-Sa, containing the 1.6-kb insert was used for in situ chromosome hybridization.
Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. 503048. cw3-7543&3$3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
revised
48
HEMOPEXIN
Somatic Cell HybricEs and DNA Preparation A series of human-rodent cell hybrids used in the present study was derived from several fusions involving various Chinese hamster ovary (CHO) auxotrophic mutants and human fibroblasts or lymphocytes. The human chromosome content in these hybrids and the methods of analyses have been described (Law et al., 1986; Morse et al., 1982). Human cell line HT1080 (Croce, 1976) and the CHO auxotrophic mutant Gly-A (Law and Kao, 1978) were used as genomic DNA controls for human and Chinese hamster, respectively. The probe used in Southern blot studies of cell hybrids was XHx-1. The procedures for DNA preparation from the cultured cells and the cell hybrids have been described (Gusella et al., 1979). In Situ Chromosome Hybridization The procedures used for in situ hybridization to metaphase chromosomes were similar to those described by Harper and Saunders (1981). Briefly, human chromosomes were prepared from phytohemagglutinin-stimulated peripheral blood lymphocytes, synchronized with BrdU and thymidine, and incubated with colcemid (0.1 fig/ml) for 10 min prior to harvest. The chromosome spreads were aged for 5 days, treated with RNase, and denatured with formamide. The hemopexin cDNA clone MHx-5a was labeled with [3H]dCTP, [3H]TTP, and [3H]dATP to a specific activity of 1.1 X 1Oa cpmlrg DNA by the random-primer labeling technique of Feinberg and Vogelstein (1983,1984). Hybridization was carried out at 37°C for 18 h in the presence of formamide and dextran sulfate. After hybridization and extensive washing, the slides were coated with Kodak NTB2 nuclear track emulsion and stored at 4°C for 14 or 21 days before developing. The slides were then stained with Hoechst H33258 for 15 min, placed under uv light for 75 min, restained with Giemsa, and examined under the microscope. Southern Blot Analysis of Genomic DNAs from Different Species Genomic DNAs were prepared from lymphocytes of human, bovine, sheep, rabbit, guinea pig, mouse, dog, pig, chicken, monkey, and rat. Blood (10 ml) from each species was centrifuged at 3000 rpm for 20 min. The supernatant was removed and the cell pellet was washed and centrifuged twice with PBS. Five and a half packed cell volumes of hemolytic buffer (0.131 it4 ammonium chloride, 0.91 m&f ammonium carbonate) was added to each sample. After incubation in an ice bath for 20 min, the supernatant, which contained erythrocyte lysate, was removed by centrifugation. The pellet was resuspended in 10 ml of STE (10 mM Tris-Cl, pH 7.6, 1 mM EDTA, 0.1 M NaCl), treated
GENE
49
MAPPING
with 0.25 ml of proteinase K (2 mg/ml) and 0.25 ml of SDS (20%) at 37°C overnight, extracted with phenolchloroform, precipitated with ethanol, and redissolved in TE buffer. EcoRI or HindIII restriction endonuclease was used to digest the DNAs to completion. A Southern blot of these digested DNAs electrophoresed on 0.8% agarose gel was hybridized to the 32P-labeled 1.6-kb hemopexin cDNA fragment released from XHx-5 by the method of Wahl et al. (1979). RESULTS
Assignment of the Hemopexin Chromosome llp15.4-~15.5
Gene to Human
Upon HindIII digestion, human genomic DNA exhibited an 11-kb band that hybridized to the human hemopexin cDNA clone XHx-1 (Fig. 1). HindIII-digested Chinese hamster genomic DNA produced two hybridizing bands at different positions, 6.0 and 2.8 kb (Fig. 1, lane 12), indicating homology between the hamster and the human hemopexin gene sequences. Table 1 presents hybridization results from 10 cell hybrids. Synteny analysis based on the human chromosome content in the hybrids localized the hemopexin gene to human chromosome 11. Definitive evidence came from the positive hybridization of hybrid Jl (Fig. 1, lane ll), which contains human chromosome 11 as its only human genomic material (Kao et al., 1976). In situ hybridization of 3H-labeled MHx-5a to metaphase chromosomes prepared from human lympho1 2 3 4 5 6 7 8 9 101112
Kb
-
7.1 6.1
-
5.1
-
4.1
-
3.1
-
2.0
DNA8 from FIG. 1. Southern blot analysis of HindHI-digested human, hamster, and cell hybrids using 32P-labeled XHx-1 a8 probe: Lane 1, human (HT1030); lane 2, CP3-1; lane 3, CP5-1; lane 4, CP12-1; lane 5, CP14-1; lane 6, CP15-1; lane 7, CP17-1; lane 8, CPU%1; lane 9, CP20-1; lane 10, CP%-1; lane 11, Jl; lane 12, hamster (Gly-A). The 1-kb DNA ladder (BRL) is shown on the far right.
50
LAW
Assignment
of the Human Hemopexin
ET
AL.
TABLE
1
Gene to Chromosome Human
Hybrid
1
CP3-1 CP5-1
(i Presence
3
4
5
6
7
8
9
11
10
20
60
40
(+) or absence
(-)
40
and Synteny Analysis
12
13
14
15
16
17
18
19
20
21
22
X
Human Hx gene + + + _ +
-----
-
-+++---
+
2644344445 (%)
Hybridization
chromosome
---++-----++-+-++++++-+ +---++-+++-++++++++++++-+---+++++++-------++ ---+++----------++--+++ ---+++----+++-++++----+--++------+----+-----+------+--+--++-+++----+--++--+-++----++--++--+ +--++--++-----------++-----------
CP12-1 CP14-1 CP15-1 CP17-1 CPM-1 CP20-1 CP2f3-1 Jl Concordant hybrids Concordant frequency
2
11 by Molecular
30
40
40
of the hybridizing
40
40
human
50
bands
10
5
5
5
5
6
5
5
4
4
4
3
5
100
50
50
50
50
60
50
50
40
40
40
30
60
using
cytes further localized the hemopexin gene to the distal portion of the short arm of chromosome 11. In 52 random spreads, 21 had grains on chromosome 11. Among 216 grains in the 45 spreads that were analyzed in detail, 65 grains (30%) were on chromosome 11, 37 grains (57%) of which were clustered in the region p15.4-~15.5 (Fig. 2).
the human
hemopexin
probe.
Southern Blot Analysis Figure 3 shows that hemopexin cDNA cross-hybridizes to DNAs extracted from lymphocytes of human, bovine, sheep, rabbit, guinea pig, mouse, dog, pig, monkey, and rat (lanes l-8,10 and ll), but not from chicken lymphocytes (lane 9). The human and mon-
11 FIG. 2. Grain distribution on human a probe. Grains are mainly in the region
chromosome llp16.4-~15.5.
11 after
in situ hybridization
to metaphase
chromosomes
using
‘H-labeled
MHx-5a
as
HEMOPEXIN
123456789lOll
51
GENE MAPPING
1234567891011
*-
6.1 5.1 4.1
-
20
-
1.6
-
1.0
FIG. 8. Southern blot analysis of restriction endonuclease-digested DNAs from various animal species. Lane 1, human; lane 2, bovine; lane 3, sheep; lane 4, rabbit; lane S, guinea pig; lane 6, mouse; lane ‘7, dog, lane 8, pig; lane 9, chicken; lane 10, monkey; lane 11, rat. Restriction enzymes used: (A) EcoRI; (B) HindHI. DNAs from all species cross-hybridized with the hemopexin cDNA except that from chicken. The 1-kb DNA ladder (BRL) is shown on the far right.
key hemopexin genomic sequences have HindI restriction fragments of approximately 11 kb which cross-hybridized to the cDNA probe. DISCUSSION
Previously we have reported preliminary results on the localization of hemopexin gene to human chromosome 11 (Cai and Law, 1986). Naylor et al. (1987) regionally mapped the gene to the short arm of the chromosome. Further assignment of hemopexin gene to the llp15.4-~15.5 region as shown in the present study implies close linkage between the hemopexin gene and the b-globin gene cluster, which was also localized to the same region (Lin et al., 1985). Hemopexin and p-globin have the common feature of being heme carriers, but they are expressed in different tissues. The assignment of the hemopexin gene to the same region as the @-globin gene complex may provide a basis for studying their physical proximity using pulsed-field gel electrophoresis (Schwartz and Cantor, 1984) and for further analysis of the structurefunction relationship among these related genes during development and differentiation in mammals. Although DNA sequences undergo changes during evolution, the hemopexin gene appears to contain a very conserved region that is present in the genomes of various mammalian species as shown by their cross-hybridization to the cDNA probe in Southern blot analysis (Fig. 3). The relatively simple hybridization pattern using either EcoRI or Hi&II implies that there is probably a single gene encoding hemo-
pexin in the mammalian haploid genome and that no pseudogenes are present. To date, no genetic disease is known to result from mutations of the hemopexin gene. However, there are disorders such as hemolytic anemias in which the level of hemopexin is reduced because of increased hemopexin catabolism (Wochner et al., 1974), and other disorders such as chronic neuromuscular diseases and acute intermittent porphyria in which the level of hemopexin is elevated due to increased rates of synthesis (Adornato et al., 1978; Foidart et aZ., 1983). Elevated levels of hemopexin were also found in mice with certain solid tumors (Ishiguro et aZ., 1984). Interestingly, CAMP can dramatically increase the rate of hemopexin synthesis in cultured hepatoma cells (McCracken et al., 1984). The physical map of the hemopexin gene in the human genome provides a genetic basis which will facilitate the studies on the mechanisms regulating hemopexin synthesis in these pathways, as well as the gene structure and its developmental biology in mammals. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health (HD17449, HD02080, GM33903) and the National Science Foundation (PCM-8306832) and by a grant from R. J. Reynolds Industries. REFERENCES 1.
ADORNATO,
B. T., ENGEL,
W. K., AND FOIDART-DESALLE,
(1978). Elevations of hemopexin levels in neuromuscular ease. Arch. Neural. 35: 577-530.
M.
dis-
LAW ET AL.
52
2. ALTRUDA, F., POLI, V., RESTAGNO, G., ARGOS, P., CORTESE, R., AND SILENGO, L. (1985). The primary structure of human hemopexin deduced from cDNA sequence: Evidence for internal, repeating homology. Nucleic Acids Res. 13: 3841-3859. 3. BREMNER, K. C. (1964). Studies on hapotoglobin and haemopexin in the plasma of cattle. Au& J. Exp. Med. Sci. 42: 643-656. 4. CAI, G.-Y., AND LAW, M. L. (1986). Cloning and characterization of a human gene coding for hemopexin. Amer. J. Hum. Genet. 39: A191. 5. CAI, G.-Y., SUHAN, J., BLOSE, G. A., AND BLOSE, S. H. (1983). Detection of hemopexin (a heme-binding plasma fl-glycoprotein) in the lysosomes of cultured cells using a monoclonai antibody against hemopexin. In “Cold Spring Harbor Laboratory Annual Report 1983,” pp. 25-27, Cold Spring Harbor, NY. 6. CROCE, C. M. (1976). Loss of chromosomes in somatic cell hybrids between HT-1080 human fibrosarcoma cells and mouse peritoneal macrophages. Proc. Natl. Acad. Sci. USA 73: 3248-3252. 7. DAVIES, G. A., SMITH, A., AND MULLER-EBERHARD, U. (1979). Hepatic subcellular metabolism of heme from hemehemopexin: Incorporation of iron into ferritin. Biochem. Biophys. Res. Commun. 91: 1504-1511. 8. FEINBERG, A. P., AND VOGELSTEIN, B. (1983). A technique for radiolabeiing DNA restriction endonuclease fragments to high specific activity. AnaL B&hem. 132: 6-13. 9. FEINBERG, A. P., AND VOGELSTEIN, B. (1984). Addendum: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137: 266-267. 10. FOIDART, M., LIEM, H. H., ADORNATO, B. T., ENGEL, W. K., AND MULLER-EBERHARD, U. (1983). Hemopexin metabolism in patients with altered serum levels. J. Lab. Clin. Med. 102: 838-846. 11. GUSELLA, J., VARSANYI-BREINER, A., KAO, F. T., JONES, C., PUCK, T. T., KEYS, C., ORKIN, S., AND HOUSEMAN, D. (1979). Precise localization of human j3-globin gene complex on chromosome 11. Proc. NatL Acad. Sci. USA 76: 5239-5243. 12. HARPER, M. E., AND SAUNDERS, G. F. (1981). Localization of single copy DNA sequence on G-banded human chromosomes by in situ hybridization. Chromosoma 83: 431-439. 13. HRKAL, Z., VODRAZKA, Z., AND KALOUSEK, I. (1974). Transfer of heme from ferrihemoglobin and ferrihemoglobin isolated chains to hemopexin. Eur. J. Biochem. 43: 73-78. 14. ISHIGURO, T., IMANISHI, K., AND SUZUKI, I. (1984). Hemopexin levels in mice. Znt. J. Zmmunopharmacol. 6: 241-244. 15. KAO, F. T., JONES, C., AND PUCK, T. T. (1976). Genetics of somatic mammalian cells: Genetic, immunologic, and biochemical analysis with Chinese hamster cell hybrids containing selected human chromosomes. Pmt. Natl. Acad. Sci. USA 73: 193-197. 16. LAW, M. L., CAI, G.-Y., LIN, F-K., WEI, Q., HUANG, S. Z., HARTZ, J. H., MORSE, H., LIN, C-H., JONES, C., AND KAO, F.-T. (1986). Chromosomal assignment of the erythropoietin gene and its DNA polymorphism. Proc. NatL Acad. Sci. USA 83: 6920-6924. 17. LAW, M. L., AND KAO, F.-T. (1978). Induced segregation of human syntenic genes by 5-bromodeoxyuridine + near-visible light. Somat. Cell Genet. 4: 465-476. 18. LIN, C. C., DRAPER, P. N., AND DE BRAEKELEER, M. (1985). High-resolution chromosomal localization of the &gene of the human @-globin gene complex by in situ hybridization. Cytogenet.
Cell Genet.
39:
269-274.
19. 20. 21. 22. 23.
24. 25.
26. 27. 28. 29. 30.
31.
32.
33.
MCCRACKEN, A. A., EMMETT, M., CROWLE, A. J., AND BROWN, J. L. (1984). Studies on the secretion of serum proteins from rat hepatoma cells. Hepatology 4: 715-721. MORGAN, W. T., LIEM, H. H., SUTOR, R. P., AND MULLEREBERHARD, U. (1976). Transfer of heme from heme-albumin to hemopexin. Bicchem. Biophys. Acta 444: 435-445. MORSE, H. G., PATTERSON, P., AND JONES, C. (1982). Giemsa-11 technique: Applications in basic research. Mamm. Chromosomes News 23: 127-133. MULLER-EBERHARD, U., AND ENGLISH, E. C. (1967). Purification and partial characterization of human hemopexin. J. Lab. Clin. Med. 70: 619-626. MULLER-EBERHARD, U., BOSMAN, C., AND LIEM, H. H. (1970). Tissue localization of the heme-hemopexin complex in the rabbit and the rat as studied by light microscope with use of radioisotopes. J. Lab. Clin. Med. 76: 426-431. NAYLOR, S. L., ALTRUDA, F., MARSHALL, A., SILENGO, L., AND BOWMAN, B. H. (1987). Hemopexin is localized to human chromosome 11. Somat. Cell Genet. 13: 355-358. NEALE, F. C., ABER, G. M., AND NORTHAM, B. E. (1958). The demonstration of intravascular haemolysis by means of serum paper electrophoresis and modification of Schumm’s reaction. J. Clin. Pathol. 11: 206-219. SCHWARTZ, D. C., AND CANTOR, C. R. (1984). Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37: 67-75. SMITH, A., AND MORGAN, W. T. (1978). Transport of heme by hemopexin to the liver: Evidence for receptor-mediated uptake. Biochem. Biophys. Res. Commun. 04: 151-157. SMITH, A., AND MORGAN, W. T. (1979). Haem transport to the liver by haemopexin, receptor-mediated uptake with recycling of the protein. B&hem. J. 162: 47-54. SMITH, A., AND MORGAN, W. T. (1981). Hemopexin-mediated transport of heme into isolated rat hepatocytes. J. BioL Chem. 256: 10902-10909. STRATIL, A., GLASNAK, V., TOMASEK, V., WILLIAMS, J., AND CLAMP, J. R. (1984). Haemopexin in sheep, mouflon and goat: Genetic polymorphism, heterogeneity and partial characterization. Anim. Blood Groups B&hem. Genet. 15: 285-297. TAKAHASHI, N., TAKAHASHI, Y., AND PUTNAM, F. W. (1985). Complete amino acid sequence of human hemopexin, the heme-binding protein of serum. Proc. NatL Acad. Sci. USA 02: 73-77. TAKETANI, S., KOHNO, H., AND TOKUNAGA, R. (1986). Receptor-mediated heme uptake from hemopexin by human erythroleukemia K562 cells. Biochem. Znt. 13: 307-312. TAKETANI, S., KONNO, H., AND TOKUNAGA, R. 0987). Cell surface receptor for hemopexin in human leukemia HL60 cells, specific binding, affinity labeling, and fate of heme. J. Biol.
Chem.
262:
4639-4643.
34. THORBECKE, G. J., LIEM, H. H., KNIGHT, S., Cox, K., AND MULLER-EBERHARD, U. (1973). Sites of formation of the serum proteins transfer& and hemopexin. J. Clin. Invest. 52: 725-731. 35. WAHL, G. M., STERN, M., AND STARK, G. R. (1979). Efficient transfer of large DNA fragments from agarose gels to diaxobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc. Natl. Acad. Sci. USA 76: 3683-3687. 36. WITZ, I., AND GROSS, J. (1965). Purification and partial characterization of mouse hemopexin (Beta S-111 globulin). Proc. Sot. Ezp. Biol. Med. 121: 111-116. 37. WOCHNER, R. D., SPILBERG, I., 110, A., LIEM, H. H., AND MULLER-EBERHARD, U. (1974). Hemopexin metabolism in sickle-cell disease, porphyrias and control subjects-Effects of heme injection. N. EngL J. Med. 290: 822-826.