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Subregional mapping of 13 single-copy genes on the long arm of chromosome 12 by fluorescence in Situ hybridization
GENOMICS
14, 775-779 (1992)
Subregional Mapping of 13 Single-Copy Genes on the Long Arm of Chromosome 12 by Fluorescence in Situ Hybridization SUSAN MATHEW,* V. V. V. S. MURTY,*W. HUNZIKER,I AND R. S. K. CHAGANTI* *Laboratory of Cancer Genetics and the Department of Pathology, Memorial 51oan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021; and tHoffman-La Roche, Basel, Switzerland Received March 5, 1992; revised July 2. 1992 S u b r e g i o n a l l o c a l i z a t i o n o f 1 3 s i n g l e - c o p y D N A seq u e n c e s p r e v i o u s l y a s s i g n e d to t h e l o n g a r m o f c h r o m o some 12 has been performed using the fluorescence in situ h y b r i d i z a t i o n (FISH) technique. T h e f o l l o w i n g o r d e r is s u g g e s t e d f o r t h e 1 3 m a p p e d g e n e s : c e n -~ COL2A1 -~ (VDR-D12815) --~ ( D 1 2 S 1 7 - D 1 2 8 4 D12S14-D12S6) --~ D 1 2 S 8 --~ ( I A P P - M G F - D 1 2 S 7 D 1 2 S 1 2 ) -~ I G F 1 - ~ q t e r . E i g h t o f t h e m a p p e d g e n e s c l u s t e r e d at t w o r e g i o n s , o n e at 1 2 q 1 3 ( D 1 2 S 1 7 D12S4-D12S14-D12S6) a n d t h e o t h e r at 1 2 q 2 2 (IAPP-MGF-D12S7-D12S12). Our results show that s i n g l e - c o p y D N A s e q u e n c e s a s s m a l l a s 5 0 0 bp c a n b e s u c c e s s f u l l y m a p p e d b y F I S H . © 1992 AcademicPress, Inc.
INTRODUCTION
Systematic efforts to map the human genome have resulted in primary genetic and linkage maps for each human chromosome (Human Gene Mapping 11, 1991; Stephens et al., 1990). The gene map of chromosome 12 is of interest because of its involvement in nonrandom abnormality in a variety of tumor types, e.g., deletions and other rearrangements in germ cell tumors (Rodriguez et al., 1992) and leiomyomas (Pandis et al., 1990) and nondisjunction in lymphoid malignancies, in particular chronic lymphocytic lymphoma (Gahrton and Robeft, 1982). Based on an analysis of loss of constitutional heterozygosity at eight polymorphic loci, two candidate tumor suppressor genes specific to germ cell tumors have been identified (Murty et al., 1992). To enhance the fine map of chromosome 12 and to facilitate the eventual identification of the putative tumor suppressor gene(s) and other genes perturbed in tumors, we have assigned 13 independently derived probes previously assigned to 12q to specific bands of metaphase chromosomes using the fluorescence in situ hybridization (FISH) technique. We further show t h a t the FISH technique can be used for mapping DNA sequences as small as 500 bp derived from single-copy genes. MATERIALS AND METHODS
cell hybrid analysis and, in addition, three have been mapped subregionally by in situ hybridization of radiolabeled probes to metaphase chromosomes. In the present FISH analysis, the vectors containing the probes were labeled with b i o t i n - l l - d U T P or biotin-14-dATP by nick-translation using E s c h e r i c h i a coli DNA polymerase I (Maniatis et al., 1982). Labeled probes were purified on Sephadex G-50 chromatography columns, ethanol precipitated, and dissolved in TE (10 m M Tris-HC1, 1 m M EDTA, p H 8.0). Chromosome preparations were made from human lymphocyte cultures synchronized with 5-bromodeoxyuridine as described (Zabel et al., 1983). I n situ hybridization was performed by a modification of the protocol described by Neel et al. (1982). Biotin-labeled probes at a concentration of 100-200 ng of DNA in 30 ttl of hybridization mixture (50% formamide/2X SSC/10% dextran sulfate/IX Denhardt's/0.1% SDS/ 25 m M sodium phosphate, and 200 ~g/ml of sheared salmon sperm DNA) were denatured at 75°C for 7 min. Suppression of repeated sequences was performed whenever necessary by preannealing the probe with 1-2 #g of placental DNA at 37°C for 5-20 min. Chromosomal DNA was denatured at 70°C for 2 min in 70% formamide/2x SSC. Overnight hybridization was carried out at 37°C. Posthybridization washes were performed at 42°C in 50% formamide/2X SSC for 20 min, followed by several washes in 2X SSC. Hybridized probes were detected by indirect immunofluorescenceusing avidin-conjugated fluorescein isothiocyanate (FITC-avidin) and biotinylated anti-avidin (Pinkel et al., 1986; Fan et al., 1990). The slides were stained with propidium iodide (PI) (Sigma) and counterstained with 4',6'-diamidino-2-phenylindole (DAPI, Sigma). To obtain G-banding, slides were stained with 33258 Hoechst, exposed to UV, and counterstained with Giemsa. Informative metaphases exhibiting fluorescent hybridization signals were photographed using a Nikon fluorescence microscope and the filter combinations B2A for FITC and UV-2A for DAPI to sequentially visualize hybridization signals and banding patterns on the chromosomes. Microphotographs were made sequentially, keeping the settings on the microscope and camera constant and using Kodak Ektachrome 400 film. The assignment of a signal to a specific chromosome band was made in the following manner. Kodachromes of the PI- and DAPI-stained individual metaphases were loaded in sequence in a Kodak carousel tray and projected onto the screen of a Kodak Ektagraphic projector. The end of the arm of the H - s t a i n e d chromosome exhibiting hybridization signal was marked on the screen, the distance between the chromosome end and the middle of the signal was measured using a dial caliper (Mitutoya), and the caliper setting was retained unchanged. The PI image was replaced by the DAPI image on the screen. The point of one arm of the caliper was placed on the end of the chromosome (marked from the PI-stained image) and the point of the other arm was placed on the chromosome, which then identified the specific band in the DAPI image at which hybridization occurred. RESULTS
The probes utilized in this study, the loci from which they were derived, their size, and the source(s) from which they were obtained are shown in Table 1. Insert sizes of the probes ranged from 0.5 to 4.0 kb. All these probes have previously been assigned to 12q by somatic
We used the FISH assay for the subregional localization of 10 probes for the first time and confirmation of 775
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TABLE 1 1 2 q P r o b e s U s e d for M a p p i n g b y F I S H Probe
Locus
Insert size (kb)
Source
Reference
p9Fll 11-1-17 pDL32B p9Gll pAC230 pFED33.2 pCMM1.2 pYNH15 pH-KITL phigfl WV38
D12S4 D12S6 D12S7 D12S8 D12S12 D12S14 D12S15 D12S17 MGF IGFI COL2A1
1.2 0.5 3.2 1.0 2.2 3.2 1.9 3.2 0.8 0.6 4.0
ATCC M. Litt ATCC ATCC I. Balazs ATCC ATCC ATCC W. Hunziker ATCC E.J. Weaver
scIAP-8 pill3
IAPP VDR
0.6 2.1
J. Hoppener J. Shine
O'Connell et aL, 1987 Buroker et al., 1986 Tsui et al., 1985 O'Connell et al., 1987 Balazs et al., 1987 Nakamura et al., 1988a Martin et al., 1988 Nakamura et al., 1988b Unpublished Hoppener et al., 1985 Sangiorgi et al., 1985; Law et al., 1986 Mosselman et al., 1988 Faraco et al., 1989
N o t e . I G F 1 , insulin-like growth factor 1; C O L 2 A 1, procollagen, type II, alpha; V D R , v i t a m i n D receptor; I A P P , Islet amyloid polypeptide; M G F , m a s t cell growth factor.
previous assignments of three probes from among a panel of 13 single-copy DNA sequences previously assigned to the long arm of chromosome 12. Since these probes have previously been assigned to 12q, the analysis was confined only to cells exhibiting signals on 12q with each probe. For each probe, 25-100 metaphases with fluorescent hybridization signals on chromosome 12 were scored. Hybridization signals were observed either as single (on one chromatid) or double (on both chromatids) dots. The percentage of single or double hybridization signals varied from probe to probe. The signals always clustered in a probe-specific manner, thus allowing unambiguous assignment of the 13 probes to specific bands on 12q (Fig. 1). The data from hybridization results are summarized in Table 2 and the positions of the 13 mapped probes are shown on an idiogram of chromosome 12 in Fig. 2. Based on the data discussed above, the following order is suggested for the 13 mapped genes: cen -~ C O L 2 A I --~ ( V D R - D 1 2 S 1 5 ) --~ (D12S17D12S4-D12S14-D12S6) --~ D12S8 --~ ( I A P P - M G F D12S7-D12S12) --~ IGF1 --~ qter.
Mapping 11, 1991) is confirmed in the present study. C O L 2 A 1 has previously been mapped to 12q13.1-q13.2 using 8H-labeled probe DNA for in situ hybridization (Huerre-Jeanpierre et al., 1986). In another study, Law et al. (1986) assigned C O L 2 A 1 to 12q14.3 using a panel of hamster-human somatic cell hybrids. The map position of this gene was recently revised to 12q13 (Human Gene Mapping 11, 1991). Using FISH, we now map C O L 2 A 1 to 12q12 (Table 2, Fig. 2). Kit-ligand, otherwise referred to as mast cell growth factor (MGF) or stem cell factor (SCF), is the product of the steel (S1) locus in the mouse and comprises the ligand for the K I T protooncogene tyrosine kinase receptor (Zsebo et al., 1990; Huang et al., 1990; Martin et al., 1990). The human chromosomal localization of M G F was reported by two groups. Geissler et al. (1991), using a panel of somatic cell hybrids, assigned this gene to 12q14.3-qter, while Anderson et al. (1991), using both somatic cell hybrid and in situ hybridization of radiolabeled probe, mapped M G F to 12q22-q24. In the present study, using FISH, we localize M G F to 12q22. Somatic cell hybrid analysis showed that the human I A P P gene is located in the region 12pll.2-p12.3 (Hoppener et al., 1985; Mosselman et al., 1988). Buckle et al. (1989) and Fan et al. (1989) assigned this gene to 12pll.2-p12.3 by in situ hybridization, whereas Cockburn et al. (1989) provided evidence for another hybridization site at 12cen-q21.1. In another study, also by in situ hybridization of radiolabeled probe, 12p12-q13 and 12q13-q14 were suggested to be the sites of localization of this gene (Christmanson et al., 1990). In HMG 11 (1991) the map position is listed as 12p12.3-p13 and 12q13-q21. However, in an analysis of 37 cells with FISH signals on chromosome 12, we saw signals consistently only in the 12q21.3-q22 region; no hybridization signal was seen on 12p. TABLE 2 A s s i g n m e n t o f 13 D N A P r o b e s on the L o n g A r m o f C h r o m o s o m e 12 b y F l u o r e s c e n c e in S i t u H y b r i d i z a t i o n
DISCUSSION To enhance the fine map and to facilitate the investigation of genetic alterations affecting 12q in tumors, we have attempted to subregionally localize 13 probes on the long arm of chromosome 12 by FISH. With the exception of D12S6, IGF1, and C O L 2 A 1 , which were previously mapped to specific bands, the remaining probes mapped by us were previously assigned only to broad regions of 12q (Craig and McBride, 1991). Buroker et al. (1986) assigned D12S6 to the 12cen-q13 region by in situ hybridization of a radiolabeled cosmid probe, whereas O'Connell et al. (1987) assigned the same marker to 12q14 by linkage analysis. Our FISH mapping assigns this probe to 12q13.2-q13.3 (Table 2, Fig. 2). The previous localization of IGF1 to 12q23 (Human Gene
Locus COL2A 1 VDR
D12S15 D12S17 D12S4 D12S14 D12S6 D12S8
IAPP MGF
D12S7 D12S12 IGF1
No. of c h r o m o s o m e s 12 with
No. of metaphases with signal on 12q
Single signal
Double signal
Signal position
100 28 40 46 49 33 25 41 37 28 29 72 26
80 8 30 38 40 13 20 29 21 17 18 33 2
48 20 16 20 12 23 6 17 20 12 12 43 25
12q12 12q12-13.1 12q12-13.1 12q13.1 12q13 12q13 12q13.2-13.3 12q14-15 12q21.3-22 12q22 12q22 12q22 12q23
MAPPING OF SINGLE-COPY GENES ON 12q BY FISH
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D12S4
D12S6
D12S7
D12S8
D12S12
D12S14
D12S15
D12S17
MGF
IGF1
COL2A1
lAPP
VDR
FIG. 1. In situ hybridization mapping of 13 single-copy genes on chromosome 12. Partial metaphases stained with propidium iodide (left) and DAPI (right). In the case of D12S7, the preparation was G-banded following fluorescence microscopy of the hybridized signal. The G-banded picture of the chromosome is shown in the right panel. Arrows indicate fluorescent signals and arrowheads indicate band positions.
T h e v i t a m i n D r e c e p t o r (VDR) gene has previously b e e n assigned to c h r o m o s o m e 12 b y s o m a t i c cell h y b r i d analysis (Faraco et al., 1989). W e now localize it to 12q12-q13.1 b y F I S H . D12S7 has b e e n m a p p e d to 12q14.3-qter b y s o m a t i c cell h y b r i d analysis, a n d we now assign this p r o b e to 12q22. D12S8 has previously b e e n assigned to 1 2 q 1 4 - q t e r ( H u m a n G e n e M a p p i n g 11, 1991). In t h e p r e s e n t study, we h a v e assigned this p r o b e to 12q14-q15. D12S4 was p r e v i o u s l y assigned to 1 2 c e n q14, a n d our results assign it to 12q13. D12S15 ( M a r t i n et al., 1988), D12S14 ( N a k a m u r a et al., 1988a), D12S17
( N a k a m u r a et al., 1988b), a n d D12S12 (Balazs et al., 1987) have previously b e e n assigned only to the long a r m of c h r o m o s o m e 12; we now localize these p r o b e s to 12q12-q13.1, 12q13, 12q13.1, a n d 12q22 regions, respectively, using F I S H . T h e signal resolution a n d m a p p i n g precision are higher with F I S H t h a n with s o m a t i c cell h y b r i d analysis a n d in s i t u h y b r i d i z a t i o n of radiolabeled probes. T h e precise identification of h u m a n c h r o m o s o m e s e g m e n t s in s o m a t i c cell hybrids t e n d s to be difficult, a n d regional s c a t t e r of silver grains f r o m radiolabeled p r o b e s used in
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COL2A1 VDR, D12S15
D12S17 D12S4, D12S14 D12S6 D12S8
I IAPP
MGF D12S7 D12S12
IGF1
F I G . 2. Idiogram of chromosome 12 showing the map positions of the 13 sing!e-copy genes mapped in this study.
autoradiography of in situ hybridized probes also makes specific assignment difficult. Probe signals do not scatter in FISH, and both chromatids usually take part in hybridization, resulting in "twin" or double hybridization spots within the chromosome and thus enhancing the precision of mapping. In the present study, we determined the map positions of 13 DNA sequences on 12q by FISH. Furthermore, our results clearly demonstrate the feasibility of mapping single-copy DNA sequences as small as 500 bp, as in the case of D12S6. The current map of 12q generated by us also correlates well with the linear order of the existing linkage map for chromosome 12 (O'Connell et al., 1987). Our data on the 12q map showed clustering of loci on two regions, one at 12q13 (D12S17-D12S4-D12S14-D12S6) and the other at 12q22 ( I A P P - M G F - D 1 2 S 7 - D 1 2 S 1 2 ) . ACKNOWLEDGMENTS We thank Drs. M. Litt, I. Balazs, J. Shine, E. J. Weaver, and J. Hoppener for providing some of the probes used in this study (Table 1). We thank Dr. Jane Houldsworth for help with preparation of probes. This investigation was supported in part by National Institutes of Health Research Grants CA-34775 and CA-05826. REFERENCES Anderson, D. M., Williams, D. E., Tushinski, R., Gimpel, S., Eisenman, J., Cannizzaro, L. A., Aronson, M., Croce, C. M., Huebner, K., Cosman, D., and Lyman, S. D. (1991). Alternate splicing of mRNAs encoding human mast cell growth factor and localization of the gene to chromosome 12q22-q24. Cell Growth & Differ. 2: 373-378.
Balazs, I., Nicholas, L., and Grzeschick, K.-H. (1987). Isolation and characterization of a cDNA clone detecting a RFLP in chromosome 12: Human Gene Mapping 9. Cytogenet. Cell Genet. 46: 574. Buckle, V. J., Marsland, A. M., Mosselman, S., and Hoppener, J. W. M. (1989). Localization of the human islet amyloid polypeptide (IAPP) gene to 12pli.2-p12.3: Human Gene Mapping 10. Cytogenet. Cell Genet. 51: 972. Buroker, N. E., Magenis, R. E., Weliky, K., Bruns, G., and Litt, M. (1986). Four restriction fragment length polymorphisms revealed by probes from a single cosmid map to human chromosomes. Hum. Genet. 72: 86-94. Christmanson, L., Russman, F., Stenman, G., Westermark, P., and Bestholz, C. (1990). The human islet amyloid polypeptide (IAPP) gene: Organization, chromosomal localization and functional identification of promoter region. F E B S Lett. 2 6 2 : 160-166. Cockburn, D. C., Holt, S. M., Roberts, A. N., Cooper, G. J. S., Reid, K. B. M., and Boyd, Y. (1989). Localization of the amylin locus to chromosome 12: Human Gene Mapping 10. Cytogenet. Cell Genet. 51: 997. Craig, I. W., and McBride, O. W. (1991). Report of the committee on the genetic constitution of chromosome 12: Human Gene Mapping 11. Cytogenet. Cell Genet. 58: 555-579. Fan, Y. S., Eddy, R. L., Byers, M. G., Haley, L. L., Henry, W. M., Sanke, T., Steiner, D. F., Shows, T. B., and Bell, G. I. (1989). Localization of the human islet amyloid polypeptide gene (IAP) to chromosome 12p12.3: Human Gene Mapping 10. Cytogenet. Cell Genet. 51: 997. Fan, Y. S., Davis, L. M., and Shows, T. B. (1990). Mapping small DNA sequences by fluorescence in situ hybridization directly on banded metaphase chromosomes. Proc. Natl. Acad. Sci. USA 87: 62236227. Faraco, J. H., Morrison, N. A., Baker, A., Shine, J., and Frossard, P. M. (1989). Apa I dimorphism at the human vitamin D receptor gene locus. Nucleic Acids Res. 17: 2150. Gahrton, G., and Robert, K.-H. (1982). Chromosomal aberrations in chronic B-cell lymphocytic leukemia. Cancer Genet. Cytogenet. 6: 171-181. Geissler, E. N., Liao, M., Brook, J. D., Martin, F. H., Zsebo, K. M., Houseman, D. E., and Galli, S. J. (1991). Stem cell factor (SCF), a novel hematopoietic growth factor and ligand for c-kit tyrosine kinase receptor, maps on human chromosome 12 between 12q14.3 and 12qter. Somat. Cell Mol. Genet. 17: 207-214. Hoppener, J. W. M., de Pagter-Holthuizen, P., Geurts van Kessel, A. H. M., Jansen, M., Kittur, S. D., Antonarakis, S. E., Lips, C. J. M., and Sussenbach, J. S. (1985). The human gene encoding insulin-like growth factor I is located on chromosome 12. Hum. Genet. 69: 157-160. Huang, E., Nocka, K., Beier, D. R., Chu, T-Y., Buck, J., Lahm, H-W., Wellner, D., Leder, P., and Besmer, P. (1990). The hematopoietic growth factor KL is encoded by the $1 locus and is the ligand of the c-Kit receptor, the gene product of the W locus. Cell 63: 225-233. Huerre-Jeanpierre, C., Mattei, M. G., Weil, D., Grzeschik, K. H., Chu, M. L., Sangiorgi, F. 0., Sobel, M. E., Ramirez, F., and Junien, C. (1986). Further evidence for the dispersion of the fibrillar collagen genes. Am. J. Hum. Genet. 38: 26-37. Human Gene Mapping 11 (1991). Eleventh International Workshop on Human Gene Mapping. Cytogenet. Cell Genet. 58: 1-2200. Law, M. L., Tung, L., Morse, H. G., Berger, R., Jones, C., Cheah, K. S. E., and Solomon, E. (1986). The human type II collagen gene (COL2A1) assigned to 12q14.3. Ann. Hum. Genet. 59: 131-137. Maniatis, T., Fritseh, F. F., and Sambrook, J. (1982). "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Martin, C., Nakamura, Y., Hoff, M., O'Connell, P., Leppert, M., Lathrop, G. M., Lalouel, J.-M., and White, R. (1988). Isolation and
MAPPING OF SINGLE-COPY GENES ON 12q BY FISH mapping of a polymorphic DNA sequence (pCMM1.2) on chromosome 12q (D12S15). Nucleic Acids Res. 16: 3596. Martin, F. H., Suggs, S. V., Langley, K. E., Lu, H. S., Ting, J., Okino, K. H., Morris, C. F., McNiece, I. K., Jacobsen, F. W., Mendiaz, E. A., Birkett, N. C., Smith, K. A., Johnson, M. J., Parker, V. P., Flores, J. C., Patel, A. C., Fisher, E. F., F,rjavec, H. 0., Herrera, C. J., Wypych, J., Sachdev, R. K., Pope, J. A., Leslie, I., Wen, D., Lin, C-H., Cupples, R. L., and Zsebo, K. M., (1990). Primary structure and functional expression of rat and human stem cell factor DNAs. Cell 63: 203-211. Mosselman, S., Hoppener, J. W. M., Zandberg, J., van Mansfeld, A. D. M., Geurts van Kessel, A. H. M., Lips, C. J. M., and Jansz, H. S. (1988). Islet amyloid polypeptide: Identification and chromosomal localization of the human gene. F E B S Lett. 239: 227-232. Murty, V. V. V. S., Houldsworth, J., Baldwin, S., Reuter, V., Hunziker, W., Besmer, P., Bosl, G., and Chaganti, R. S. K. (1992). Allelic deletions in the long arm of chromosome 12 identify sites of candidate tumor suppressor genes in male germ cell tumors. Proc. Natl. Acad. Sci. USA, in press. Nakamura, Y., Fujimoto, E., Martin, C., O'Connell, P., Leppert, M., Lathrop, G. M., Lalouel, J.-M., and White, R. (1988a). Isolation and mapping of a polymorphic DNA sequence pEFD33.2 on chromosome 12 (D12S14). Nucleic Acids Res. 16- 778. Nakamura, Y., Ballard, L., O'Connell, P., Leppert, M., Lathrop, G. M., Lalouel, J.-M., and White, R. (1988b). Isolation and mapping of a polymorphic DNA sequence pYNH15 on chromosome 12q (D12S17). Nucleic Acids Res. 16: 779. Neel, B. G., Jhanwar, S. C., Chaganti, R. S. K., and Hayward, W. S. {1982). Two human c-onc genes are located on the long arm of chromosome 8. Proc. Natl. Acad. Sci. USA 79: 7842-7846. O'Connell, P., Lathrop, M., Law, M., Leppert, M., Nakamura, Y., Hoff, M., Kumlin, E., Thomas, W., Elsner, T., Ballard, L., Goodman, P., Azen, E., Sadler, J. E., Cai, G. Y., Lalouel, J.-M., and
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White, R. (1987). A primary genetic linkage map for human chromosome 12. Genomics 1-" 93-102. Pandis, N., Heim, S., Bandi, G., Mandahl, N., and Mitelman, F. {1990). High resolution mapping of consistent leiomyoma breakpoints in chromosomes 12 and 14 to 12q15 and 14q24.1. Genes Chrom. Cancer 2: 227-230. Pinkel, D., Straume, T., and Gray, J. W. {1986). Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. USA 83: 2934-2938. Rodriguez, E., Mathew, S., Reuter, V., Ilson, D. H., Bosl, G. J., and Chaganti, R. S. K. (1992). Cytogenetic analysis of 124 prospectively ascertained male germ cell tumors. Cancer Res. 52: 2285-2291. Sangiorgi, F. O., Benson-Chanda, V., de Wet, W. J., Sobel, M. E., Tsipouras, P., and Ramirez, F. (1985). Isolation and partial characterization of the entire human pro a 1 (II) collagen gene. Nucleic Acids Res. 13: 2207-2225. Stephens, J. C., Cavanaugh, M. L., Gradie, M. I., Mador, M. L., and Kidd, K. K. (1990). Mapping the human genome" Current status. Science 250: 237-244. Tsui, L.-C., Buchwald, M., Markiewicz, D., Kao, F.-T., Cai, G.-Y., and Law, M. L. (1985). Identification of a polymorphic DNA marker pDL32B (D12S7) and its localization to the long arm of chromosome 12, region q14.3-qter. Cytogenet. Cell Genet. 40: 763. Zabel, B. U., Naylor, S. L., Sakaguchi, A. Y., Bell, G. I., and Shows, T. B. (1983). High-resolution chromosomal localization of human genes for amylase, proopiomelanocortin, somatostatin, and a DNA fragment (D3S1) by in situ hybridization. Proc. Natl. Acad. Sci. USA 80: 6932-6936. Zsebo, K. M., Williams, D. A., Geissler, E. N., Broudy, V. C., Martin, F. H., Atkins, H. L., Hsu, R-Y., Birkett, N. C., Okino, K. H., Murdock, D. C., Jacobsen, F. W., Langley, K. E., Smith, K. A., Takeishi, T., Cattanach, B. M., Galli, S. J., and Suggs, S. V. (1990). Stem cell factor is encoded at the Sl of the mouse and is the ligand for the c-kit tyrosine kinase receptor. Cell 63: 213-224.
14, 775-779 (1992)
Subregional Mapping of 13 Single-Copy Genes on the Long Arm of Chromosome 12 by Fluorescence in Situ Hybridization SUSAN MATHEW,* V. V. V. S. MURTY,*W. HUNZIKER,I AND R. S. K. CHAGANTI* *Laboratory of Cancer Genetics and the Department of Pathology, Memorial 51oan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021; and tHoffman-La Roche, Basel, Switzerland Received March 5, 1992; revised July 2. 1992 S u b r e g i o n a l l o c a l i z a t i o n o f 1 3 s i n g l e - c o p y D N A seq u e n c e s p r e v i o u s l y a s s i g n e d to t h e l o n g a r m o f c h r o m o some 12 has been performed using the fluorescence in situ h y b r i d i z a t i o n (FISH) technique. T h e f o l l o w i n g o r d e r is s u g g e s t e d f o r t h e 1 3 m a p p e d g e n e s : c e n -~ COL2A1 -~ (VDR-D12815) --~ ( D 1 2 S 1 7 - D 1 2 8 4 D12S14-D12S6) --~ D 1 2 S 8 --~ ( I A P P - M G F - D 1 2 S 7 D 1 2 S 1 2 ) -~ I G F 1 - ~ q t e r . E i g h t o f t h e m a p p e d g e n e s c l u s t e r e d at t w o r e g i o n s , o n e at 1 2 q 1 3 ( D 1 2 S 1 7 D12S4-D12S14-D12S6) a n d t h e o t h e r at 1 2 q 2 2 (IAPP-MGF-D12S7-D12S12). Our results show that s i n g l e - c o p y D N A s e q u e n c e s a s s m a l l a s 5 0 0 bp c a n b e s u c c e s s f u l l y m a p p e d b y F I S H . © 1992 AcademicPress, Inc.
INTRODUCTION
Systematic efforts to map the human genome have resulted in primary genetic and linkage maps for each human chromosome (Human Gene Mapping 11, 1991; Stephens et al., 1990). The gene map of chromosome 12 is of interest because of its involvement in nonrandom abnormality in a variety of tumor types, e.g., deletions and other rearrangements in germ cell tumors (Rodriguez et al., 1992) and leiomyomas (Pandis et al., 1990) and nondisjunction in lymphoid malignancies, in particular chronic lymphocytic lymphoma (Gahrton and Robeft, 1982). Based on an analysis of loss of constitutional heterozygosity at eight polymorphic loci, two candidate tumor suppressor genes specific to germ cell tumors have been identified (Murty et al., 1992). To enhance the fine map of chromosome 12 and to facilitate the eventual identification of the putative tumor suppressor gene(s) and other genes perturbed in tumors, we have assigned 13 independently derived probes previously assigned to 12q to specific bands of metaphase chromosomes using the fluorescence in situ hybridization (FISH) technique. We further show t h a t the FISH technique can be used for mapping DNA sequences as small as 500 bp derived from single-copy genes. MATERIALS AND METHODS
cell hybrid analysis and, in addition, three have been mapped subregionally by in situ hybridization of radiolabeled probes to metaphase chromosomes. In the present FISH analysis, the vectors containing the probes were labeled with b i o t i n - l l - d U T P or biotin-14-dATP by nick-translation using E s c h e r i c h i a coli DNA polymerase I (Maniatis et al., 1982). Labeled probes were purified on Sephadex G-50 chromatography columns, ethanol precipitated, and dissolved in TE (10 m M Tris-HC1, 1 m M EDTA, p H 8.0). Chromosome preparations were made from human lymphocyte cultures synchronized with 5-bromodeoxyuridine as described (Zabel et al., 1983). I n situ hybridization was performed by a modification of the protocol described by Neel et al. (1982). Biotin-labeled probes at a concentration of 100-200 ng of DNA in 30 ttl of hybridization mixture (50% formamide/2X SSC/10% dextran sulfate/IX Denhardt's/0.1% SDS/ 25 m M sodium phosphate, and 200 ~g/ml of sheared salmon sperm DNA) were denatured at 75°C for 7 min. Suppression of repeated sequences was performed whenever necessary by preannealing the probe with 1-2 #g of placental DNA at 37°C for 5-20 min. Chromosomal DNA was denatured at 70°C for 2 min in 70% formamide/2x SSC. Overnight hybridization was carried out at 37°C. Posthybridization washes were performed at 42°C in 50% formamide/2X SSC for 20 min, followed by several washes in 2X SSC. Hybridized probes were detected by indirect immunofluorescenceusing avidin-conjugated fluorescein isothiocyanate (FITC-avidin) and biotinylated anti-avidin (Pinkel et al., 1986; Fan et al., 1990). The slides were stained with propidium iodide (PI) (Sigma) and counterstained with 4',6'-diamidino-2-phenylindole (DAPI, Sigma). To obtain G-banding, slides were stained with 33258 Hoechst, exposed to UV, and counterstained with Giemsa. Informative metaphases exhibiting fluorescent hybridization signals were photographed using a Nikon fluorescence microscope and the filter combinations B2A for FITC and UV-2A for DAPI to sequentially visualize hybridization signals and banding patterns on the chromosomes. Microphotographs were made sequentially, keeping the settings on the microscope and camera constant and using Kodak Ektachrome 400 film. The assignment of a signal to a specific chromosome band was made in the following manner. Kodachromes of the PI- and DAPI-stained individual metaphases were loaded in sequence in a Kodak carousel tray and projected onto the screen of a Kodak Ektagraphic projector. The end of the arm of the H - s t a i n e d chromosome exhibiting hybridization signal was marked on the screen, the distance between the chromosome end and the middle of the signal was measured using a dial caliper (Mitutoya), and the caliper setting was retained unchanged. The PI image was replaced by the DAPI image on the screen. The point of one arm of the caliper was placed on the end of the chromosome (marked from the PI-stained image) and the point of the other arm was placed on the chromosome, which then identified the specific band in the DAPI image at which hybridization occurred. RESULTS
The probes utilized in this study, the loci from which they were derived, their size, and the source(s) from which they were obtained are shown in Table 1. Insert sizes of the probes ranged from 0.5 to 4.0 kb. All these probes have previously been assigned to 12q by somatic
We used the FISH assay for the subregional localization of 10 probes for the first time and confirmation of 775
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TABLE 1 1 2 q P r o b e s U s e d for M a p p i n g b y F I S H Probe
Locus
Insert size (kb)
Source
Reference
p9Fll 11-1-17 pDL32B p9Gll pAC230 pFED33.2 pCMM1.2 pYNH15 pH-KITL phigfl WV38
D12S4 D12S6 D12S7 D12S8 D12S12 D12S14 D12S15 D12S17 MGF IGFI COL2A1
1.2 0.5 3.2 1.0 2.2 3.2 1.9 3.2 0.8 0.6 4.0
ATCC M. Litt ATCC ATCC I. Balazs ATCC ATCC ATCC W. Hunziker ATCC E.J. Weaver
scIAP-8 pill3
IAPP VDR
0.6 2.1
J. Hoppener J. Shine
O'Connell et aL, 1987 Buroker et al., 1986 Tsui et al., 1985 O'Connell et al., 1987 Balazs et al., 1987 Nakamura et al., 1988a Martin et al., 1988 Nakamura et al., 1988b Unpublished Hoppener et al., 1985 Sangiorgi et al., 1985; Law et al., 1986 Mosselman et al., 1988 Faraco et al., 1989
N o t e . I G F 1 , insulin-like growth factor 1; C O L 2 A 1, procollagen, type II, alpha; V D R , v i t a m i n D receptor; I A P P , Islet amyloid polypeptide; M G F , m a s t cell growth factor.
previous assignments of three probes from among a panel of 13 single-copy DNA sequences previously assigned to the long arm of chromosome 12. Since these probes have previously been assigned to 12q, the analysis was confined only to cells exhibiting signals on 12q with each probe. For each probe, 25-100 metaphases with fluorescent hybridization signals on chromosome 12 were scored. Hybridization signals were observed either as single (on one chromatid) or double (on both chromatids) dots. The percentage of single or double hybridization signals varied from probe to probe. The signals always clustered in a probe-specific manner, thus allowing unambiguous assignment of the 13 probes to specific bands on 12q (Fig. 1). The data from hybridization results are summarized in Table 2 and the positions of the 13 mapped probes are shown on an idiogram of chromosome 12 in Fig. 2. Based on the data discussed above, the following order is suggested for the 13 mapped genes: cen -~ C O L 2 A I --~ ( V D R - D 1 2 S 1 5 ) --~ (D12S17D12S4-D12S14-D12S6) --~ D12S8 --~ ( I A P P - M G F D12S7-D12S12) --~ IGF1 --~ qter.
Mapping 11, 1991) is confirmed in the present study. C O L 2 A 1 has previously been mapped to 12q13.1-q13.2 using 8H-labeled probe DNA for in situ hybridization (Huerre-Jeanpierre et al., 1986). In another study, Law et al. (1986) assigned C O L 2 A 1 to 12q14.3 using a panel of hamster-human somatic cell hybrids. The map position of this gene was recently revised to 12q13 (Human Gene Mapping 11, 1991). Using FISH, we now map C O L 2 A 1 to 12q12 (Table 2, Fig. 2). Kit-ligand, otherwise referred to as mast cell growth factor (MGF) or stem cell factor (SCF), is the product of the steel (S1) locus in the mouse and comprises the ligand for the K I T protooncogene tyrosine kinase receptor (Zsebo et al., 1990; Huang et al., 1990; Martin et al., 1990). The human chromosomal localization of M G F was reported by two groups. Geissler et al. (1991), using a panel of somatic cell hybrids, assigned this gene to 12q14.3-qter, while Anderson et al. (1991), using both somatic cell hybrid and in situ hybridization of radiolabeled probe, mapped M G F to 12q22-q24. In the present study, using FISH, we localize M G F to 12q22. Somatic cell hybrid analysis showed that the human I A P P gene is located in the region 12pll.2-p12.3 (Hoppener et al., 1985; Mosselman et al., 1988). Buckle et al. (1989) and Fan et al. (1989) assigned this gene to 12pll.2-p12.3 by in situ hybridization, whereas Cockburn et al. (1989) provided evidence for another hybridization site at 12cen-q21.1. In another study, also by in situ hybridization of radiolabeled probe, 12p12-q13 and 12q13-q14 were suggested to be the sites of localization of this gene (Christmanson et al., 1990). In HMG 11 (1991) the map position is listed as 12p12.3-p13 and 12q13-q21. However, in an analysis of 37 cells with FISH signals on chromosome 12, we saw signals consistently only in the 12q21.3-q22 region; no hybridization signal was seen on 12p. TABLE 2 A s s i g n m e n t o f 13 D N A P r o b e s on the L o n g A r m o f C h r o m o s o m e 12 b y F l u o r e s c e n c e in S i t u H y b r i d i z a t i o n
DISCUSSION To enhance the fine map and to facilitate the investigation of genetic alterations affecting 12q in tumors, we have attempted to subregionally localize 13 probes on the long arm of chromosome 12 by FISH. With the exception of D12S6, IGF1, and C O L 2 A 1 , which were previously mapped to specific bands, the remaining probes mapped by us were previously assigned only to broad regions of 12q (Craig and McBride, 1991). Buroker et al. (1986) assigned D12S6 to the 12cen-q13 region by in situ hybridization of a radiolabeled cosmid probe, whereas O'Connell et al. (1987) assigned the same marker to 12q14 by linkage analysis. Our FISH mapping assigns this probe to 12q13.2-q13.3 (Table 2, Fig. 2). The previous localization of IGF1 to 12q23 (Human Gene
Locus COL2A 1 VDR
D12S15 D12S17 D12S4 D12S14 D12S6 D12S8
IAPP MGF
D12S7 D12S12 IGF1
No. of c h r o m o s o m e s 12 with
No. of metaphases with signal on 12q
Single signal
Double signal
Signal position
100 28 40 46 49 33 25 41 37 28 29 72 26
80 8 30 38 40 13 20 29 21 17 18 33 2
48 20 16 20 12 23 6 17 20 12 12 43 25
12q12 12q12-13.1 12q12-13.1 12q13.1 12q13 12q13 12q13.2-13.3 12q14-15 12q21.3-22 12q22 12q22 12q22 12q23
MAPPING OF SINGLE-COPY GENES ON 12q BY FISH
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D12S4
D12S6
D12S7
D12S8
D12S12
D12S14
D12S15
D12S17
MGF
IGF1
COL2A1
lAPP
VDR
FIG. 1. In situ hybridization mapping of 13 single-copy genes on chromosome 12. Partial metaphases stained with propidium iodide (left) and DAPI (right). In the case of D12S7, the preparation was G-banded following fluorescence microscopy of the hybridized signal. The G-banded picture of the chromosome is shown in the right panel. Arrows indicate fluorescent signals and arrowheads indicate band positions.
T h e v i t a m i n D r e c e p t o r (VDR) gene has previously b e e n assigned to c h r o m o s o m e 12 b y s o m a t i c cell h y b r i d analysis (Faraco et al., 1989). W e now localize it to 12q12-q13.1 b y F I S H . D12S7 has b e e n m a p p e d to 12q14.3-qter b y s o m a t i c cell h y b r i d analysis, a n d we now assign this p r o b e to 12q22. D12S8 has previously b e e n assigned to 1 2 q 1 4 - q t e r ( H u m a n G e n e M a p p i n g 11, 1991). In t h e p r e s e n t study, we h a v e assigned this p r o b e to 12q14-q15. D12S4 was p r e v i o u s l y assigned to 1 2 c e n q14, a n d our results assign it to 12q13. D12S15 ( M a r t i n et al., 1988), D12S14 ( N a k a m u r a et al., 1988a), D12S17
( N a k a m u r a et al., 1988b), a n d D12S12 (Balazs et al., 1987) have previously b e e n assigned only to the long a r m of c h r o m o s o m e 12; we now localize these p r o b e s to 12q12-q13.1, 12q13, 12q13.1, a n d 12q22 regions, respectively, using F I S H . T h e signal resolution a n d m a p p i n g precision are higher with F I S H t h a n with s o m a t i c cell h y b r i d analysis a n d in s i t u h y b r i d i z a t i o n of radiolabeled probes. T h e precise identification of h u m a n c h r o m o s o m e s e g m e n t s in s o m a t i c cell hybrids t e n d s to be difficult, a n d regional s c a t t e r of silver grains f r o m radiolabeled p r o b e s used in
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COL2A1 VDR, D12S15
D12S17 D12S4, D12S14 D12S6 D12S8
I IAPP
MGF D12S7 D12S12
IGF1
F I G . 2. Idiogram of chromosome 12 showing the map positions of the 13 sing!e-copy genes mapped in this study.
autoradiography of in situ hybridized probes also makes specific assignment difficult. Probe signals do not scatter in FISH, and both chromatids usually take part in hybridization, resulting in "twin" or double hybridization spots within the chromosome and thus enhancing the precision of mapping. In the present study, we determined the map positions of 13 DNA sequences on 12q by FISH. Furthermore, our results clearly demonstrate the feasibility of mapping single-copy DNA sequences as small as 500 bp, as in the case of D12S6. The current map of 12q generated by us also correlates well with the linear order of the existing linkage map for chromosome 12 (O'Connell et al., 1987). Our data on the 12q map showed clustering of loci on two regions, one at 12q13 (D12S17-D12S4-D12S14-D12S6) and the other at 12q22 ( I A P P - M G F - D 1 2 S 7 - D 1 2 S 1 2 ) . ACKNOWLEDGMENTS We thank Drs. M. Litt, I. Balazs, J. Shine, E. J. Weaver, and J. Hoppener for providing some of the probes used in this study (Table 1). We thank Dr. Jane Houldsworth for help with preparation of probes. This investigation was supported in part by National Institutes of Health Research Grants CA-34775 and CA-05826. REFERENCES Anderson, D. M., Williams, D. E., Tushinski, R., Gimpel, S., Eisenman, J., Cannizzaro, L. A., Aronson, M., Croce, C. M., Huebner, K., Cosman, D., and Lyman, S. D. (1991). Alternate splicing of mRNAs encoding human mast cell growth factor and localization of the gene to chromosome 12q22-q24. Cell Growth & Differ. 2: 373-378.
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