- Email: [email protected]
An Integrated Physical and Genetic Map Spanning Chromosome Band 10q24
SHORT COMMUNICATION An Integrated Physical and Genetic Map Spanning Chromosome Band 10q24 Ian C. Gray,*,1,2 Joanne Fallowfield,* Sally Ford,* Carlo Nobile,† Emanuela V. Volpi,† and Nigel K. Spurr*,1 *Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertsfordshire EN6 3LD, England; and †Consiglio Nazionale delle Ricerche, Istituto di Genetica Molecolare, Alghero (Sassari), Italy Received December 11, 1996; accepted May 6, 1997
markers from loci other than 10q23–q25 or hybridizing to cell hybrid DNA with a human complement other than chromosome 10) were avoided. The cytogenetic location of each YAC was confirmed by fluorescence in situ hybridization (FISH) to metaphase spreads of human lymphocytes; 9/42 YACs (21%) showed chimerism detectable by FISH, a level somewhat lower than the estimated mega-YAC library average of 40% (4) and probably reflecting our biased selection procedure (Table 1). Ge´ne´thon CA repeat genetic markers from 10q23– q25 (3, 4) were assigned to YACs by PCR amplification, allowing us to confirm the presence of markers previously assigned by Ge´ne´thon (3, 4), make novel assignments not reported by Ge´ne´thon, and position several new previously unmapped markers. Establishing the physical order of these markers should resolve any discrepancies in genetic map order. The YACs were also screened using PCR primers derived from genes listed as mapping to 10q23–q26 in the Genome Database (Johns Hopkins University, Baltimore, MD). Sequencetagged sites (STSs) derived from nine known gene loci (IFI56, IDE, PDE6C, RBP4, CYP2C, CD39, DNTT, GOT1, WNT8B, and PAX2) were mapped to one or more YACs, together with one expressed sequence tag (EST), D10S181E. To identify additional 10q24 genes, YACs were screened with 144 ESTs derived from chromosome 10 (Ref. 1; Dr. Charles Auffray, Paris, pers. comm., 1996). Seven proved positive: D10S1326E, D10S1360E, D10S1363E D10S1319E, D10S1396E, D10S1403E, and D10S1399E. We were also able to localize 3 further ESTs from the Whitehead Institute database (10) previously mapped to 10q24 YACs: IB3311, NIB1686, and IB3259. The assignment of CA markers, genes, ESTs, and a further 4 Whitehead Institute STSs (D10S1442, D10S1571, D10S2269, and D10S2172; Ref. 10) allowed the majority of YACs to be linked to one or more overlapping clones by a common STS. The contig is summarized in Table 1. Two overlaps are equivocal: according to the Ge´ ne´ thon data (4), D10S577 is common to YACs 756-G-3 and 941-F-5; however, we were unable to confirm the presence of this marker on YAC 941-F-5. In addition,
Chromosome band 10q24 is rich in genes involved in development, tumorigenesis, neurological disorders, hormone metabolism, and environmentally induced disease susceptibility. We have constructed an STSbased integrated physical and genetic map of 10q24 derived from the CEPH–Ge´ne´thon mega-YAC contig data for this region. This map consists of 42 fluorescence in situ hybridization-mapped overlapping CEPH mega-YACs spanning approximately 15 Mb to which 49 STS markers have been assigned, including 24 Ge´ne´thon CA repeat genetic markers, 10 known gene loci from the 10q24 region (IFI56, IDE, PDE6C, RBP4, CYP2C, CD39, DNTT, GOT1, WNT8B, and PAX2) and 11 additional expressed sequences of unknown function. q 1997 Academic Press
The chromosomal location 10q24 bears a number of genes of fundamental biological significance, including the developmental genes PAX2, HOX11, and WNT8B (13, 15, 18) and the cytochrome P450IIC gene cluster involved in the metabolism of steroid hormones and environmental agents (17). Linkage analysis implies that this region also contains genes involved in epilepsy (21), spinocerebellar ataxia (20), and Cowden disease (19). Allele loss studies also suggest the presence of a prostate tumor suppressor gene at proximal 10q24 (7, 12, 14, 26). As part of an effort to identify this tumor suppressor gene, we have constructed a mega-YAC contig of 10q24 between the CA repeat genetic markers D10S579 and D10S1268, a distance of 20 cM (5). This contig combines genetic markers with known 10q24 genes and further anonymous expressed sequences. To provide a scaffold for contig construction, 42 mega-YACs derived from the 10q23–q25 region were selected from the CEPH–Ge´ne´thon Quickmap database (3, 4). Potentially chimeric YACs (those bearing 1 Present address: SmithKline Beecham Pharmaceuticals, Biopharmaceutical R&D, New Frontiers Science Park (North), Harlow, Essex CM10 5AW, UK. 2 To whom correspondence should be addressed. Telephone: 01279 627225. Fax: 01279 622500. E-mail: [email protected].
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0888-7543/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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10q24 10q24 10q24 10q24 / 11q 10q24 10q24 10q24 / 10q21 10q24 / 2q 10q24 10q24 10q24 10q24 10q24 – q25 / A 10q24 – q25 10q24 – q25
912C4 912C7 805A9 784F4 853A4 744D4 789B10 946E3 857E10 756G3 941F5 845H7 963C11 926D8 954E2
/ / 10p
/ /
/5
10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q24 10q24
Cytolocation
738B12 921F8 746H8 821D2 831E5 796D5 829E1 734B4 927H12 934D3 906D1 682G1 794H1 944C1 750H1 847D4 759C9 950A1 885H11 773F6 890G6 761B1 958A7 745D8 791C3 912C4 912C7
YAC / / / / /
D10S 579 / / / / /
D10S 215
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/ /
/ /
/ / /
PDE6C RBP4 CYP2C
/
D10S 1326E
R Centromere
/ / / / /
D10S 571
/ / / / /
D10S 1765
/ / / / / / / / /
D10S 541
/ / / / /
D10S 1403E
/ / / /
/ / / /
D10S 1571
/
/ / /
D10S 181E
/ / / /
/ /
D10S 1442
/
/
/ / /
D10S 1399E CD39
/ /
/ /
/ / /
/
/ / /
/ /
/ /
/ /
/
/ / / /
D10S 208
STS
/ /
/ /
AFM22 5yd12
/ (/) / /
/ /
AFMa0 48wb9
EST D10S IB3259 2269
IFI56
D10S 1680
D10S 1363E
/ /
/ /
D10S 1360E
STS
/ /
/
DNTT
/ /
(/) / /
D10S 1753
YAC Contig Spanning 10q24
TABLE 1
/ / /
D10S 564
/
/ / /
/
/ (/)
/
/ /
D10S 1755
/ /
/ /
/ /
/ /
/ / /
IDE
D10S 603
D10S 1319E
D10S 198 GOT1
EST IB3311
D10S D10S D10S 574 577 2172
/ /
D10S 536
/ / /
WNT8B
/ / /
D10S 583
/ / /
D10S 185
/ /
/ /
D10S 200
Telomere r
EST NIB 1686
(/) / (/)
(/)
/
/
/ /
D10S D10S D10S D10S 1266 1265 192 PAX2 1268
/ / /
D10S 1396E
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our data exclude any overlap between YAC 885-H-11 and YACs 761-B-1 and 958-A-7, although such an overlap has been suggested previously by YAC crosshybridization and L1 fingerprinting analysis (4). These discrepancies and others between our marker assignments and those reported by Ge´ ne´ thon (3, 4) together with further inconsistencies in our STS assignments between YACs suggest that at least five clones (847-D-4, 885-H-11, 941-F-5, 845-H-7, and 926-D-8) have undergone internal rearrangements during propagation. YAC 784-F-4 also gives an anomalous marker profile, but as this YAC is chimeric it is unclear whether rearrangement of the 10q24 complement occurred during library construction or subsequent propagation. An excess of rearranged YACs are clustered at the distal end of the contig, beyond D10S574. Four of the five YACs from this region (941F-5, 845-H-7, 963-C-11, and 926-D-8) show some evidence of rearrangement. Immediately downstream of this region is a sector of DNA for which we have been unable to isolate YAC clones, despite repeated library screens with CA repeat markers known to reside there (data not shown). It therefore appears that this genomic region is highly unstable when cloned in a yeast host. This may explain our inability to assign the developmental gene HOX11 to our contig (data not shown), despite its proximity to PAX2 (within 200 kb; Ref. 16). Perhaps significantly, HOX11 is the site of a translocation breakpoint in acute lymphoblastic T-cell leukemia (8). Furthermore, a BrdU-inducible fragile site has been identified in 10q24 (23), suggesting that this region may be inherently unstable. It may be possible to overcome some of the clone rearrangement problems experienced with YACs by using clones derived from bacterial and P1-derived artificial chromosome vector libraries (11, 24). We envisage the contig presented here being used as a framework for the construction of more refined physical maps of selected 10q24 areas using such clones. These maps should provide a substrate for further transcript identification, particularly in those areas of 10q24 shown to harbor disease genes by linkage or deletion studies and for large-scale DNA sequencing projects.
ACKNOWLEDGMENTS We thank Dr. Charles Auffay for providing chromosome 10 EST primer sequences and Iain Goldsmith and colleagues for oligonucleotide synthesis. YAC clones were provided by the UK Human Genome Mapping Project Resource Centre. This work was supported by a grant from the European Commission under Biomed 1 contract GENE CT93 0032. Note added in proof. We and others have recently identified a candidate tumor suppressor gene, designated PTEN or MMAC1, at the 10q23–q24 boundary (8, 16, 25). Mutations in this gene have been identified in tumors derived from a wide range of tissue types. PTEN/ MMAC1 lies between markers D10S1765 and D10S541 and is present on YACs 746-H-8, 796-D-5, 821-D-2, 831-D-5, and 921-F-8.
REFERENCES 1. Auffray, C., Behar, G., Bois, F., Bouchier, C., Da Silva, C., Devignes, M. D., Duprat, S., Houlgatte, R., Jumeau, M. N., Lamy, B., Lorenzo, F., Mitchell, H., Mariagesamson, R., Pietu, G., Pouliot, Y., Sebastianikabaktchis, C., and Tessier, A. (1995). IMAGE: Molecular integration of the analysis of the human genome and its expression. C. R. Acad. Sci. III 318: 263–272. 2. Baldini, A., Ross, M., Nizetic, D., Vatcheva, R., Lindsay, E. A., Lehrach, H., and Siniscalco, M. (1992). Chromosomal assignment of human YAC clone by fluorecence in situ hybridization: Use of single-yeast-colony PCR and multiple labeling. Genomics 14: 181–184. 3. Chumakov, I. M., Rigault, P., Le Gall, I., Bellanne-Chantelot, C., Billault, A., Guillou, S., Soularue, P., Guasconi, G., Poullier, E., Gros, I., Belova, M., Sambucy, J. L., Susini, L., Gervy, P., Glibert, F., Beaufils, S., Bui, H., Massart, C., Detand, M. F., Dukasz, F., Lecoulant, S., Ougen, P., Perrot, V., Saumler, M., Soravito, C., Bahouayila, R., Cohenakenine, A., Barillot, E., Bertrand, S., Codani, J. J., Caterina, D., Georges, I., Lacroix, B., Lucotte, G., Sahbatou, M., Schmit, C., Sangouard, M., Tubacher, E., Dib, C., Faure, C., Fizames, C., Gyapay, G., Millasseau, P., NGuyen, S., Muselet, D., Vignal, A., Morissette, J., Menninger, J., Lieman, J., Desai, T., Banks, A., Bray-Ward, P., Ward, D., Hudson, T., Gerety, S., Foote, S., Stein, L., Page, D. C., Lander, L. S., Weissenbach, J., Le Paslier, D., and Cohen, D. (1995). A yac contig map of the human genome. Nature 377(Suppl.): 175–297. 4. Cohen, D., Chumakov, I., and Weissenbach, J. (1993). A first generation physical map of the human genome. Nature 366: 698–701. 5. Dib, C., Faure, S., Fizames, C., Samson, D., Drouot, N., Vignal, A., Millasseau, P., Marc, S., Hazan, J., Seboun, E., Lathrop, M., Gyapay, G., Morissette, J., and Weissenbach, J. (1996). A
Note. ‘‘/’’ following a cytolocation indicates a second FISH signal elsewhere in the genome, implying chimerism. The second chromosomal location is given where known. FISH analyses were performed as described in Refs. 2 and 22. Ge´ne´thon CA repeat genetic markers are shown in boldface type. ‘‘/’’ in a marker column indicates positive marker assignment to a given YAC; marker order is based primarily on YAC assignments and secondarily on Ge´ne´thon linkage data (5). Where the order is equivocal, markers are boxed together. ‘‘(/)’’ indicates a Ge´ne´thon assignment that we were unable to duplicate, probably due to clone rearrangment during propagation. Although there is no common STS linking YAC 885-H-11 to YACs 761-B-1 and 958-A-7, this overlap has been established previously by clone cross-hybridization and L1 fingerprinting (4). However, the absence of EST IB3311 from YAC 885 H-11 suggests some clone rearrangment in this region. Further inconsistencies in marker assignment are evident for YAC 847-D-4 between markers D10S1442 and D10S1753 and for YAC784F-4 between markers D10S1403E and D10S1680. YAC PCR primers for STS assignment were taken from the Ge´ne´thon Quickmap database (3, 4), Whitehead Institute database (10), Genome database (Johns Hopkins University, Baltimore, MD) and Refs. 6 and 15; primer sequences derived from IMAGE consortium ESTs were provided by Dr. Charles Auffray (1). PCR was performed in 25-ml reactions containing 30 ng template YAC DNA, 11 PCR buffer (Boehringer Mannheim), 10 pmol primer, 200 mM dNTPs (Boehringer Mannheim), and 1 unit of Taq polymerase (Boehringer Mannheim) in a Gene-Amp 9600 thermal cycler (Perkin–Elmer). Thirty cycles of 30 s at 947C, 30 s at 557 C, and 30 s at 727C were preceded by a 3-min hot start at 947C. Presence or absence of a PCR product of the correct size was determined by electrophoresis through a 2% agarose gel. YAC sizes can be found on the CEPH–Ge´ne´thon database (3, 4).
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SHORT COMMUNICATION comprehensive genetic-map of the human genome based on 5,264 microsatellites. Nature 380: 152–154.
6. Gray, I. C., Nobile, C., Moresu, R., Ford, S., and Spurr, N. K. (1995). A 2.4 megabase physical map spanning the CYP2C gene cluster on chromosome 10q24. Genomics 28: 328–332. 7. Gray, I. C., Phillips, S. M. A., Lee, S. J., Neoptolemos, J. P., Weissenbach, J., and Spurr, N. K. (1995). Loss of the chromosomal region 10q23–25 in prostate cancer. Cancer Res. 55: 4800–4803. 8. Gray, I. C., Phillips, S. M. A., Stewart, L. M. D., Snary, D., and Spurr, N. K (1997). The putative tumour suppressor gene PTEN is mutated in primary prostate tumours. [Submitted for publication]
17.
18.
19.
9. Hatano, M., Roberts, C. W. M., Minden, M., Crist, W. M., and Korsmeyer, S. J. (1991). Deregulation of a homeobox gene, HOX11, by the t(10;14) in T cell leukemia. Science 253: 79–82. 10. Hudson, T., Stein, L., Gerety, S., Ma, J., Castle, A., Silva, J., Slonim, D., Baptista, R., Kruglyak, L., Xu, S., Hu, X., Colbert, A., Rosenberg, C., Reeve-Daly, M., Rozen, S., Hui, L., Wu, X., Vestergaard, C., Wilson, K., Bae, J., Maitra, S., Ganiatsas, S., Evans, C., DeAngelis, M., Ingalls, K., Nahf, R., Horton, L., Oskin, M., Collymore, A., Ye, W., Kouyoumjian, V., Zernsteva, I., Tarn, J., Devine, R., Courtney, D., Renaud, M., Nguyen, H., O’Connor, T., Fizames, C., Faure, S., Gyapay, G., Dib, C., Morissette, J., Orlin, J., Birren, B., Goodman, N., Weissenbach, J., Hawkins, T., Foote, S., Page, D., and Lander, E. (1995). An STS-based map of the human genome. Science 270: 1945–1954. 11. Ionnou, P. A., Amemiya, C. T., Garnes, J., Kroisel, P. M., Hiroaki, S., Chen, C., Batzer, M. A., and de Jong, P. J. (1994). A new bacteriophage P1-derived vector for the propagation of large human DNA fragments. Nature Genet. 6: 84–89. 12. Ittmann, M. (1996). Allelic loss on chromosome 10 in prostate adenocarcinoma. Cancer Res. 56: 2143–2147. 13. Kennedy, M. A., Gonzalez-Sarmiento, R., Kees, U. R., Lampert, F., Dear, N., Boehm, T., and Rabbitts, T. H. (1991). HOX11, a homeobox-containing T-cell oncogene on human chromosome 10q24. Proc. Natl. Acad. Sci. USA 88: 8900–8904. 14. Lacombe, L., Orlow, I., Reuter, V. E., Fair, W. R., Dalbagni, G., Zhang, Z. F., and Cordoncardo, C. (1996). Microsatellite instability and deletion analysis of chromosome-10 in human prostate-cancer. Int. J. Cancer 69: 110–113.
20.
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15. Lako, M., Strachan, T., Curtis, A. R. J., and Lindsay, S. (1996). Isolation and characterization of wnt8b, a novel human wnt gene that maps to 10q24. Genomics 35: 386–388. 16. Li, J., Yen, C., Liaw, Y., Podyspanina, K., Bose, S., Wang, S. I., Puc, J., Miliaresis, C., Rodgers, L., McCombie, R., Bigner, S. H., Giovanella, B. C., Ittmann, M., Tycko, B., Hibshoosh, H., Wigler, M. H., and Parsons, R. (1997). PTEN, a putative protein
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26.
tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275: 1943–1947. Meehan, R. R., Gosden, J. R., Rout, D., Hastie, N. D., Friedberg, T., Adesnik, M., Buckland, R., Heyningen, V. v., Fletcher, J., Spurr, N. K., Sweeney, J., and Wolf, C. R. (1988). Human cytochrome P-450 PB-I: A multigene family involved in mephenytoin and steroid oxidations that maps to human chromosome 10. Am. J. Hum. Genet. 42: 26–37. Moschonas, N. K., Spurr, N. K., and Mao, J. (1996). Report of the first international workshop on human-chromosome-10 mapping 1995. Cytogenet. Cell Genet. 72: 100–110. Nelen, M. R., Padberg, G. W., Peeters, E. A. J., Lin, A. Y., van den Helm, B., Frants, R. R., Coulon, V., Goldstein, A. M., van Reen, M. M. M., Easton, D. F., Eeles, R. A., Hodgson, S., Mulvihill, J. J., Murday, V. A., Tucker, M. A., Mariman, E. C. M., Starink, T. M., Ponder, B. A. J., Ropers, H. H., Kremer, H., Longy, M., and Eng, C. (1996). Localization of the gene for Cowden disease to chromosome 10q22–23. Nature Genet. 13: 114– 116. Nikali, K., Suomalainen, A., Terwilliger, J., Koskinen, T., Weissenbach, J., and Peltonen, L. (1995). Random search for shared chromosomal regions in 4 affected individuals—The assignment of a new hereditary ataxia locus. Am. J. Hum. Genet. 56: 1088–1095. Ottman, R., Risch, N., Hauser, W. A., Pedley, T. A., Lee, J. H., Barker-Cummings, C., Lustenberger, A., Nagle, K. J., Lee, K. S., Scheuer, M. L., Neystat, M., Susser, M., and Wilhelmsen, K. C. (1995). Localization of a gene for partial epilepsy to chromosome 10q. Nature Genet. 10: 55–60. Ragoussis, J., Monaco, A., Mockridge, I., Kendall, E., Campbell, R. D., and Trowsdale, J. (1991). Cloning of the HLA class II region in yeast artificial chromosomes. Proc. Natl. Acad. Sci. USA 88: 3753–3757. Scheres, J. M., and Hustinx, T. W. (1980). Heritable fragile sites and lymphocyte culture medium containing BrdU. Am. J. Hum. Genet. 32: 628–629. Shizuya, H., Birren, B., Kim, U., Mancino, V., Slepak, T., Tachiri, Y., and Simon, M. (1992). Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. USA 89: 8794–8797. Steck, P. A., Pershouse, M. A., Jasser, S. A., Yung, W. K. A., Lin, H., Ligon, A. H., Langford, L. A., Baurngard, M. L., Hattier, T., Davis, T., Frye, C., Hu, R., Swedlund, B., Teng, D. H. F., and Tavtigian, S. V. (1997). Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nature Genet. 15: 356–362. Trybus, T., Burgess, A. C., Wojno, K. J., Glover, T. W., and Macoska, J. A. (1996). Distinct areas of allelic loss on chromosomal regions 10p and 10q in human prostate cancer. Cancer Res. 56: 2263–2267.
gnmxa
markers from loci other than 10q23–q25 or hybridizing to cell hybrid DNA with a human complement other than chromosome 10) were avoided. The cytogenetic location of each YAC was confirmed by fluorescence in situ hybridization (FISH) to metaphase spreads of human lymphocytes; 9/42 YACs (21%) showed chimerism detectable by FISH, a level somewhat lower than the estimated mega-YAC library average of 40% (4) and probably reflecting our biased selection procedure (Table 1). Ge´ne´thon CA repeat genetic markers from 10q23– q25 (3, 4) were assigned to YACs by PCR amplification, allowing us to confirm the presence of markers previously assigned by Ge´ne´thon (3, 4), make novel assignments not reported by Ge´ne´thon, and position several new previously unmapped markers. Establishing the physical order of these markers should resolve any discrepancies in genetic map order. The YACs were also screened using PCR primers derived from genes listed as mapping to 10q23–q26 in the Genome Database (Johns Hopkins University, Baltimore, MD). Sequencetagged sites (STSs) derived from nine known gene loci (IFI56, IDE, PDE6C, RBP4, CYP2C, CD39, DNTT, GOT1, WNT8B, and PAX2) were mapped to one or more YACs, together with one expressed sequence tag (EST), D10S181E. To identify additional 10q24 genes, YACs were screened with 144 ESTs derived from chromosome 10 (Ref. 1; Dr. Charles Auffray, Paris, pers. comm., 1996). Seven proved positive: D10S1326E, D10S1360E, D10S1363E D10S1319E, D10S1396E, D10S1403E, and D10S1399E. We were also able to localize 3 further ESTs from the Whitehead Institute database (10) previously mapped to 10q24 YACs: IB3311, NIB1686, and IB3259. The assignment of CA markers, genes, ESTs, and a further 4 Whitehead Institute STSs (D10S1442, D10S1571, D10S2269, and D10S2172; Ref. 10) allowed the majority of YACs to be linked to one or more overlapping clones by a common STS. The contig is summarized in Table 1. Two overlaps are equivocal: according to the Ge´ ne´ thon data (4), D10S577 is common to YACs 756-G-3 and 941-F-5; however, we were unable to confirm the presence of this marker on YAC 941-F-5. In addition,
Chromosome band 10q24 is rich in genes involved in development, tumorigenesis, neurological disorders, hormone metabolism, and environmentally induced disease susceptibility. We have constructed an STSbased integrated physical and genetic map of 10q24 derived from the CEPH–Ge´ne´thon mega-YAC contig data for this region. This map consists of 42 fluorescence in situ hybridization-mapped overlapping CEPH mega-YACs spanning approximately 15 Mb to which 49 STS markers have been assigned, including 24 Ge´ne´thon CA repeat genetic markers, 10 known gene loci from the 10q24 region (IFI56, IDE, PDE6C, RBP4, CYP2C, CD39, DNTT, GOT1, WNT8B, and PAX2) and 11 additional expressed sequences of unknown function. q 1997 Academic Press
The chromosomal location 10q24 bears a number of genes of fundamental biological significance, including the developmental genes PAX2, HOX11, and WNT8B (13, 15, 18) and the cytochrome P450IIC gene cluster involved in the metabolism of steroid hormones and environmental agents (17). Linkage analysis implies that this region also contains genes involved in epilepsy (21), spinocerebellar ataxia (20), and Cowden disease (19). Allele loss studies also suggest the presence of a prostate tumor suppressor gene at proximal 10q24 (7, 12, 14, 26). As part of an effort to identify this tumor suppressor gene, we have constructed a mega-YAC contig of 10q24 between the CA repeat genetic markers D10S579 and D10S1268, a distance of 20 cM (5). This contig combines genetic markers with known 10q24 genes and further anonymous expressed sequences. To provide a scaffold for contig construction, 42 mega-YACs derived from the 10q23–q25 region were selected from the CEPH–Ge´ne´thon Quickmap database (3, 4). Potentially chimeric YACs (those bearing 1 Present address: SmithKline Beecham Pharmaceuticals, Biopharmaceutical R&D, New Frontiers Science Park (North), Harlow, Essex CM10 5AW, UK. 2 To whom correspondence should be addressed. Telephone: 01279 627225. Fax: 01279 622500. E-mail: [email protected].
GENOMICS
85
43, 85–88 (1997) GE974809
ARTICLE NO.
0888-7543/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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GENO 4809
/
6r3b$$$$$1
06-05-97 01:15:29
gnmxa
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GENO 4809
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10q24 10q24 10q24 10q24 / 11q 10q24 10q24 10q24 / 10q21 10q24 / 2q 10q24 10q24 10q24 10q24 10q24 – q25 / A 10q24 – q25 10q24 – q25
912C4 912C7 805A9 784F4 853A4 744D4 789B10 946E3 857E10 756G3 941F5 845H7 963C11 926D8 954E2
/ / 10p
/ /
/5
10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q23 – q24 10q24 10q24
Cytolocation
738B12 921F8 746H8 821D2 831E5 796D5 829E1 734B4 927H12 934D3 906D1 682G1 794H1 944C1 750H1 847D4 759C9 950A1 885H11 773F6 890G6 761B1 958A7 745D8 791C3 912C4 912C7
YAC / / / / /
D10S 579 / / / / /
D10S 215
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gnmxa
/ /
/ /
/ / /
PDE6C RBP4 CYP2C
/
D10S 1326E
R Centromere
/ / / / /
D10S 571
/ / / / /
D10S 1765
/ / / / / / / / /
D10S 541
/ / / / /
D10S 1403E
/ / / /
/ / / /
D10S 1571
/
/ / /
D10S 181E
/ / / /
/ /
D10S 1442
/
/
/ / /
D10S 1399E CD39
/ /
/ /
/ / /
/
/ / /
/ /
/ /
/ /
/
/ / / /
D10S 208
STS
/ /
/ /
AFM22 5yd12
/ (/) / /
/ /
AFMa0 48wb9
EST D10S IB3259 2269
IFI56
D10S 1680
D10S 1363E
/ /
/ /
D10S 1360E
STS
/ /
/
DNTT
/ /
(/) / /
D10S 1753
YAC Contig Spanning 10q24
TABLE 1
/ / /
D10S 564
/
/ / /
/
/ (/)
/
/ /
D10S 1755
/ /
/ /
/ /
/ /
/ / /
IDE
D10S 603
D10S 1319E
D10S 198 GOT1
EST IB3311
D10S D10S D10S 574 577 2172
/ /
D10S 536
/ / /
WNT8B
/ / /
D10S 583
/ / /
D10S 185
/ /
/ /
D10S 200
Telomere r
EST NIB 1686
(/) / (/)
(/)
/
/
/ /
D10S D10S D10S D10S 1266 1265 192 PAX2 1268
/ / /
D10S 1396E
86 SHORT COMMUNICATION
87
SHORT COMMUNICATION
our data exclude any overlap between YAC 885-H-11 and YACs 761-B-1 and 958-A-7, although such an overlap has been suggested previously by YAC crosshybridization and L1 fingerprinting analysis (4). These discrepancies and others between our marker assignments and those reported by Ge´ ne´ thon (3, 4) together with further inconsistencies in our STS assignments between YACs suggest that at least five clones (847-D-4, 885-H-11, 941-F-5, 845-H-7, and 926-D-8) have undergone internal rearrangements during propagation. YAC 784-F-4 also gives an anomalous marker profile, but as this YAC is chimeric it is unclear whether rearrangement of the 10q24 complement occurred during library construction or subsequent propagation. An excess of rearranged YACs are clustered at the distal end of the contig, beyond D10S574. Four of the five YACs from this region (941F-5, 845-H-7, 963-C-11, and 926-D-8) show some evidence of rearrangement. Immediately downstream of this region is a sector of DNA for which we have been unable to isolate YAC clones, despite repeated library screens with CA repeat markers known to reside there (data not shown). It therefore appears that this genomic region is highly unstable when cloned in a yeast host. This may explain our inability to assign the developmental gene HOX11 to our contig (data not shown), despite its proximity to PAX2 (within 200 kb; Ref. 16). Perhaps significantly, HOX11 is the site of a translocation breakpoint in acute lymphoblastic T-cell leukemia (8). Furthermore, a BrdU-inducible fragile site has been identified in 10q24 (23), suggesting that this region may be inherently unstable. It may be possible to overcome some of the clone rearrangement problems experienced with YACs by using clones derived from bacterial and P1-derived artificial chromosome vector libraries (11, 24). We envisage the contig presented here being used as a framework for the construction of more refined physical maps of selected 10q24 areas using such clones. These maps should provide a substrate for further transcript identification, particularly in those areas of 10q24 shown to harbor disease genes by linkage or deletion studies and for large-scale DNA sequencing projects.
ACKNOWLEDGMENTS We thank Dr. Charles Auffay for providing chromosome 10 EST primer sequences and Iain Goldsmith and colleagues for oligonucleotide synthesis. YAC clones were provided by the UK Human Genome Mapping Project Resource Centre. This work was supported by a grant from the European Commission under Biomed 1 contract GENE CT93 0032. Note added in proof. We and others have recently identified a candidate tumor suppressor gene, designated PTEN or MMAC1, at the 10q23–q24 boundary (8, 16, 25). Mutations in this gene have been identified in tumors derived from a wide range of tissue types. PTEN/ MMAC1 lies between markers D10S1765 and D10S541 and is present on YACs 746-H-8, 796-D-5, 821-D-2, 831-D-5, and 921-F-8.
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Note. ‘‘/’’ following a cytolocation indicates a second FISH signal elsewhere in the genome, implying chimerism. The second chromosomal location is given where known. FISH analyses were performed as described in Refs. 2 and 22. Ge´ne´thon CA repeat genetic markers are shown in boldface type. ‘‘/’’ in a marker column indicates positive marker assignment to a given YAC; marker order is based primarily on YAC assignments and secondarily on Ge´ne´thon linkage data (5). Where the order is equivocal, markers are boxed together. ‘‘(/)’’ indicates a Ge´ne´thon assignment that we were unable to duplicate, probably due to clone rearrangment during propagation. Although there is no common STS linking YAC 885-H-11 to YACs 761-B-1 and 958-A-7, this overlap has been established previously by clone cross-hybridization and L1 fingerprinting (4). However, the absence of EST IB3311 from YAC 885 H-11 suggests some clone rearrangment in this region. Further inconsistencies in marker assignment are evident for YAC 847-D-4 between markers D10S1442 and D10S1753 and for YAC784F-4 between markers D10S1403E and D10S1680. YAC PCR primers for STS assignment were taken from the Ge´ne´thon Quickmap database (3, 4), Whitehead Institute database (10), Genome database (Johns Hopkins University, Baltimore, MD) and Refs. 6 and 15; primer sequences derived from IMAGE consortium ESTs were provided by Dr. Charles Auffray (1). PCR was performed in 25-ml reactions containing 30 ng template YAC DNA, 11 PCR buffer (Boehringer Mannheim), 10 pmol primer, 200 mM dNTPs (Boehringer Mannheim), and 1 unit of Taq polymerase (Boehringer Mannheim) in a Gene-Amp 9600 thermal cycler (Perkin–Elmer). Thirty cycles of 30 s at 947C, 30 s at 557 C, and 30 s at 727C were preceded by a 3-min hot start at 947C. Presence or absence of a PCR product of the correct size was determined by electrophoresis through a 2% agarose gel. YAC sizes can be found on the CEPH–Ge´ne´thon database (3, 4).
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