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Chromosomal Mapping of the Mouse and RatbrtGenes
SHORT COMMUNICATION Chromosomal Mapping of the Mouse and Rat brt Genes YOICHI MATSUDA,*,1 TOSHIYUKI SAITO,* JIRO FUJIMOTO,†
AND
TADASHI YAMAMOTO†,2
*Genome Research Group, National Institute of Radiological Sciences, 4-9-1 Anagawa, Chiba 263, Japan; and †Department of Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, 4-6-1 Shirokanedai, Tokyo 108, Japan Received August 16, 1996; accepted November 18, 1996
brt encodes a receptor-type protein-tyrosine kinase and was isolated from fetal mouse brain by using a PCR-mediated cloning procedure; this gene is expressed preferentially in the brain of both embryo and adult. Chromosomal locations of the mouse and rat brt genes were determined by fluorescence in situ hybridization using a mouse cDNA fragment as a probe. The brt gene was localized to mouse Chromosome 2F1 and rat Chromosome 3q36.1, where a conserved linkage homology has been identified between the two species. Interspecific backcross analysis genetically demonstrated no recombination between the mouse BRT locus and D2Mit63. q 1997 Academic Press
The frequent appearance of protein tyrosine kinases (PTKs) as products of oncogenes and growth factor receptors suggests that tyrosine phosphorylation plays an important role in the signal transduction pathway in cell growth and differentiation. There have been two reports on loss-of-function mutations of protein-tyrosine kinase in mice. In the W/W mutation, normal development of hematopoietic cell lineage is affected by alteration of the c-kit gene (2, 5). The missense mutation of Bruton agammaglobulinemia tyrosine kinase (btk) causes a defect of B cell development in the Xlinked immunodeficiency (xid ) mutation (21, 26). These results show important roles of PTKs in normal processes of development and the importance of the chromosomal assignment of PTK genes to known mutation loci. A feature common to all members of the protein kinase family is a highly conserved catalytic domain of 200–300 amino acids (9). Many efforts have been made to clone protein kinase genes by using the PTK-conserved motif, and a large number of PTK genes have been cloned and characterized (30). Using the polymerase chain reaction-mediated cloning procedure (28) in conjunction with the subtractive subcloning method, we cloned a novel protein tyrosine kinase gene, brt (for brain tyrosine kinase), from fetal mouse brain tissue (4). This gene is also known as tyro3/rse/sky (12, 17, 1
Present address: Laboratory of Animal Genetics, School of Agricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-01, Japan. 2 To whom correspondence and reprint requests should be addressed. Telephone: 81-3-5449-5301. Fax: 81-3-5449-5413.
19). The brt protein contains a transmembrane domain and is closely related to the axl/ufo/ark (10, 18, 22), mer (7), and eyk (11) proteins. Northern blot and in situ hybridization analyses showed that Brt is expressed preferentially in the brain of both embryo and adult (4, 12, 17, 19). A possible function mediating cell aggregation by homophilic binding of axl/ufo/ark protein molecules (1) and a common candidate ligand for both axl/ ufo/ark and brt/rse/sky/tyro3 proteins (6, 20, 27) were very recently reported. This accumulating evidence suggests that Brt may play an important role in the development, function, and maintenance of the central nervous system. To determine whether the brt gene might be associated with known mutations in the mouse and rat, we mapped brt to mouse and rat chromosomes by fluorescence in situ hybridization (FISH) and interspecific backcross analysis. Preparation of R-banded chromosomes and FISH were performed as described by Matsuda et al. (14) and Matsuda and Chapman (15). Mitogen-stimulated lymphocyte cultures were synchronized by thymidine block, and differential replication staining was made by using 5-bromodeoxyuridine. The 4.6kb mouse full-length cDNA fragment inserted in pBluescript II SK(/) (4) was biotinylated, hybridized, and stained with anti-biotin antibody (Vector Laboratories) and subsequently with fluorescein-antigoat IgG (Nordic Immunology). The chromosomal localization was firmly determined based on observation of 200 metaphase spreads. Of 200 metaphase spreads of the mouse and rat, 67 and 78, respectively, exhibited twin spots. The signals were localized to the R-positive F1 band of mouse Chromosome 2 (14, 23) and the R-positive q36.1 band of rat Chromosome 3 (24) (Fig. 1). A conserved linkage homology has been identified in these regions between the two species (29). Fine linkage mapping of the mouse brt was made by interspecific backcross analysis using progeny derived from the mating of (C57BL/6J 1 Mus spretus)F1 1 M. spretus mice (16). For localizing mouse brt, we used Southern blot hybridization with genomic DNAs of the interspecific backcross mice. Genomic DNAs of C57BL/ 6 and M. spretus were separately digested with six different restriction endonucleases, ApaI, BamHI, EcoRI, HindIII, KpnI, and PstI, and analyzed by Southern blot hybridization with a mouse 4.6-kb brt cDNA fragment GENOMICS
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FIG. 1. Chromosomal localization of the Brt gene on mouse and rat R-banded chromosomes using a mouse 4.6-kb cDNA fragment as a biotinylated probe. The hybridization signals are indicated by arrows. The signals are localized to the F1 band of mouse Chromosome 2 (a, c, and d) and the q36.13 band of rat Chromosome 5 (e and f). The metaphase spreads were photographed with Nikon B-2A (a, c –f) and UV-2A (b) filters. R-band and G-band patterns are demonstrated in (a, c –f) and (b), respectively.
to determine restriction fragment length variants (RFLVs). A comparison of C57BL/6 and M. spretus identified RFLVs between the two Mus species for ApaI, BamHI, EcoRI, KpnI, and PstI. Among these restriction endonuclease digestions, ApaI digestion identified the following DNA fragments: 9.4- and 7.4-kb fragments in C57BL/6, 7.4-, 7.0-, and 6.5-kb fragments in M. spretus, and heterozygous patterns in the F1 mice. Segregation of a 9.4-kb C57BL/6-specific ApaI fragment was used to follow the segregation of the BRT locus in a total number of 146 backcross mice. Four microsatellite DNA markers (D2Mit43, D2Mit58, D2Mit63, and D2Mit19) were chosen for fine-mapping of mouse brt on the data of cytogenetic localization by FISH (3). The PCR primers for the DNA markers were purchased from Research Genetics (Huntsville, AL). All PCRs were carried out in 15 ml containing 75 ng of genomic DNA and 15 pmol of each
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oligonucleotide primer. Amplification conditions were 947C for 5 min; 30 cycles of 947C for 0.5 min, 557C for 0.5 min, and 727C for 5 min; and 727C for 5 min. Each locus was analyzed in pairwise combination for recombination frequencies, and gene order was determined by minimizing the number of double recombination events. The gene order and recombination frequencies for each pair of loci in 146 backcross mice, expressed as genetic distances in centimorgans { the standard error, and the number of recombinants for each pair of loci are centromere (Hc 2)–D2Mit43–6.2 { 2.0 (9/146)– D2Mit58–4.8 { 1.8 (7/146) –Brt–0 { 0 (0/146)– D2Mit63–4.8 { 1.8 (7/146) –D2Mit19–telomere (Fig. 2) (25). Comparing the present results with recent composite linkage maps (13, 25), the gene order and map distance obtained from the cross in this study are in good agreement with those in the composite maps,
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Chr 3 suggests from the human–rat –mouse synteny that the human homologue will be located on the short arm of human chromosome 11 or the long arm of human chromosome 15 (25, 29). This expectation from the present comparative genomic analysis is in good accordance with the previous chromosome mapping data using a human–hamster hybrid panel (17), in which the human homologue was localized to human chromosome 15. We have compared our interspecific linkage map of mouse Chromosome 2 with a recent composite mouse linkage map showing the map location of uncloned mouse mutations (25). The comparison between the maps revealed that three spontaneous mutations, whose genes have not been identified yet, lst (Strong’s luxoid), anx (anorexia), and ro (rough), have been mapped to the region around the BRT locus. lst is located mostly near the position of the BRT locus. The lst mutation shows skeletal deformity and developmental abnormality (summarized in Ref. 8). anx is characterized by reduction of body weight and abnormal behavior, which might be caused by anorexia. Homozygous ro mice have wavy vibrisse, apparent a few days after birth. The possible association between the brt gene and these mutations localized near the BRT locus remains to be clarified. MGD Accession Nos. MGD-INEX-35 and MGD-CREX-728 for FISH and genetic linkage data, respectively, have been assigned to our mapping data. ACKNOWLEDGMENTS We thank Ms. T. Maeda for technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, and Science and Technology Agency, Japan.
FIG. 2. Location of mouse brt on Chromosome 2. The segregation patterns of mouse brt with flanking microsatellite DNA markers D2Mit43, D2Mit58, D2Mit63, and D2Mit19, in the backcross mice between (C57BL/6 1 M. spretus)F1 females and M. spretus males, are shown at the top. Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/ 6 1 M. spretus)F1 parent. The open squares represent the presence of the M. spretus allele, and the closed squares represent the presence of the C57BL/6 allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. The partial linkage map of Chromosome 2 at the bottom shows the location of mouse brt in relation to the flanking DNA markers. Recombination distances between loci are shown in centimorgans to the right of the chromosome.
except that the distance between D2Mit63 and D2Mit19 is much shorter than that reported in Siracusa and Abbott (25). Our present results are additional evidence of a conserved linkage homology between mouse and rat chromosomes and support the idea that the region of mouse Chromosome 2 and rat Chromosome 3 evolved from an ancestral mammalian chromosome. Though the precise human BRT locus has not been mapped, the position of brt in the mid-distal region of mouse Chr 2 and rat
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REFERENCES 1. Bellosta, P., Costa, M., Lin, D. A., and Basilico, C. (1995). The receptor tyrosine kinase ARK mediates cell aggregation by homophilic binding. Mol. Cell. Biol. 15: 614– 625. 2. Chabot, B., Stephenson, D. A., Chapman, V. M., Besmer, P., and Bernstein, A. (1988). The proto-oncogene c-kit encoding transmembrane tyrosine kinase receptor maps to the mouse W locus. Nature 335: 88– 89. 3. Dietrich, W., Katz, H., Lincoln, S. E., Shin, H.-S, Friedman, J., Dracopoli, N. C., and Lander, E. S. (1992). A genetic map of the mouse suitable for typing intraspecific crosses. Genetics 131: 423– 447. 4. Fujimoto, J., and Yamamoto, T. (1994). Brt, a mouse encoding a novel receptor-type protein-tyrosine kinase, is preferentially expressed in the brain. Oncogene 9: 693– 698. 5. Geissler, E. N., Ryan, M. A., and Housman, D. E. (1988). The dominant-white spotting (W ) locus of the mouse encodes the ckit proto-oncogene. Cell 55: 185– 192. 6. Godowski, P. J., Mark, M. R., Chen, J., Sadick, M. D., Raab, H., and Hammonds, R. G. (1995). Reevaluation of the roles of protein S and gas6 as ligands for the receptor tyrosine kinase Rse/ Tyro 3. Cell 82: 355–358. 7. Graham, D. K., Dawson, T. L., Mullaney, D. L., Snodgrass, H. R., and Earp, H. S. (1994). Cloning and mRNA expression analysis of a novel human protooncogene, c-mer. Cell Growth Differ. 5: 647– 657.
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8. Green, M. C. (1989). Catalog of mutant genes and polymorphic loci. In ‘‘Genetic Variants and Strains of the Laboratory Mouse’’ (M. F. Lyon and A. G. Searle, Eds.), pp. 12 –403. Oxford Univ. Press, Oxford. 9. Hanks, S. K., Quinn, A. M., and Hunter, T. (1988). The protein kinase family: Conserved features and deduced phylogeny of the catalytic domain. Science 241: 42 –52. 10. Janssen, J. W. G., Schulz, A. S., Steenvoorden, A. C. M., Schmidberger, M., Strehl, S., Ambros, P. F., and Bartram, C. R. (1991). A novel putative tyrosine kinase receptor with oncogenic potential. Oncogene 6: 2113 – 2120. 11. Jia, R., and Hanafusa, H. (1994). The proto-oncogene of v-eyk (v-ryk) is a novel receptor-type protein tyrosine kinase with extracellular Ig/FN-III domains. J. Biol. Chem. 269: 1839 – 1844. 12. Lai, C., Gore, M., and Lemke, G. (1994). Structure, expression and activity of Tyro 3, a neural adhesion-related receptor tyrosine kinase. Oncogene 9: 693– 698. 13. Lyon, M. F., and Kirby, M. C. (1994). Mouse chromosome atlas. Mouse Genome 92: 19–61. 14. Matsuda, Y., Harada, Y.-N., Natsuume-Sakai, S., Lee, K., Shiomi, T., and Chapman, V. M. (1992). Location of the mouse complement factor H gene (cfh) by FISH analysis and replication R-banding. Cytogenet. Cell Genet. 61: 282 –285. 15. Matsuda, Y., and Chapman, V. M. (1995). Application of fluorescence in situ hybridization in genome analysis of the mouse. Electrophoresis 16: 261– 272.
20.
21.
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24. 25.
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27.
16. Matsuda, Y., Imai, T., Shiomi, T., Saito, T., Yamauchi, M., Fukao, T., Akao, Y., Seki, N., Ito, H., and Hori, T. (1996). Comparative genome mapping of ataxia telangiectasia region in the mouse, rat and hamster. Genomics 34: 347 –352. 17. Melanie, R. M., David, T. S., Zhengyu, W., Qimin, G., Audrey, G., and Paul, J. G. (1994). rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain. J. Biol. Chem. 269: 10720 –10728. 18. O’Bryan, J. P., Frye, R. A., Cogswell, P. C., Neubauer, A., Kitch, B., Prokop, C., Espinosa, R., Le Beau, M. M., Earop, H. S., and Liu, E. T. (1991). axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase. Mol. Cell. Biol. 11: 5016 – 5031. 19. Ohashi, K., Mizuno, K., Kuma, K., Miyata, T., and Nakamura, T. (1994). Cloning of the cDNA for a novel receptor tyrosine
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28.
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kinase, sky, predominantly expressed in brain. Oncogene 9: 699– 705. Ohashi, K., Nagata, K., Toshima, J., Nakano, T., Arita, H., Tsuda, H., Suzuki, K., and Mizuno, K. (1995). Stimulation of Sky receptor tyrosine kinase by the product of growth arrestspecific gene 6. J. Biol. Chem. 270: 22681 –22684. Rawlings, D. J., Saffran, D. C., Tsukada, S., Largaespada, D. A., Grimaldi, J. C., Cohen, L., Mohr, R. N., Bazan, J. F., Howard, M., Copeland, N. G., Jenkins, N. A., and Witte, O. N. (1993). Mutation of unique region of Bruton’s tyrosine kinase in immunodeficient XID mice. Science 261: 358–361. Rescigno, J., Mansukhani, A., and Basilico, C. (1991). A putative receptor tyrosine kinase with unique structural topology. Oncogene 6: 1909 –1913. Satoh, H., Yoshida, M. C., and Sasaki, M. (1989). High resolution chromosome banding in the Norway rat, Rattus norvegicus. Cytogenet. Cell Genet. 50: 151– 154.5 Siracusa, L. D., and Abbott, C. M. (1994). Mouse Chromosome 2. Mamm. Genome 5: S22–S39. Somssich, I. E., and Hameister, H. (1989). Standard karyotype of early replicating bands (RBG-banding). In ‘‘Genetic Variants and Strains of the Laboratory Mouse’’ (M. F. Lyon and A. G. Searle, Eds.), pp. 580– 581, Oxford Univ. Press, Oxford. Thomas, J. D., Sideras, P., Smith, C. I. E., Vorechovsky, I., Chapman, V. M., and Paul, W. E. (1993). Colocalization of Xlinked agammaglobulinemia and X-linked immunodeficiency genes. Science 261: 355–358. Varnum, B. C., Young, C., Elliott, G., Garcia, A., Bartley, T. D., Fridell, Y.-W., Hunt, R. W., Trall, G., Clogston, C., Toso, R. J., Yanagihara, D., Bennett, L., Sylber, M., Merewether, L. A., Tseng, A., Escobar, E., Liu, E. T., and Yamane, H. K. (1995). Axl receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growth-arrested-specific gene 6. Nature 373: 623– 626. Wilks, A. M. (1989). Two putative protein-tyrosine kinases identified by application of the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 86: 1603 –1607. Yamada, J., Kuramoto, T., and Serikawa, T. (1994). A rat genetic linkage map and comparative maps for mouse or human homologous rat genes. Mamm. Genome 5: 63 –83. Yamamoto, T., and Kadowaki, Y. (1995). Superfamilies of protooncogenes: Homology cloning and characterization of related members. In ‘‘Methods in Enzymology,’’ Vol. 254, pp. 169–183, Academic Press, San Diego.
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TADASHI YAMAMOTO†,2
*Genome Research Group, National Institute of Radiological Sciences, 4-9-1 Anagawa, Chiba 263, Japan; and †Department of Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, 4-6-1 Shirokanedai, Tokyo 108, Japan Received August 16, 1996; accepted November 18, 1996
brt encodes a receptor-type protein-tyrosine kinase and was isolated from fetal mouse brain by using a PCR-mediated cloning procedure; this gene is expressed preferentially in the brain of both embryo and adult. Chromosomal locations of the mouse and rat brt genes were determined by fluorescence in situ hybridization using a mouse cDNA fragment as a probe. The brt gene was localized to mouse Chromosome 2F1 and rat Chromosome 3q36.1, where a conserved linkage homology has been identified between the two species. Interspecific backcross analysis genetically demonstrated no recombination between the mouse BRT locus and D2Mit63. q 1997 Academic Press
The frequent appearance of protein tyrosine kinases (PTKs) as products of oncogenes and growth factor receptors suggests that tyrosine phosphorylation plays an important role in the signal transduction pathway in cell growth and differentiation. There have been two reports on loss-of-function mutations of protein-tyrosine kinase in mice. In the W/W mutation, normal development of hematopoietic cell lineage is affected by alteration of the c-kit gene (2, 5). The missense mutation of Bruton agammaglobulinemia tyrosine kinase (btk) causes a defect of B cell development in the Xlinked immunodeficiency (xid ) mutation (21, 26). These results show important roles of PTKs in normal processes of development and the importance of the chromosomal assignment of PTK genes to known mutation loci. A feature common to all members of the protein kinase family is a highly conserved catalytic domain of 200–300 amino acids (9). Many efforts have been made to clone protein kinase genes by using the PTK-conserved motif, and a large number of PTK genes have been cloned and characterized (30). Using the polymerase chain reaction-mediated cloning procedure (28) in conjunction with the subtractive subcloning method, we cloned a novel protein tyrosine kinase gene, brt (for brain tyrosine kinase), from fetal mouse brain tissue (4). This gene is also known as tyro3/rse/sky (12, 17, 1
Present address: Laboratory of Animal Genetics, School of Agricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-01, Japan. 2 To whom correspondence and reprint requests should be addressed. Telephone: 81-3-5449-5301. Fax: 81-3-5449-5413.
19). The brt protein contains a transmembrane domain and is closely related to the axl/ufo/ark (10, 18, 22), mer (7), and eyk (11) proteins. Northern blot and in situ hybridization analyses showed that Brt is expressed preferentially in the brain of both embryo and adult (4, 12, 17, 19). A possible function mediating cell aggregation by homophilic binding of axl/ufo/ark protein molecules (1) and a common candidate ligand for both axl/ ufo/ark and brt/rse/sky/tyro3 proteins (6, 20, 27) were very recently reported. This accumulating evidence suggests that Brt may play an important role in the development, function, and maintenance of the central nervous system. To determine whether the brt gene might be associated with known mutations in the mouse and rat, we mapped brt to mouse and rat chromosomes by fluorescence in situ hybridization (FISH) and interspecific backcross analysis. Preparation of R-banded chromosomes and FISH were performed as described by Matsuda et al. (14) and Matsuda and Chapman (15). Mitogen-stimulated lymphocyte cultures were synchronized by thymidine block, and differential replication staining was made by using 5-bromodeoxyuridine. The 4.6kb mouse full-length cDNA fragment inserted in pBluescript II SK(/) (4) was biotinylated, hybridized, and stained with anti-biotin antibody (Vector Laboratories) and subsequently with fluorescein-antigoat IgG (Nordic Immunology). The chromosomal localization was firmly determined based on observation of 200 metaphase spreads. Of 200 metaphase spreads of the mouse and rat, 67 and 78, respectively, exhibited twin spots. The signals were localized to the R-positive F1 band of mouse Chromosome 2 (14, 23) and the R-positive q36.1 band of rat Chromosome 3 (24) (Fig. 1). A conserved linkage homology has been identified in these regions between the two species (29). Fine linkage mapping of the mouse brt was made by interspecific backcross analysis using progeny derived from the mating of (C57BL/6J 1 Mus spretus)F1 1 M. spretus mice (16). For localizing mouse brt, we used Southern blot hybridization with genomic DNAs of the interspecific backcross mice. Genomic DNAs of C57BL/ 6 and M. spretus were separately digested with six different restriction endonucleases, ApaI, BamHI, EcoRI, HindIII, KpnI, and PstI, and analyzed by Southern blot hybridization with a mouse 4.6-kb brt cDNA fragment GENOMICS
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FIG. 1. Chromosomal localization of the Brt gene on mouse and rat R-banded chromosomes using a mouse 4.6-kb cDNA fragment as a biotinylated probe. The hybridization signals are indicated by arrows. The signals are localized to the F1 band of mouse Chromosome 2 (a, c, and d) and the q36.13 band of rat Chromosome 5 (e and f). The metaphase spreads were photographed with Nikon B-2A (a, c –f) and UV-2A (b) filters. R-band and G-band patterns are demonstrated in (a, c –f) and (b), respectively.
to determine restriction fragment length variants (RFLVs). A comparison of C57BL/6 and M. spretus identified RFLVs between the two Mus species for ApaI, BamHI, EcoRI, KpnI, and PstI. Among these restriction endonuclease digestions, ApaI digestion identified the following DNA fragments: 9.4- and 7.4-kb fragments in C57BL/6, 7.4-, 7.0-, and 6.5-kb fragments in M. spretus, and heterozygous patterns in the F1 mice. Segregation of a 9.4-kb C57BL/6-specific ApaI fragment was used to follow the segregation of the BRT locus in a total number of 146 backcross mice. Four microsatellite DNA markers (D2Mit43, D2Mit58, D2Mit63, and D2Mit19) were chosen for fine-mapping of mouse brt on the data of cytogenetic localization by FISH (3). The PCR primers for the DNA markers were purchased from Research Genetics (Huntsville, AL). All PCRs were carried out in 15 ml containing 75 ng of genomic DNA and 15 pmol of each
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oligonucleotide primer. Amplification conditions were 947C for 5 min; 30 cycles of 947C for 0.5 min, 557C for 0.5 min, and 727C for 5 min; and 727C for 5 min. Each locus was analyzed in pairwise combination for recombination frequencies, and gene order was determined by minimizing the number of double recombination events. The gene order and recombination frequencies for each pair of loci in 146 backcross mice, expressed as genetic distances in centimorgans { the standard error, and the number of recombinants for each pair of loci are centromere (Hc 2)–D2Mit43–6.2 { 2.0 (9/146)– D2Mit58–4.8 { 1.8 (7/146) –Brt–0 { 0 (0/146)– D2Mit63–4.8 { 1.8 (7/146) –D2Mit19–telomere (Fig. 2) (25). Comparing the present results with recent composite linkage maps (13, 25), the gene order and map distance obtained from the cross in this study are in good agreement with those in the composite maps,
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Chr 3 suggests from the human–rat –mouse synteny that the human homologue will be located on the short arm of human chromosome 11 or the long arm of human chromosome 15 (25, 29). This expectation from the present comparative genomic analysis is in good accordance with the previous chromosome mapping data using a human–hamster hybrid panel (17), in which the human homologue was localized to human chromosome 15. We have compared our interspecific linkage map of mouse Chromosome 2 with a recent composite mouse linkage map showing the map location of uncloned mouse mutations (25). The comparison between the maps revealed that three spontaneous mutations, whose genes have not been identified yet, lst (Strong’s luxoid), anx (anorexia), and ro (rough), have been mapped to the region around the BRT locus. lst is located mostly near the position of the BRT locus. The lst mutation shows skeletal deformity and developmental abnormality (summarized in Ref. 8). anx is characterized by reduction of body weight and abnormal behavior, which might be caused by anorexia. Homozygous ro mice have wavy vibrisse, apparent a few days after birth. The possible association between the brt gene and these mutations localized near the BRT locus remains to be clarified. MGD Accession Nos. MGD-INEX-35 and MGD-CREX-728 for FISH and genetic linkage data, respectively, have been assigned to our mapping data. ACKNOWLEDGMENTS We thank Ms. T. Maeda for technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, and Science and Technology Agency, Japan.
FIG. 2. Location of mouse brt on Chromosome 2. The segregation patterns of mouse brt with flanking microsatellite DNA markers D2Mit43, D2Mit58, D2Mit63, and D2Mit19, in the backcross mice between (C57BL/6 1 M. spretus)F1 females and M. spretus males, are shown at the top. Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/ 6 1 M. spretus)F1 parent. The open squares represent the presence of the M. spretus allele, and the closed squares represent the presence of the C57BL/6 allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. The partial linkage map of Chromosome 2 at the bottom shows the location of mouse brt in relation to the flanking DNA markers. Recombination distances between loci are shown in centimorgans to the right of the chromosome.
except that the distance between D2Mit63 and D2Mit19 is much shorter than that reported in Siracusa and Abbott (25). Our present results are additional evidence of a conserved linkage homology between mouse and rat chromosomes and support the idea that the region of mouse Chromosome 2 and rat Chromosome 3 evolved from an ancestral mammalian chromosome. Though the precise human BRT locus has not been mapped, the position of brt in the mid-distal region of mouse Chr 2 and rat
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REFERENCES 1. Bellosta, P., Costa, M., Lin, D. A., and Basilico, C. (1995). The receptor tyrosine kinase ARK mediates cell aggregation by homophilic binding. Mol. Cell. Biol. 15: 614– 625. 2. Chabot, B., Stephenson, D. A., Chapman, V. M., Besmer, P., and Bernstein, A. (1988). The proto-oncogene c-kit encoding transmembrane tyrosine kinase receptor maps to the mouse W locus. Nature 335: 88– 89. 3. Dietrich, W., Katz, H., Lincoln, S. E., Shin, H.-S, Friedman, J., Dracopoli, N. C., and Lander, E. S. (1992). A genetic map of the mouse suitable for typing intraspecific crosses. Genetics 131: 423– 447. 4. Fujimoto, J., and Yamamoto, T. (1994). Brt, a mouse encoding a novel receptor-type protein-tyrosine kinase, is preferentially expressed in the brain. Oncogene 9: 693– 698. 5. Geissler, E. N., Ryan, M. A., and Housman, D. E. (1988). The dominant-white spotting (W ) locus of the mouse encodes the ckit proto-oncogene. Cell 55: 185– 192. 6. Godowski, P. J., Mark, M. R., Chen, J., Sadick, M. D., Raab, H., and Hammonds, R. G. (1995). Reevaluation of the roles of protein S and gas6 as ligands for the receptor tyrosine kinase Rse/ Tyro 3. Cell 82: 355–358. 7. Graham, D. K., Dawson, T. L., Mullaney, D. L., Snodgrass, H. R., and Earp, H. S. (1994). Cloning and mRNA expression analysis of a novel human protooncogene, c-mer. Cell Growth Differ. 5: 647– 657.
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