- Email: [email protected]
Mapping of guanylin to murine chromosome 4 and human chromosome 1p34–p35
BRIEF REPORTS 5. Jakes, R., Spillantini, M. G., and Goedert, M. (1994). Identification of two distinct synucleins from human brain. FEBS Lett. 345: 27-32. 6. Ma, J., Yee, A., Brewer, H. B., Jr., Das, S., and Potter, H. (1994). Amyloid-associated proteins al-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer/%protein into filaments. Nature 372: 92-94. 7. Saitoh, T., Xia, Y., Chen, X., Masliah, E., Galasko, D., Shults, D., Thai, L. J., Hansen, L., and Katzman, R. (1995). The CYP2D6B mutant allele is overrepresented in the Lewy body variant of Alzheimer's disease. Ann. Neurol. 37: 110-112. 8. Suzuki, T., Cheung, T. T., Cai, X. D., Odaka, A., Otvos, L., Jr., Eckman, C., Golde, T. E., and Younkin, S. G. (1994). An increased percentage of long amyloid/~ protein secreted by familial amyloid/~ protein precursor (~APP717)mutants. Science 264: 1336-1340. 9. U4da, K., Fukushima, H., Masliah, E., Xia, Y., Iwai, A., Yoshimoto, M., Otero, D. A., Kondo, J., Ihara, Y., and Saltoh, T. (1993). Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc. Natl. Acad. Sci. USA 90: 11282-11286.
427
crypt axes (13, 14). G u a n y l i n m R N A a b u n d a n c e is m a x i m a l in t h e d i s t a l s m a l l i n t e s t i n e a n d p r o x i m a l colon, w h e r e t h e m R N A is detected m a i n l y in differentiated villus epithelial cells a n d superficial colonic epithelial cells, respectively (11, 13, 14, 19, 20). The routine g u a n y l i n gene (Guca2) h a s been isolated a n d sequenced (16); the gene is 1.7 k b a n d consists of 3 exons. We r e p o r t h e r e t h e m a p p i n g of Guca2 to mouse chromosome 4 by l i n k a g e a n a l y s i s a n d to h u m a n chromosome region l p 3 4 - p 3 5 using fluorescence in situ h y b r i d i z a t i o n (FISH). The m u r i n e chromosomal location of Guca2 was determ i n e d b y interspecific backcross a n a l y s i s u s i n g progeny derived from m a t i n g s of [(C57BL/6J x M u s spretus)F1 x C57BL/
Cyp4alom[-I D m
c
rmi-1 35 56
Mapping of Guanylin to Murine Chromosome 4 and Human Chromosome 1p34-p35 Daniela Sciaky,* Nancy A. Jenkins, t Debra J. Gilbert, t Neal G. Copeland, t Gonosuke Sonoda,* Joseph R. Testa,* and Mitchell B. Cohen *'l
Dm mD
mD mD 0
Dm Dm Dm
0
1
0
5
1
4
(
1.2 0.9
Cyp4alO GlutI Guca2
lp34-p12 lp35-p31.3
Csfgr
lp35-p34.3
7.3 *Children's Hospital Medical Center and The University of Cincinnati, Cincinnati, Ohio; tMammalian Genetics Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland; and *Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania
ReceivedOctober24, 1994; acceptedJanuary13, 1995
G u a n y l i n is a 15-amino-acid peptide s i m i l a r in s t r u c t u r e a n d in function to STa, the h e a t stable enterotoxin of enterotoxigenic Escherichia coli (4). Both g u a n y l i n a n d STa bind g u a n y l y l cyclase-C (GC-C), r e s u l t i n g in i n c r e a s e d levels of i n t r a c e l l u l a r cGMP a n d induction of C1- secretion (4) via the cystic fibrosis t r a n s m e m b r a n e r e g u l a t o r (CFTR) (2). Guanylin is a highly r e g u l a t e d i n t e s t i n a l gene t h a t is differentially expressed along t h e duodenal-to-colonic a n d villus-to1To whom correspondence should be addressed at the Division of Pediatric Gastroenterology and Nutrition, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. Telephone: (513) 559-8051. Fax: (513) 559-7805. GENOMICS26, 427--429 (1995) 0888-7543/95 $6.00 Copyright © 1995 by AcademicPress, Inc. All rights of reproduction in any form reserved.
¢, FIG. 1. Guca2 maps to the middle region of mouse chromosome 4. Guca2 was mapped to mouse chromosome 4 by interspecific backcross analysis. The segregation patterns of Guca2 and flanking genes
in 98 backcross animals are shown at the top of the figure. For some individual pairs of loci, more than 98 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J x M. spretus)F1 parent. The black boxes represent the presence of a C57BL/6J allele, while the white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 4 linkage map showing the location of Guca2 in relation to linked genes is shown at the bottom of the figure. Recombination distances between loci in centimorgans are shown to the left of the chromosome, and the positions of loci in human chromosomes are shown to the right. References for human map positions can be obtained from GDB (Genome Data Base), a computerized database for human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).
428
BRIEF REPORTS
FIG. 2. Localization of human Guca2 to human chromosome 1p34-p35 by FISH. Arrows identify fluorescent signals resulting from hybridization of human gnanylin cDNA to human chromosome 1 at position p34-p35. 6J] mice. This interspecific backcross mapping panel has been typed for over 1600 loci that are well distributed among all mouse autosomes and the X chromosome (3; Copeland and Jenkins, unpublished results). C57BL/6J and M. spretus DNAs were digested with several restriction enzymes and analyzed by Southern blot hybridization for informative restriction fragment length polymorphisms (RFLPs) using a mouse genomic Guca2 probe (16). DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization were performed essentially as described (10). All blots were prepared with Hybond-N ÷ nylon membrane (Amersham). The probe, a 2.8-kb EcoRI fragment of mouse genomic DNA (16), was labeled with [a-32p]dCTP using a random prime labeling kit (Stratagene); washing was performed to a final stringency of 0.1× SSCP, 0.1% SDS, 65°C. A 4.8-kb fragment was detected in HincII-digested C57BL/6J DNA, and a 6.4-kb fragment was detected in M. spretus DNA. The presence or absence of the 6.4-kb M. spretus-specific HincII RFLP was used to follow the segregation of the Guca2 locus in backcross DNA. A description of the probes and RFLPs for the loci linked to Guca2 including cytochrome P450, al0 (Cyp4alO), glucose transporter 1, erythrocyte (Glut1), and colony stimulating factor granulocyte receptor (Csfgr) has been given previously (1, 8, 9). Recombination distances were calculated as described by Green (7) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of double and multiple recombination events across the chromosome.
The mapping results indicate that Guca2 is located in the middle region of mouse chromosome 4 (Fig. 1). Although 98 mice were analyzed for every marker shown in the haplotype analysis (Fig. 1), up to 161 mice were typed for some pairs of markers. Each locus was analyzed in pairwise combinations for recombination frequencies using the additional data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are: centromereCyp4a l O- 2/161- Glut1-1/112-Guca2-8/110-Csfgr. The recombination frequencies {expressed as genetic distances in centimorgans _+ the standard error) are: centromereCyp4alO-1.2 +_ 0.9-Glut1-0.9 +_ 0.9-Guca2-7.3 +_ 2.5Csfgr. The distal half of mouse chromosome 4 shares a region of homology with human chromosome 1 (Fig. 1). In particular, Guca2 was found to localize between two loci, Glut1 and Csfgr, that have been mapped to human chromosome l p 3 5 p31.3 and lp35-p34.3, respectively. These comparative mapping results suggested that the human homolog of Guca2 would map to human lp as well. To map human Guca2, human metaphase spreads were prepared according to the method of Fan et al. (6). Human lymphocytes were cultured for 72 h at 37°C in RPMI 1640 medium containing phytohemagglutinin and 10% fetal bovine serum. The cells were synchronized by treatment with 5-bromodeoxyuridine (0.18 mg/ml, Sigma) for 16 h, followed by release from the block by incubation in fresh medium containing thymidine (2.5 pg/ml) for 6 h. Metaphase cells
BRIEF REPORTS
were h a r v e s t e d a n d chromosome s p r e a d s were p r e p a r e d according to s t a n d a r d procedures. Slide p r e p a r a t i o n s were b a k e d for 1 h a t 75°C before hybridization. The h u m a n guanylin gene probe used for F I S H was a 500-bp full-length cDNA clone d e s i g n a t e d pMON22305 (gift of M. Currie). Uncut p l a s m i d DNA (total molecular weight 3485 bp) was labeled with b i o t i n y l a t e d 16-dUTP by n i c k - t r a n s l a t i o n (Boehr i n g e r Mannheim). U n i n c o r p o r a t e d nucleotides were separ a t e d u s i n g a S e p h a d e x G-50 column. F I S H a n d detection of immunofluorescence were carried out according to the technique of P i n k e l et al. (15), with m i n o r modifications (18). Chromosomes were c o u n t e r s t a i n e d with diamidino-2-phenylindole (DAPI) a n d p r o p i d i u m iodide in a n t i f a d e solution (Oncor). M e t a p h a s e s p r e a d s were observed using a Zeiss Axiophot microscope. I m a g e s were c a p t u r e d by a cooled CCD c a m e r a connected to a c o m p u t e r workstation. Digitized images of DAPI s t a i n i n g a n d fluorescein signals were m e r g e d as described elsewhere (18). F I S H h y b r i d i z a t i o n of t h e pMON22305 cDNA probe to m e t a p h a s e s p r e a d s revealed specific labeling on h u m a n chromosome 1 (Fig. 2). F l u o r e s c e n t signals were detected on chromosome 1 in 32 of 37 m e t a p h a s e s examined. A l t h o u g h nonspecific h y b r i d i z a t i o n was r e l a t i v e l y high, 59 of 190 signals (31%) were located on chromosome l p 3 4 - p 3 5 (Fig. 2). Labeling of both chromosome 1 homologues a t l p 3 4 - p 3 5 was observed in 9 of 37 (24%) m e t a p h a s e spreads. Guca2, a gene t h a t is h i g h l y expressed in the rat, mouse, a n d h u m a n colon (11, 13, 14, 19, 20), m a p s to t h e middle region of mouse chromosome 4 a n d to h u m a n chromosome l p 3 4 - p 3 5 . This observation confirms the expected linkage conservation between mouse a n d h u m a n genomes. It is also of i n t e r e s t because h u m a n colorectal cancers d e m o n s t r a t e a high frequency of deletions a t chromosome l p 3 5 (12), while introduction of a n o r m a l chromosome l p 3 6 region can suppress the t u m o r i g e n i c i t y of h u m a n colon carcinoma cells (17). Recently a modifier of i n t e s t i n a l t u m o r formation (Morn-l) in t h e m u l t i p l e i n t e s t i n a l n e o p l a s i a mouse (Min) was also m a p p e d to mouse chromosome 4 in a region of syntenic conservation with h u m a n chromosome 1 p 3 5 - p 3 6 (5). These observations r a i s e t h e possibility t h a t in addition to t h e pres u m e d role of g u a n y l i n in i n t e s t i n a l secretion, Guca2 could be linked to colonic t u m o r formation or repression.
ACKNOWLEDGMENTS We thank Debbie Barnhart for excellent technical assistance. This research was supported, in part, by the NCI, DHHS, under Contract NO1-CO-74101 with ABL, NCI Grants CA-06927 and CA-45745, and a grant from DHHS, NIDDK (01908).
REFERF~CES 1. Bell, D. R., Plant, N. J., Rider, C. G., Na, L., Brown, S., Ateitalla, I., Acharya, S. I~, Davies, M. H., Elias, E., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., and Elcombe, C. R. (1993). Species specific induction of cytochrome P450A RNAs: PCR cloning of partial guinea-pig, human and mouse CYP4A cDNAs. Biochem. J. 294: 173-180. 2. Chao, A. C., de Sauvage, F. J., Dong, Y.-J., Wagner, J. A., Goeddel, D. V., and Gardner, P. (1994). Activation of intestinal CFTR C1- channel by heat-stable enterotoxin and guanylin via cAMPdependent protein kinase. EMBO J. 13: 1065-1072.
429
3. Copeland, N. G., and Jenkins, N. A. (1991). Development and applications of a molecular genetic linkage map of the mouse genome. Trends Genet. 7: 113-118. 4. Currie, M. G., Fok, K. F., Kato, J., Moore, R. J., Hamra, F. I~, Duifm, K. L., and Smith, C. E. (1992). Guanylin: An endogenous activator of intestinal guanylate cyclase. Proc. Natl. Acad. Sci. USA 89: 947-951. 5. Dietrich, W. F., Lander, E. S., Smith, J. S., Moser, A. R., Gould, K. A., Luongo, C., Borenstein, N., and Dove, W. (1993). Genetic identification of Morn-l, a major modifier locus affecting Mininduced intestinal neoplasia in the mouse. Cell 75: 631-639. 6. 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: 6223-6227. 7. Green, E. L. (1981). Linkage, recombination, and mapping. In "Genetics and Probability in Animal Breeding Experiments," pp. 77-113, Oxford Univ. Press, New York. 8. Hogan, A., Heyner, S., Charron, M. J., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Thorens, B., and Schultz, G. A. (1991). Glucose transporter gene expression in early mouse embryos. Development 113: 363-372. 9. Ito, Y., Seto, Y., Brannan, C. I., Copeland, N. G., Jenkins, N. A., Fukunaga, R., and Nagata, S. (1994). Structural analysis of the functional gene and processed pseudogene for murine granulocyte colony-stimulating factor receptor. Eur. J. Biochem. 220: 881-891. 10. Jenkins, N. A., Copeland, N. G., Taylor, B. A., and Lee, B. K. (1982). Organization, distribution, and stability of endogenous ecotropic murine leukemia virus DNA sequences in chromosomes of Mus musculus. J. Virol. 43: 26-36. 11. Kate, J., Wiegand, and Currie, M. G. (1993). Characterization of the structure of preproguanylin. In "Advances in Second Messenger and Phosphoprotein Research" (B. L. Brown and P. R. M. Dobson, Eds.), Vol. 28, pp. 139-142, Raven Press, New York. 12. Leister, I., Weith, A., Briiderlein, S., Cziepluch, C., Kangwanpong, D., Schlag, P., and Schwab, M. (1990). Human colorectal cancer: High frequency of deletions at chromosome lp35. Cancer Res. 50: 7232-7235. 13. Lewis, L. G., Witte, D. P., Laney, D. W., Currie, M. G., and Cohen, M. B. (1993). Guanylin mRNA is expressed in villous enterocytes of the rat small intestine and superficial epithelia of the rat colon. Biochem. Biophys. Res. Commun. 196: 553560. 14. Li, Z., and Gey, M. F. (1993). Peptide-regulated guanylate cyclase pathways in rat colon: In situ localization of GCA, GCC, and guanylin mRNA. Am. J. Physiol. 265: G394-G402. 15. 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. 16. Sciaky, D., Kasiba, J. L., and Cohen, M. B. (1994). Genomic sequence of the routine guanylin gene. Genomics 24: 583-587. 17. Tanaka, I~, Yanoshita, R., Konishi, M., Oshimura, M., Maeda, Y., Mori, T., and Michiko, M. (1993). Suppression of tumourigenicity in human colon carcinoma cells by introduction of norreal chromosome lp36. Oncogene 8: 2253-2258. 18. Testa, J. R., Taguchi, T., Knudson, A. G., and Hino, O. (1992). Localization of the interferon-a gene cluster to rat chromosome bands 5q31 -* q33 by fluorescence in situ hybridization. Cytogenet. Cell Genet. 60: 247-249. 19. Wiegand, R. C., Kato, J., and Currie, M. G. (1992). Rat guanylin cDNA: Characterization of the precursor of an endogenous activator of intestinal guanylate cyclase. Biochem. Biophys. Res. Commun. 185: 812-817. 20. Wiegand, R. C., Kato, J., Huang, M. D., Fok, K. F., Kachur, J. F., and Currie, M. G. (1992). Human guanylin: cDNA isolation, structure, and activity. FEBS Lett. 311: 150-154.
427
crypt axes (13, 14). G u a n y l i n m R N A a b u n d a n c e is m a x i m a l in t h e d i s t a l s m a l l i n t e s t i n e a n d p r o x i m a l colon, w h e r e t h e m R N A is detected m a i n l y in differentiated villus epithelial cells a n d superficial colonic epithelial cells, respectively (11, 13, 14, 19, 20). The routine g u a n y l i n gene (Guca2) h a s been isolated a n d sequenced (16); the gene is 1.7 k b a n d consists of 3 exons. We r e p o r t h e r e t h e m a p p i n g of Guca2 to mouse chromosome 4 by l i n k a g e a n a l y s i s a n d to h u m a n chromosome region l p 3 4 - p 3 5 using fluorescence in situ h y b r i d i z a t i o n (FISH). The m u r i n e chromosomal location of Guca2 was determ i n e d b y interspecific backcross a n a l y s i s u s i n g progeny derived from m a t i n g s of [(C57BL/6J x M u s spretus)F1 x C57BL/
Cyp4alom[-I D m
c
rmi-1 35 56
Mapping of Guanylin to Murine Chromosome 4 and Human Chromosome 1p34-p35 Daniela Sciaky,* Nancy A. Jenkins, t Debra J. Gilbert, t Neal G. Copeland, t Gonosuke Sonoda,* Joseph R. Testa,* and Mitchell B. Cohen *'l
Dm mD
mD mD 0
Dm Dm Dm
0
1
0
5
1
4
(
1.2 0.9
Cyp4alO GlutI Guca2
lp34-p12 lp35-p31.3
Csfgr
lp35-p34.3
7.3 *Children's Hospital Medical Center and The University of Cincinnati, Cincinnati, Ohio; tMammalian Genetics Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland; and *Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania
ReceivedOctober24, 1994; acceptedJanuary13, 1995
G u a n y l i n is a 15-amino-acid peptide s i m i l a r in s t r u c t u r e a n d in function to STa, the h e a t stable enterotoxin of enterotoxigenic Escherichia coli (4). Both g u a n y l i n a n d STa bind g u a n y l y l cyclase-C (GC-C), r e s u l t i n g in i n c r e a s e d levels of i n t r a c e l l u l a r cGMP a n d induction of C1- secretion (4) via the cystic fibrosis t r a n s m e m b r a n e r e g u l a t o r (CFTR) (2). Guanylin is a highly r e g u l a t e d i n t e s t i n a l gene t h a t is differentially expressed along t h e duodenal-to-colonic a n d villus-to1To whom correspondence should be addressed at the Division of Pediatric Gastroenterology and Nutrition, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. Telephone: (513) 559-8051. Fax: (513) 559-7805. GENOMICS26, 427--429 (1995) 0888-7543/95 $6.00 Copyright © 1995 by AcademicPress, Inc. All rights of reproduction in any form reserved.
¢, FIG. 1. Guca2 maps to the middle region of mouse chromosome 4. Guca2 was mapped to mouse chromosome 4 by interspecific backcross analysis. The segregation patterns of Guca2 and flanking genes
in 98 backcross animals are shown at the top of the figure. For some individual pairs of loci, more than 98 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J x M. spretus)F1 parent. The black boxes represent the presence of a C57BL/6J allele, while the white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 4 linkage map showing the location of Guca2 in relation to linked genes is shown at the bottom of the figure. Recombination distances between loci in centimorgans are shown to the left of the chromosome, and the positions of loci in human chromosomes are shown to the right. References for human map positions can be obtained from GDB (Genome Data Base), a computerized database for human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).
428
BRIEF REPORTS
FIG. 2. Localization of human Guca2 to human chromosome 1p34-p35 by FISH. Arrows identify fluorescent signals resulting from hybridization of human gnanylin cDNA to human chromosome 1 at position p34-p35. 6J] mice. This interspecific backcross mapping panel has been typed for over 1600 loci that are well distributed among all mouse autosomes and the X chromosome (3; Copeland and Jenkins, unpublished results). C57BL/6J and M. spretus DNAs were digested with several restriction enzymes and analyzed by Southern blot hybridization for informative restriction fragment length polymorphisms (RFLPs) using a mouse genomic Guca2 probe (16). DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization were performed essentially as described (10). All blots were prepared with Hybond-N ÷ nylon membrane (Amersham). The probe, a 2.8-kb EcoRI fragment of mouse genomic DNA (16), was labeled with [a-32p]dCTP using a random prime labeling kit (Stratagene); washing was performed to a final stringency of 0.1× SSCP, 0.1% SDS, 65°C. A 4.8-kb fragment was detected in HincII-digested C57BL/6J DNA, and a 6.4-kb fragment was detected in M. spretus DNA. The presence or absence of the 6.4-kb M. spretus-specific HincII RFLP was used to follow the segregation of the Guca2 locus in backcross DNA. A description of the probes and RFLPs for the loci linked to Guca2 including cytochrome P450, al0 (Cyp4alO), glucose transporter 1, erythrocyte (Glut1), and colony stimulating factor granulocyte receptor (Csfgr) has been given previously (1, 8, 9). Recombination distances were calculated as described by Green (7) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of double and multiple recombination events across the chromosome.
The mapping results indicate that Guca2 is located in the middle region of mouse chromosome 4 (Fig. 1). Although 98 mice were analyzed for every marker shown in the haplotype analysis (Fig. 1), up to 161 mice were typed for some pairs of markers. Each locus was analyzed in pairwise combinations for recombination frequencies using the additional data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are: centromereCyp4a l O- 2/161- Glut1-1/112-Guca2-8/110-Csfgr. The recombination frequencies {expressed as genetic distances in centimorgans _+ the standard error) are: centromereCyp4alO-1.2 +_ 0.9-Glut1-0.9 +_ 0.9-Guca2-7.3 +_ 2.5Csfgr. The distal half of mouse chromosome 4 shares a region of homology with human chromosome 1 (Fig. 1). In particular, Guca2 was found to localize between two loci, Glut1 and Csfgr, that have been mapped to human chromosome l p 3 5 p31.3 and lp35-p34.3, respectively. These comparative mapping results suggested that the human homolog of Guca2 would map to human lp as well. To map human Guca2, human metaphase spreads were prepared according to the method of Fan et al. (6). Human lymphocytes were cultured for 72 h at 37°C in RPMI 1640 medium containing phytohemagglutinin and 10% fetal bovine serum. The cells were synchronized by treatment with 5-bromodeoxyuridine (0.18 mg/ml, Sigma) for 16 h, followed by release from the block by incubation in fresh medium containing thymidine (2.5 pg/ml) for 6 h. Metaphase cells
BRIEF REPORTS
were h a r v e s t e d a n d chromosome s p r e a d s were p r e p a r e d according to s t a n d a r d procedures. Slide p r e p a r a t i o n s were b a k e d for 1 h a t 75°C before hybridization. The h u m a n guanylin gene probe used for F I S H was a 500-bp full-length cDNA clone d e s i g n a t e d pMON22305 (gift of M. Currie). Uncut p l a s m i d DNA (total molecular weight 3485 bp) was labeled with b i o t i n y l a t e d 16-dUTP by n i c k - t r a n s l a t i o n (Boehr i n g e r Mannheim). U n i n c o r p o r a t e d nucleotides were separ a t e d u s i n g a S e p h a d e x G-50 column. F I S H a n d detection of immunofluorescence were carried out according to the technique of P i n k e l et al. (15), with m i n o r modifications (18). Chromosomes were c o u n t e r s t a i n e d with diamidino-2-phenylindole (DAPI) a n d p r o p i d i u m iodide in a n t i f a d e solution (Oncor). M e t a p h a s e s p r e a d s were observed using a Zeiss Axiophot microscope. I m a g e s were c a p t u r e d by a cooled CCD c a m e r a connected to a c o m p u t e r workstation. Digitized images of DAPI s t a i n i n g a n d fluorescein signals were m e r g e d as described elsewhere (18). F I S H h y b r i d i z a t i o n of t h e pMON22305 cDNA probe to m e t a p h a s e s p r e a d s revealed specific labeling on h u m a n chromosome 1 (Fig. 2). F l u o r e s c e n t signals were detected on chromosome 1 in 32 of 37 m e t a p h a s e s examined. A l t h o u g h nonspecific h y b r i d i z a t i o n was r e l a t i v e l y high, 59 of 190 signals (31%) were located on chromosome l p 3 4 - p 3 5 (Fig. 2). Labeling of both chromosome 1 homologues a t l p 3 4 - p 3 5 was observed in 9 of 37 (24%) m e t a p h a s e spreads. Guca2, a gene t h a t is h i g h l y expressed in the rat, mouse, a n d h u m a n colon (11, 13, 14, 19, 20), m a p s to t h e middle region of mouse chromosome 4 a n d to h u m a n chromosome l p 3 4 - p 3 5 . This observation confirms the expected linkage conservation between mouse a n d h u m a n genomes. It is also of i n t e r e s t because h u m a n colorectal cancers d e m o n s t r a t e a high frequency of deletions a t chromosome l p 3 5 (12), while introduction of a n o r m a l chromosome l p 3 6 region can suppress the t u m o r i g e n i c i t y of h u m a n colon carcinoma cells (17). Recently a modifier of i n t e s t i n a l t u m o r formation (Morn-l) in t h e m u l t i p l e i n t e s t i n a l n e o p l a s i a mouse (Min) was also m a p p e d to mouse chromosome 4 in a region of syntenic conservation with h u m a n chromosome 1 p 3 5 - p 3 6 (5). These observations r a i s e t h e possibility t h a t in addition to t h e pres u m e d role of g u a n y l i n in i n t e s t i n a l secretion, Guca2 could be linked to colonic t u m o r formation or repression.
ACKNOWLEDGMENTS We thank Debbie Barnhart for excellent technical assistance. This research was supported, in part, by the NCI, DHHS, under Contract NO1-CO-74101 with ABL, NCI Grants CA-06927 and CA-45745, and a grant from DHHS, NIDDK (01908).
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