- Email: [email protected]
The human CD53 gene, coding for afour transmembrane domain protein, maps to chromosomal region 1p13
725
BRIEF REPORTS Research Grant from the March of Dimes National Foundation (C.V.D.).
genes for amylase, proopiomelanocortin, somatostatin and a DNA fragment (D3S1) by in situ hybridization. Proc. Natl. Acad. Sci. USA 80: 6932-6936.
REFERENCES 1.
2.
3.
4.
5. 6. 7.
8.
9.
10.
11.
12.
13.
14.
15. 16.
17.
18.
Blatt, C., Eversole-Cire, P., Cohn, V. H., ZoUman, S., Fournier, R. E. K., Mohandas, T., Nesbitt, M., Lugo, T., Jones, D. T., Reed, R. R., Weiner, L. P., Sparkes, R. S., and Simon, M. I. (1988). Chromosomal localization of genes encoding G protein subunits in mouse and human. Proc. Natl. Acad. Sci. USA 8 5 : 7642 7646. Fung, B. K. K. (1983). Characterization of transducin from bovine retinal rod outer segments: Separation and reconstitution of the subunits. J. Biol. Chem. 2 5 8 : 10495-10502. Fung, B. K. K., Hurley, J. B., and Stryer, L. (1981). Flow of information in the light-triggered cyclic nucleotide cascade of vision. Proc. Natl. Acad. Sci. USA 78: 152-156. Fung, B. K. K., and Stryer, L. (1980). Photolyzed rhodopsin catalyzes the exchange of GTP for GDP in retinal rod outer segment membranes. Proc. Natl. Acad. Sci. USA 77: 2500-2504. Gilman, A. G. (1984). G Proteins and dual control of adenylate cyclase. Cell 36: 814-816. Gilman, A. G. (1987). G proteins: Transducers of receptor-generated signals. Annu. Rev. Biochem. 56: 615-650. Harper, M., and Saunders, G. (1981). Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization. Chromosoma 83: 431-439. Kahn, R. A., and Gilman, A. G. (1984). ADP-ribosylation of Gs promotes the dissociation of ADP-ribosylation of its alpha and beta subunits. J. Biol. Chem. 2 5 9 : 6235-6240. Kanaho, Y., Tsai, S. C., Admik, R., Hewlett, E. L., Moss, J., and Vaugban, M. (1984). Rhodopsin-enhanced GTPase activity of the inhibitory GTP-binding protein of adenylate cyclase. J. Biol. Chem. 2 5 9 : 7 3 7 8 7381. Kataoka, T., Powers, S., Cameron, S., Fasano, O., Goldfarb, M., Broach, J., and Wigler, M. (1985). Functional homology of mammalian and yeast RAS genes. Cell 40: 19-26. Lefkowitz, R. J., Caron, M. G., and Stiles, G. L. (1984). Mechanisms of membrane-receptor regulation: Biochemical, physiological, and clinical insights derived from studies of the adrenergic receptors. N. Engl. J. Med. 3 1 0 : 1570-1579. Lerea, C. L., Somers, D. E., Hurley, J. B., Klock, I. B., and BuntMilam, A. H. (1986). Identification of specific transducin alpha subunits in retinal rod and cone receptors. Science 2 3 4 : 77-80. Rocchi, M., Colantuoni, V., and Romeo, G. (1987). Assignment of RBP to 10 and subregional mapping of CRBP on 3q21-3qter. Cytogenet. Cell Genet. 46: 683. Sparkes, R. S., Klisak, I., Kaufman, D., Mohandas, T., Tobin, A. J., and McGinnis, J. F. (1986). Assignment of the rhodopsin gene to human chromosome three, region 3q21-3q24 by in situ hybridization studies. Curr. Eye Res. 5: 797-798. Stryer, L., and Bourne, H. R. (1986). G proteins: A family of signal transducers. Annu. Rev. Cell Biol. 2: 391-419. Sung, C. H., Schneider, B. G., Agarwal, N., Papermaster, D. S., and Nathans, J. (1991). Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa. Proc. Natl. Acad. Sci. USA 88: 8840-8844. Van Dop, C., Medynski, D. C., and Apone, L. M. (1989). Nucleotide sequence for a cDNA encoding the alpha subunit of retinal transducin (GNAT1) isolated from the human eye. Nucleic Acids Res. 17: 4887. Zabel, B., Naylor, S., Sakaguchi, A., Bell, G., and Shows, T. (1983). High-resolution chromosomal localization of human
The Human CD53 Gene, Coding for a Four Transmembrane Domain Protein, Maps to Chromosomal Region lp13 M. Eugenia Gonzalez,* Fernando Pardo-Manuel de Villena, t Elena Fernandez-Ruiz,$ Santiago Rodriguez de Cordoba, t and Pedro A. Lazo*'1 *Unidad de Gengtica Molecular (CSIC), Centro Nacional de Biologfa Celular y Retrovirus, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain,"tCentro de Investigaciones Biol6gicas, CSIC, Velazquez 144, and ~Servicio de Inmunolog[a, Hospital de la Princesa, Universidad Aut6noma de Madrid, 28006 Madrid, Spain
ReceivedFebruary11, 1993; revisedMay 19, 1993
T h e rat O X 4 4 / C D 5 3 p r o t e i n is t h e p r o t o t y p i c m e m b e r of a " n o v e l " family of p r o t e i n s . T h e s e p r o t e i n s are c h a r a c t e r i z e d by four highly h y d r o p h o b i c t r a n s m e m b r a n e d o m a i n s , two small e x t r a c e l l u l a r d o m a i n s , one of w h i c h is e x t e n s i v e l y N - g l y cosylated, a n d b o t h t h e a m i n o a n d t h e carboxy t e r m i n u s i n t r a c y t o p l a s m i c (5). T h e f u n c t i o n of t h e s e p r o t e i n s r e m a i n s elusive a n d several possible f u n c t i o n s have b e e n suggested dep e n d i n g on the e x p e r i m e n t a l s y s t e m used (5), b u t all of t h e m are s o m e h o w i m p l i c a t e d in t h e c o n t r o l of cell p r o l i f e r a t i o n by b i n d i n g to an u n k n o w n l i g a n d (5). I n rat, CD53 a n t i g e n is d e t e c t e d o n several m a t u r e cell t y p e s of t h e h e m a t o p o i e t i c syst e m , i n c l u d i n g m a c r o p h a g e s , m o n o c y t e s , granulocytes, leukocytes, a n d B a n d T cells, as well as osteoblasts a n d osteoclasts (8, 9). In rat m a c r o p h a g e s we h a v e f o u n d t h a t t h e CD53 prot e i n also s t i m u l a t e s t h e p r o d u c t i o n of nitric oxide ( s u b m i t t e d for publication), w h i c h is i m p l i c a t e d in septic a n d h e m o r r a g h i c shocks. P r e l i m i n a r y d a t a b a s e d on s o m a t i c cell hybrids i n d i c a t e d t h a t t h e h u m a n C D 5 3 gene is located on c h r o m o s o m e 1 (13). T o c o n f i r m t h e s e d a t a a n d to d e t e r m i n e precisely t h e l o c a t i o n of h u m a n CD53 gene in c h r o m o s o m e 1, we h a v e u s e d fluorescence in situ h y b r i d i z a t i o n ( F I S H ) . U s i n g a rat O X - 4 4 c D N A probe, a h u m a n g e n o m i c clone, )~-hROX44, c o n t a i n i n g a 17-kb-long insert was isolated a n d d e m o n s t r a t e d to include t h e c o m p l e t e h u m a n O X - 4 4 / C D 5 3 gene (4). F I S H was p e r f o r m e d as p r e v i o u s l y reported. Briefly, p e r i p h e r a l blood l y m p h o c y t e s were o b t a i n e d f r o m a h u m a n
1 To whom correspondence should be addressed at Unidad de Gen~tica Molecular, CNBCR, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain. Fax: 34-1-638 82 06. GENOMICS 1 8 , 725-728 (1993)
0888-7543/93 $5.00 Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Fluorescence in situ hybridization on human chromosomes with a digoxigenin-labeled 17-kb-long genomic DNA probe of the human CD53 gene. Arrows indicate the specific site of hybridization. (A) Rhodamine detection of digoxigenin-labeled probe and DAPI counterstaining in human mitotic cells and GTG-banding of the same metaphase spread. (B) Examples of chromosome 1 from other cells.
male by Lymphoprep gradient centrifugation and stimulated with phytohemagglutinin for 72 h Colchicine (0.2 ttg/ml) was added to the culture and kept for 1 h. T h e X-hROX44 genomic clone was labeled by nick-translation with digoxigenin-11d U T P . After 1 h of suppression hybridiz~ition at 37°C, hybridization was performed in 50% formamide containing 10% dextran sulfate, 2× SSC, and 50 m M p h o s p h a t e , p H 7.0, 2 ng/#l of labeled probe, and a 500-fold excess of both Cot1 D N A (fast renaturation fraction of h u m a n genomic DNA) and sonicated h u m a n placental D N A at 37°C overnight. Washings and detection with T R I T C - c o n j u g a t e d antibodies were performed as described (14). Chromosomes were counterstained with 75 n g / ml of 4',6-diamino-2-phenylindole in antifade medium. After the fluorescence microscopy, G T G - b a n d i n g was performed as described (11). A total of 35 metaphase spreads were analyzed for the presence of fluorescent spots; 89% of t h e m showed hybridization signals on the pericentromeric region of the short arm of chromosome 1, in one (39%) or both (61%) chromatids (Fig. 1). Hybridization was highly specific, and no other chromosomes showed spots systematically. This nonrandom distribution was analyzed after G T G banding of metaphases spreads to determine the precise location of the CD53 gene (Figs. 1A and 1B). T h e results of this analysis are summarized in Fig. 2; 62 and 19% of the hybridization spots were localized on the lp13 and lp21 bands, respectively. F r o m these data we conclude t h a t the human CD53 gene maps to lp13, proximal to lp21.
Chromosomal region lp13 contains some other lymphoid genes like CD2 and its ligand CD58 (12). W h e t h e r the three lymphoid genes are linked, forming a cluster, is currently unknown. Interestingly, CD53 and. CD2 antigens coprecipitate with antibodies against CD53 in rat T lymphocytes and natural killer cells, suggesting t h a t they are associated on the cell m e m b r a n e (3). Furthermore, in these cells the response to antigenic stimulation of CD2 ÷ cells is increased severalfold if they are CD53 ÷ (3). These observations raise the possibility t h a t these two genes might have a coordinated expression in T-cells. This interaction and the pattern of OX-44/CD53 expression at different stages of hematopoietic development and in very different mature cell types make the regulation of CD53 gene transcription an interesting problem. Chromosomal translocations specific for leukemias have been very i n s t r u m e n t a l in the discovery of genes implicated in oncogenesis. There are some translocations involving the lp13 region in different types of tumors (7). Among t h e m is t(1;22) (p13;q13), which is detected in 70% of the cases of M7 acute megakaryocytic leukemia in young children and has a fatal prognosis (6). Because of the possible effects of CD53 on cell proliferation, it might be implicated in the oncogenic phenotype. Two RAS-related genes (10), N R A S and R A P I A , and other genes, such as NGFB and TSH, which might have effects on the growth properties of a cell, also map to lp13. Which genes are affected by the t(1;22) (p13;q13) translocation is not known, but a better characterization of this region should help to clarify this point.
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F I G . 2. Assignment of the CD53 gene to lp13. The idiogram of human chromosome 1 schematically indicates the intrachromosomal distribution of fluorescent spots for the CD53 locus. ACKNOWLEDGMENTS
6.
This work has been supported by grants from Fundaci6n Ram6n Areces and CICYT (SAL90-0209 and SAL91-0043) to P.A.L. and FIS (92-0889) to S.R.C. REFERENCES
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Amiot, M. (1990). Identification and analysis of cDNA clones encoding CD53. A pan-leukocyte antigen related to membrane transport proteins. J. Immunol. 145: 4322-4325. 2. Angelisova, P., Vlcek, C., Stefanova, I., Lipoldova, M., and Hotejsi, V. (1990). The human leucocyte surface antigen CD53 is a protein structurally similar to CD37 and MRC OX-44 antigens. Immunogenetics 32: 281-285. 3. Bell, G. M., Seaman, W. E., Niemi, E. C., and Imboden, J. B. (1992). The OX-44 molecule couples to signalling pathways and is associated with CD2 on rat T cells and a natural killer cell line. J. Exp. Med. 175: 527-536. 4. Bellacosa, A., Lazo, P. A., Bear, S. E., and Tsichlis, P. N. (1991). The rat leukocyte antigen MRC 0X-44 is a member of a new family of cell surface proteins which appear to be involved in growth regulation. Mol. Cell. Biol. 11: 2864-2872. 5. Horejsi, V., and Vlcek, C. (1991). Novel structurally distinct faroily of leucocyte surface glycoproteins including CD9, CD37, CD53 and CD63. F E B S Lett. 2 8 8 : 1-4.
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Lion, T., Haas, O. A., Harbott, J., Bannier, E., Ritterbach, J., Jankovic, M., Fink, F. M., Stojimirovic, A., Herrmann, J., Riehm, H. J., Lampert, F., Ritter, J., Koch, H., and Gadner, H. (1992). The translocation t(1;22) (p13C3) is a nonrandom marker specifically associated with acute megakaryocytic leukemia in young children. Blood 79: 3325-3330. Mitelman, F. (1991). "Catalog of Chromosome Aberrations in Cancer," Vol. 1, Wiley-Liss, New York. Paterson, D. J., Green, J. R., Jeffries, W. A., Puklavec, M., and Williams A. F. (1987). The MRC OX-44 antigen marks a functionally relevant subset among rat thymocytes. J. Exp. Med. 165: 1-13. Paterson, D. J., and Williams, A. F. (1987). An intermediate cell in thymocyte differentiation that expresses CD8 but not CD4 antigen. J. Exp. Med. 166: 1603-1608. Rousseau-Merck, M. F., Pizon, V., Tavitian, A., and Berger, R. (1990). Chromosome mapping of the human RAS related RAP1A, R A P I B and RAP2 genes to chromosomes 1p12-p]3, 12q14, and 13q34 respectively. Cytogenet, Cell Genet. 53: 2-4. Seabright, M. (1971). A rapid banding technique for human chromosomes. Lancet 2: 971. Sewell, W. A., Palmer, R. W., Spurr, N. K., Sheer, D., Brown, M. H., Bell, Y., and Crumpton, M. J. (1988). The human LFA-3 gene is located at the same chromosome band as the gene for its receptor CD2. Immunogenetics 28: 278-282. Taguchi et al. (1991). Mapping of the OX44 human leukocyte
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BRIEF REPORTS antigen gene to 1p12-p13 by fluorescence in situ hybridization. Cytogenet. Cell Genet. 58: 1863. [Abstract 26841] Wiegant, J., Galjart, N. J., Raap, A. K., and d'Azzo, A. (1991). The gene encoding human protective protein is on chromosome 20. Genomics 10: 345-349.
The CA Repeat Marker D17S791 Is Located within 40 kb of the WNT3 Gene on Chromosome 17q Settara C. Chandrasekharappa,*,t ,1 Stephanie E. King,* M a t t h e w L. Freedman,* Steve T. Hayes,* A n n e M. Bowcock,$ and Francis S, Collins*'t'§'¶ *Michigan Human Genome Center, Departments of tHuman Genetics and §Internal Medicine, and ¶Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, Michigan 48109, and ~Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75235
Received February 18, 1993; revised September4, 1993
Recent developments in genetic linkage mapping of the h uman genome have generated a large number of short t a n d e m repeat polymorphic markers (13). Eventual integration of these markers into a-physical map is a natural and a necessary step. A large number of genomic clones have been generated for the early-onset breast and ovarian cancer (BRCA1) region at 17q21 (1-3, 5, 7, 9, 10) and are being used to construct a physical map of this region. An a t t e m p t was made to link CA repeat markers from this chromosomal region to yeast artificial chromosome (YAC) and cosmid clones. We report here the localization of the CA repeat marker D17S791 (155xd12) to within 40 kb of W N T 3 , a h u m a n homolog of the gene activated by proviral insertion in mouse m a m m a r y tumors (12). Three YAC clones were isolated by screening the total human genomic YAC library constructed at the Center for Genetics in Medicine at W a s h i n g t o n University, St. Louis, MO (4), with a set of P C R primers generated from the mouse writ3 c D N A sequence (12). T h e primers (5'-CCA T C C T G G ACC ACA T G C AC-3' and 5'-GGT G T G CAC A T C G T A G A T GC3'), chosen from the fourth exon of wnt3, amplify the samesized (470 bp) P C R product from total human and total mouse genomic D N A templates, allowing for the screening of h u m a n W N T 3 by P C R - b a s e d methods (8, 11). Each of the three YAC clones, A236C12, B19E12, and B82F6, contained single YAC inserts of 235, 140, and 125 kb, respectively. The methods for the characterization of the YAC clones were as described earlier (6). D N A from the three YAC clones was tested by P C R with the primers for the m a r k e r D17S791. As shown in Fig. 1 (lanes 1 - 3 ) , the clones A236C12 and B19E12, but not B82F6, 1 To whom correspondence should be addressed at Human Genome Center, 2574 MSRBII, Box 0674, 1150 West Medical Center Drive, University of Michigan Medical Center, Ann Arbor, MI 48109. Telephone: (313) 764-8070. GENOMICS 18, 728-729 (1993) 0888-7543/93 $5.00 Copyright © 1993 by AcademicPress, Inc. All rights of reproduction in any form reserved.
1 2 3 4 5 6 78 91011HYN M
470 bp 193 bp FIG. 1. PCR with DNA from YAC or eosmid clones as templates and primers for WNT3 and D17S791. DNA (2 ng/tL1) from YAC clones or cells from cosmid clones were subjected to PCR reaction, separately, using primers for WNT3 and D17S791. The sequences of the PCR primers for D17S791 were 5'-GTTTTCTCC AGTTATTCCCC-3' and 5'-GCTCGTCCTTTGGAAGAGTT-3' (13). The primers for WNT3 are given in the text. The PCR reactions were carried out for 35 cycles, with each cycle consisting of 94°C for I min, 62°C for 1 min, and 72°C for 1 min for WNT3 primers, whereas the annealing temperature was 58°C for 1 rain ~br the D17S791 primers. Ten microliters from both PCR reactions for each DNA was loaded on the same lane, since the WNT3 PCR product (470 bp) resolves very well with that of D17S791 (193 bp). Lanes 1 to 3 represent YAC clones A236C12, B19E12, and B82F6; lanes 4 to 11 represent the cosmid clones 12F2, 69C8, 75A9, 89A12, 106D12, 142E3, 156A2, and 158H2; and lanes H, Y, and N were control PCR reactions with human DNA, yeast DNA, and no DNA, respectively. Lane M contains the 100-bp ladder (BRL/GIBCO) as size markers. None of three unrelated cosmids tested showed a positive PCR signal with either set of primers {data not shown). generated a P C R product (193 bp) of the expected size. Since the smaller YAC t h a t shared both the sequence-tagged sites (STSs) was only 140 kb, D17S791 and W N T 3 must be no more t h a n 140 kb apart. An arrayed chromosome 17 cosmid library, prepared and supplied by Larry Deaven of the Los Alamos N a t i o n a l Laboratory, was screened for clones containing W N T 3 . T h e 470-bp W N T 3 P C R product, amplified from h u m a n DNA, was labeled and used as probe. Eight cosmids (12F2, 69C8, 75A9, 89A12, 106D12, 142E3, 156A2, and 158H2) were identified by screening five genomic equivalents of the library. Individual colonies from these clones were tested by P C R for W N T 3 as well as for the marker D17S791, and all eight were positive for both (lanes 4 - 1 1 , Fig. 1). T h e colocalization of the marker D17S791 to the same cosmids containing an S T S for W N T 3 shows t h a t the marker is no farther t h a n 40 kb, the average size of an insert in a cosmid, from W N T 3 . Given t h a t eight of eight cosmids are positive for both, the two markers are likely to be much closer t h a n 40 kb. T h e presence of both the marker D17S791 and W N T 3 on two YAC and eight cosmid clones is in general agreement with two other studies: genetic linkage analysis places, among other markers, D17S579 (Mfd188) and H O X 2 B as the two flanking markers for D17S791 (2), and physical mapping by fluorescence in s i t u hybridization mapped W N T 3 between the same two flanking markers (7). Both W N T 3 and D17S791 have been excluded from the BRCA1 locus (2-3, 5). However, the marker D17S791, with 11 alleles, can serve as a tightly linked m a r k e r for the W N T 3 locus, which has no known polymorphism associated with it. ACKNOWLEDGMENTS We thank Laura Gross and Elizabeth Collins for technical assistance and Mary-Claire King for sharing the results of genetic linkage
BRIEF REPORTS Research Grant from the March of Dimes National Foundation (C.V.D.).
genes for amylase, proopiomelanocortin, somatostatin and a DNA fragment (D3S1) by in situ hybridization. Proc. Natl. Acad. Sci. USA 80: 6932-6936.
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Blatt, C., Eversole-Cire, P., Cohn, V. H., ZoUman, S., Fournier, R. E. K., Mohandas, T., Nesbitt, M., Lugo, T., Jones, D. T., Reed, R. R., Weiner, L. P., Sparkes, R. S., and Simon, M. I. (1988). Chromosomal localization of genes encoding G protein subunits in mouse and human. Proc. Natl. Acad. Sci. USA 8 5 : 7642 7646. Fung, B. K. K. (1983). Characterization of transducin from bovine retinal rod outer segments: Separation and reconstitution of the subunits. J. Biol. Chem. 2 5 8 : 10495-10502. Fung, B. K. K., Hurley, J. B., and Stryer, L. (1981). Flow of information in the light-triggered cyclic nucleotide cascade of vision. Proc. Natl. Acad. Sci. USA 78: 152-156. Fung, B. K. K., and Stryer, L. (1980). Photolyzed rhodopsin catalyzes the exchange of GTP for GDP in retinal rod outer segment membranes. Proc. Natl. Acad. Sci. USA 77: 2500-2504. Gilman, A. G. (1984). G Proteins and dual control of adenylate cyclase. Cell 36: 814-816. Gilman, A. G. (1987). G proteins: Transducers of receptor-generated signals. Annu. Rev. Biochem. 56: 615-650. Harper, M., and Saunders, G. (1981). Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization. Chromosoma 83: 431-439. Kahn, R. A., and Gilman, A. G. (1984). ADP-ribosylation of Gs promotes the dissociation of ADP-ribosylation of its alpha and beta subunits. J. Biol. Chem. 2 5 9 : 6235-6240. Kanaho, Y., Tsai, S. C., Admik, R., Hewlett, E. L., Moss, J., and Vaugban, M. (1984). Rhodopsin-enhanced GTPase activity of the inhibitory GTP-binding protein of adenylate cyclase. J. Biol. Chem. 2 5 9 : 7 3 7 8 7381. Kataoka, T., Powers, S., Cameron, S., Fasano, O., Goldfarb, M., Broach, J., and Wigler, M. (1985). Functional homology of mammalian and yeast RAS genes. Cell 40: 19-26. Lefkowitz, R. J., Caron, M. G., and Stiles, G. L. (1984). Mechanisms of membrane-receptor regulation: Biochemical, physiological, and clinical insights derived from studies of the adrenergic receptors. N. Engl. J. Med. 3 1 0 : 1570-1579. Lerea, C. L., Somers, D. E., Hurley, J. B., Klock, I. B., and BuntMilam, A. H. (1986). Identification of specific transducin alpha subunits in retinal rod and cone receptors. Science 2 3 4 : 77-80. Rocchi, M., Colantuoni, V., and Romeo, G. (1987). Assignment of RBP to 10 and subregional mapping of CRBP on 3q21-3qter. Cytogenet. Cell Genet. 46: 683. Sparkes, R. S., Klisak, I., Kaufman, D., Mohandas, T., Tobin, A. J., and McGinnis, J. F. (1986). Assignment of the rhodopsin gene to human chromosome three, region 3q21-3q24 by in situ hybridization studies. Curr. Eye Res. 5: 797-798. Stryer, L., and Bourne, H. R. (1986). G proteins: A family of signal transducers. Annu. Rev. Cell Biol. 2: 391-419. Sung, C. H., Schneider, B. G., Agarwal, N., Papermaster, D. S., and Nathans, J. (1991). Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa. Proc. Natl. Acad. Sci. USA 88: 8840-8844. Van Dop, C., Medynski, D. C., and Apone, L. M. (1989). Nucleotide sequence for a cDNA encoding the alpha subunit of retinal transducin (GNAT1) isolated from the human eye. Nucleic Acids Res. 17: 4887. Zabel, B., Naylor, S., Sakaguchi, A., Bell, G., and Shows, T. (1983). High-resolution chromosomal localization of human
The Human CD53 Gene, Coding for a Four Transmembrane Domain Protein, Maps to Chromosomal Region lp13 M. Eugenia Gonzalez,* Fernando Pardo-Manuel de Villena, t Elena Fernandez-Ruiz,$ Santiago Rodriguez de Cordoba, t and Pedro A. Lazo*'1 *Unidad de Gengtica Molecular (CSIC), Centro Nacional de Biologfa Celular y Retrovirus, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain,"tCentro de Investigaciones Biol6gicas, CSIC, Velazquez 144, and ~Servicio de Inmunolog[a, Hospital de la Princesa, Universidad Aut6noma de Madrid, 28006 Madrid, Spain
ReceivedFebruary11, 1993; revisedMay 19, 1993
T h e rat O X 4 4 / C D 5 3 p r o t e i n is t h e p r o t o t y p i c m e m b e r of a " n o v e l " family of p r o t e i n s . T h e s e p r o t e i n s are c h a r a c t e r i z e d by four highly h y d r o p h o b i c t r a n s m e m b r a n e d o m a i n s , two small e x t r a c e l l u l a r d o m a i n s , one of w h i c h is e x t e n s i v e l y N - g l y cosylated, a n d b o t h t h e a m i n o a n d t h e carboxy t e r m i n u s i n t r a c y t o p l a s m i c (5). T h e f u n c t i o n of t h e s e p r o t e i n s r e m a i n s elusive a n d several possible f u n c t i o n s have b e e n suggested dep e n d i n g on the e x p e r i m e n t a l s y s t e m used (5), b u t all of t h e m are s o m e h o w i m p l i c a t e d in t h e c o n t r o l of cell p r o l i f e r a t i o n by b i n d i n g to an u n k n o w n l i g a n d (5). I n rat, CD53 a n t i g e n is d e t e c t e d o n several m a t u r e cell t y p e s of t h e h e m a t o p o i e t i c syst e m , i n c l u d i n g m a c r o p h a g e s , m o n o c y t e s , granulocytes, leukocytes, a n d B a n d T cells, as well as osteoblasts a n d osteoclasts (8, 9). In rat m a c r o p h a g e s we h a v e f o u n d t h a t t h e CD53 prot e i n also s t i m u l a t e s t h e p r o d u c t i o n of nitric oxide ( s u b m i t t e d for publication), w h i c h is i m p l i c a t e d in septic a n d h e m o r r a g h i c shocks. P r e l i m i n a r y d a t a b a s e d on s o m a t i c cell hybrids i n d i c a t e d t h a t t h e h u m a n C D 5 3 gene is located on c h r o m o s o m e 1 (13). T o c o n f i r m t h e s e d a t a a n d to d e t e r m i n e precisely t h e l o c a t i o n of h u m a n CD53 gene in c h r o m o s o m e 1, we h a v e u s e d fluorescence in situ h y b r i d i z a t i o n ( F I S H ) . U s i n g a rat O X - 4 4 c D N A probe, a h u m a n g e n o m i c clone, )~-hROX44, c o n t a i n i n g a 17-kb-long insert was isolated a n d d e m o n s t r a t e d to include t h e c o m p l e t e h u m a n O X - 4 4 / C D 5 3 gene (4). F I S H was p e r f o r m e d as p r e v i o u s l y reported. Briefly, p e r i p h e r a l blood l y m p h o c y t e s were o b t a i n e d f r o m a h u m a n
1 To whom correspondence should be addressed at Unidad de Gen~tica Molecular, CNBCR, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain. Fax: 34-1-638 82 06. GENOMICS 1 8 , 725-728 (1993)
0888-7543/93 $5.00 Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Fluorescence in situ hybridization on human chromosomes with a digoxigenin-labeled 17-kb-long genomic DNA probe of the human CD53 gene. Arrows indicate the specific site of hybridization. (A) Rhodamine detection of digoxigenin-labeled probe and DAPI counterstaining in human mitotic cells and GTG-banding of the same metaphase spread. (B) Examples of chromosome 1 from other cells.
male by Lymphoprep gradient centrifugation and stimulated with phytohemagglutinin for 72 h Colchicine (0.2 ttg/ml) was added to the culture and kept for 1 h. T h e X-hROX44 genomic clone was labeled by nick-translation with digoxigenin-11d U T P . After 1 h of suppression hybridiz~ition at 37°C, hybridization was performed in 50% formamide containing 10% dextran sulfate, 2× SSC, and 50 m M p h o s p h a t e , p H 7.0, 2 ng/#l of labeled probe, and a 500-fold excess of both Cot1 D N A (fast renaturation fraction of h u m a n genomic DNA) and sonicated h u m a n placental D N A at 37°C overnight. Washings and detection with T R I T C - c o n j u g a t e d antibodies were performed as described (14). Chromosomes were counterstained with 75 n g / ml of 4',6-diamino-2-phenylindole in antifade medium. After the fluorescence microscopy, G T G - b a n d i n g was performed as described (11). A total of 35 metaphase spreads were analyzed for the presence of fluorescent spots; 89% of t h e m showed hybridization signals on the pericentromeric region of the short arm of chromosome 1, in one (39%) or both (61%) chromatids (Fig. 1). Hybridization was highly specific, and no other chromosomes showed spots systematically. This nonrandom distribution was analyzed after G T G banding of metaphases spreads to determine the precise location of the CD53 gene (Figs. 1A and 1B). T h e results of this analysis are summarized in Fig. 2; 62 and 19% of the hybridization spots were localized on the lp13 and lp21 bands, respectively. F r o m these data we conclude t h a t the human CD53 gene maps to lp13, proximal to lp21.
Chromosomal region lp13 contains some other lymphoid genes like CD2 and its ligand CD58 (12). W h e t h e r the three lymphoid genes are linked, forming a cluster, is currently unknown. Interestingly, CD53 and. CD2 antigens coprecipitate with antibodies against CD53 in rat T lymphocytes and natural killer cells, suggesting t h a t they are associated on the cell m e m b r a n e (3). Furthermore, in these cells the response to antigenic stimulation of CD2 ÷ cells is increased severalfold if they are CD53 ÷ (3). These observations raise the possibility t h a t these two genes might have a coordinated expression in T-cells. This interaction and the pattern of OX-44/CD53 expression at different stages of hematopoietic development and in very different mature cell types make the regulation of CD53 gene transcription an interesting problem. Chromosomal translocations specific for leukemias have been very i n s t r u m e n t a l in the discovery of genes implicated in oncogenesis. There are some translocations involving the lp13 region in different types of tumors (7). Among t h e m is t(1;22) (p13;q13), which is detected in 70% of the cases of M7 acute megakaryocytic leukemia in young children and has a fatal prognosis (6). Because of the possible effects of CD53 on cell proliferation, it might be implicated in the oncogenic phenotype. Two RAS-related genes (10), N R A S and R A P I A , and other genes, such as NGFB and TSH, which might have effects on the growth properties of a cell, also map to lp13. Which genes are affected by the t(1;22) (p13;q13) translocation is not known, but a better characterization of this region should help to clarify this point.
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F I G . 2. Assignment of the CD53 gene to lp13. The idiogram of human chromosome 1 schematically indicates the intrachromosomal distribution of fluorescent spots for the CD53 locus. ACKNOWLEDGMENTS
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This work has been supported by grants from Fundaci6n Ram6n Areces and CICYT (SAL90-0209 and SAL91-0043) to P.A.L. and FIS (92-0889) to S.R.C. REFERENCES
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Amiot, M. (1990). Identification and analysis of cDNA clones encoding CD53. A pan-leukocyte antigen related to membrane transport proteins. J. Immunol. 145: 4322-4325. 2. Angelisova, P., Vlcek, C., Stefanova, I., Lipoldova, M., and Hotejsi, V. (1990). The human leucocyte surface antigen CD53 is a protein structurally similar to CD37 and MRC OX-44 antigens. Immunogenetics 32: 281-285. 3. Bell, G. M., Seaman, W. E., Niemi, E. C., and Imboden, J. B. (1992). The OX-44 molecule couples to signalling pathways and is associated with CD2 on rat T cells and a natural killer cell line. J. Exp. Med. 175: 527-536. 4. Bellacosa, A., Lazo, P. A., Bear, S. E., and Tsichlis, P. N. (1991). The rat leukocyte antigen MRC 0X-44 is a member of a new family of cell surface proteins which appear to be involved in growth regulation. Mol. Cell. Biol. 11: 2864-2872. 5. Horejsi, V., and Vlcek, C. (1991). Novel structurally distinct faroily of leucocyte surface glycoproteins including CD9, CD37, CD53 and CD63. F E B S Lett. 2 8 8 : 1-4.
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Lion, T., Haas, O. A., Harbott, J., Bannier, E., Ritterbach, J., Jankovic, M., Fink, F. M., Stojimirovic, A., Herrmann, J., Riehm, H. J., Lampert, F., Ritter, J., Koch, H., and Gadner, H. (1992). The translocation t(1;22) (p13C3) is a nonrandom marker specifically associated with acute megakaryocytic leukemia in young children. Blood 79: 3325-3330. Mitelman, F. (1991). "Catalog of Chromosome Aberrations in Cancer," Vol. 1, Wiley-Liss, New York. Paterson, D. J., Green, J. R., Jeffries, W. A., Puklavec, M., and Williams A. F. (1987). The MRC OX-44 antigen marks a functionally relevant subset among rat thymocytes. J. Exp. Med. 165: 1-13. Paterson, D. J., and Williams, A. F. (1987). An intermediate cell in thymocyte differentiation that expresses CD8 but not CD4 antigen. J. Exp. Med. 166: 1603-1608. Rousseau-Merck, M. F., Pizon, V., Tavitian, A., and Berger, R. (1990). Chromosome mapping of the human RAS related RAP1A, R A P I B and RAP2 genes to chromosomes 1p12-p]3, 12q14, and 13q34 respectively. Cytogenet, Cell Genet. 53: 2-4. Seabright, M. (1971). A rapid banding technique for human chromosomes. Lancet 2: 971. Sewell, W. A., Palmer, R. W., Spurr, N. K., Sheer, D., Brown, M. H., Bell, Y., and Crumpton, M. J. (1988). The human LFA-3 gene is located at the same chromosome band as the gene for its receptor CD2. Immunogenetics 28: 278-282. Taguchi et al. (1991). Mapping of the OX44 human leukocyte
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BRIEF REPORTS antigen gene to 1p12-p13 by fluorescence in situ hybridization. Cytogenet. Cell Genet. 58: 1863. [Abstract 26841] Wiegant, J., Galjart, N. J., Raap, A. K., and d'Azzo, A. (1991). The gene encoding human protective protein is on chromosome 20. Genomics 10: 345-349.
The CA Repeat Marker D17S791 Is Located within 40 kb of the WNT3 Gene on Chromosome 17q Settara C. Chandrasekharappa,*,t ,1 Stephanie E. King,* M a t t h e w L. Freedman,* Steve T. Hayes,* A n n e M. Bowcock,$ and Francis S, Collins*'t'§'¶ *Michigan Human Genome Center, Departments of tHuman Genetics and §Internal Medicine, and ¶Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, Michigan 48109, and ~Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75235
Received February 18, 1993; revised September4, 1993
Recent developments in genetic linkage mapping of the h uman genome have generated a large number of short t a n d e m repeat polymorphic markers (13). Eventual integration of these markers into a-physical map is a natural and a necessary step. A large number of genomic clones have been generated for the early-onset breast and ovarian cancer (BRCA1) region at 17q21 (1-3, 5, 7, 9, 10) and are being used to construct a physical map of this region. An a t t e m p t was made to link CA repeat markers from this chromosomal region to yeast artificial chromosome (YAC) and cosmid clones. We report here the localization of the CA repeat marker D17S791 (155xd12) to within 40 kb of W N T 3 , a h u m a n homolog of the gene activated by proviral insertion in mouse m a m m a r y tumors (12). Three YAC clones were isolated by screening the total human genomic YAC library constructed at the Center for Genetics in Medicine at W a s h i n g t o n University, St. Louis, MO (4), with a set of P C R primers generated from the mouse writ3 c D N A sequence (12). T h e primers (5'-CCA T C C T G G ACC ACA T G C AC-3' and 5'-GGT G T G CAC A T C G T A G A T GC3'), chosen from the fourth exon of wnt3, amplify the samesized (470 bp) P C R product from total human and total mouse genomic D N A templates, allowing for the screening of h u m a n W N T 3 by P C R - b a s e d methods (8, 11). Each of the three YAC clones, A236C12, B19E12, and B82F6, contained single YAC inserts of 235, 140, and 125 kb, respectively. The methods for the characterization of the YAC clones were as described earlier (6). D N A from the three YAC clones was tested by P C R with the primers for the m a r k e r D17S791. As shown in Fig. 1 (lanes 1 - 3 ) , the clones A236C12 and B19E12, but not B82F6, 1 To whom correspondence should be addressed at Human Genome Center, 2574 MSRBII, Box 0674, 1150 West Medical Center Drive, University of Michigan Medical Center, Ann Arbor, MI 48109. Telephone: (313) 764-8070. GENOMICS 18, 728-729 (1993) 0888-7543/93 $5.00 Copyright © 1993 by AcademicPress, Inc. All rights of reproduction in any form reserved.
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470 bp 193 bp FIG. 1. PCR with DNA from YAC or eosmid clones as templates and primers for WNT3 and D17S791. DNA (2 ng/tL1) from YAC clones or cells from cosmid clones were subjected to PCR reaction, separately, using primers for WNT3 and D17S791. The sequences of the PCR primers for D17S791 were 5'-GTTTTCTCC AGTTATTCCCC-3' and 5'-GCTCGTCCTTTGGAAGAGTT-3' (13). The primers for WNT3 are given in the text. The PCR reactions were carried out for 35 cycles, with each cycle consisting of 94°C for I min, 62°C for 1 min, and 72°C for 1 min for WNT3 primers, whereas the annealing temperature was 58°C for 1 rain ~br the D17S791 primers. Ten microliters from both PCR reactions for each DNA was loaded on the same lane, since the WNT3 PCR product (470 bp) resolves very well with that of D17S791 (193 bp). Lanes 1 to 3 represent YAC clones A236C12, B19E12, and B82F6; lanes 4 to 11 represent the cosmid clones 12F2, 69C8, 75A9, 89A12, 106D12, 142E3, 156A2, and 158H2; and lanes H, Y, and N were control PCR reactions with human DNA, yeast DNA, and no DNA, respectively. Lane M contains the 100-bp ladder (BRL/GIBCO) as size markers. None of three unrelated cosmids tested showed a positive PCR signal with either set of primers {data not shown). generated a P C R product (193 bp) of the expected size. Since the smaller YAC t h a t shared both the sequence-tagged sites (STSs) was only 140 kb, D17S791 and W N T 3 must be no more t h a n 140 kb apart. An arrayed chromosome 17 cosmid library, prepared and supplied by Larry Deaven of the Los Alamos N a t i o n a l Laboratory, was screened for clones containing W N T 3 . T h e 470-bp W N T 3 P C R product, amplified from h u m a n DNA, was labeled and used as probe. Eight cosmids (12F2, 69C8, 75A9, 89A12, 106D12, 142E3, 156A2, and 158H2) were identified by screening five genomic equivalents of the library. Individual colonies from these clones were tested by P C R for W N T 3 as well as for the marker D17S791, and all eight were positive for both (lanes 4 - 1 1 , Fig. 1). T h e colocalization of the marker D17S791 to the same cosmids containing an S T S for W N T 3 shows t h a t the marker is no farther t h a n 40 kb, the average size of an insert in a cosmid, from W N T 3 . Given t h a t eight of eight cosmids are positive for both, the two markers are likely to be much closer t h a n 40 kb. T h e presence of both the marker D17S791 and W N T 3 on two YAC and eight cosmid clones is in general agreement with two other studies: genetic linkage analysis places, among other markers, D17S579 (Mfd188) and H O X 2 B as the two flanking markers for D17S791 (2), and physical mapping by fluorescence in s i t u hybridization mapped W N T 3 between the same two flanking markers (7). Both W N T 3 and D17S791 have been excluded from the BRCA1 locus (2-3, 5). However, the marker D17S791, with 11 alleles, can serve as a tightly linked m a r k e r for the W N T 3 locus, which has no known polymorphism associated with it. ACKNOWLEDGMENTS We thank Laura Gross and Elizabeth Collins for technical assistance and Mary-Claire King for sharing the results of genetic linkage