Mammalian Genome 3: 469-471, 1992
9 Springer-VerlagNew York Inc. 1992
Assignment of mouse c band F1-G3
-2-macroglobulin gene to Chromosome 6
Carl Hilliker, Lut Overbergh, Paul Petit, Fred Van Leuven, and Herman Van den Berghe Center for Human Genetics, Campus Gasthuisberg O&N, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium Received March 6, 1992; accepted April 10, 1992
a-2-macroglobulin (A2M) is a major proteinase inhibitor and scavenger in the circulation of humans (Van Leuven 1982; Sottrup-Jensen 1989). Homologous pro, teins were shown to be present in all other vertebrates examined. Despite its in-depth molecular characterization as a proteinase inhibitor, the physiological function in vivo of A2M has yet to be determined. That A2M is part of a defense system in the body is supported only by circumstantial evidence (James 1980; Van Leuven 1982; Sottrup-Jensen 1989). It is suggested by the fact that A2M is the only known plasma inhibitor of the four different classes of proteinases originating from endogenous sources as well as from parasites and pathogens. Also, A2Ms are "acute phase" proteins in some species, such as the rat (Gauthier and Mouray 1976). Furthermore, no quantitative or qualitative deficiencies have been noted in humans. In an effort to elucidate the physiology of A2M in vivo, we are characterizing the family of the macroglobulin and murinoglobulin proteinase inhibitors in the mouse. Cloning of cDNA and of the corresponding genes will eventually result in the production of transgenic mice. The results reported here are part of this effort. Structurally, proteinase inhibitors of the eL macroglobulin family (AM) are characterized by a subunit structure containing about 1500 amino acids, with three specific structural features: a bait region, an internal thioester, and a receptor-binding domain (Van Leuven 1982; Sottrup-Jensen 1989). The bait region is the actual sequence of about 60 amino acids, located in the middle of the subunit, which is typical for each type of A2M. Its sequence determines the specificity of possible proteolytic activation and thereby determines the proteinases that can be bound by each A2M type. We chose to include the bait region because it represents the most specific sequence that distinOffprint requests to: F. Van Leuven
guishes it from other members of the murinoglobulin family (Overbergh et al. 1991a,b). The 1.7-kb probe used for the isotopic in situ hybridization was generated by means of the polymerase chain reaction (PCR) essentially as described previously (Hilliker et al. 1991). The PCR product encompasses position 1741 in exon 14 to position 3517 in exon 27 of mouse A2M cDNA, including the bait region (Overbergh et al. 1991a; Overbergh and Van Leuven 1991; Van Leuven and Overbergh 1991). The nonisotopic in situ hybridization was performed with a yeast artificial chromosome (YAC) clone of 245 kb, which contains the entire A 2 M gene. The YAC clone was obtained by screening the Princeton Mouse YAC Library by PCR with specific primers. The sequences of the primers used to generate the isotropic probe are 5' TCA GCA CAG AGC CTG CCA 3'(sense strand) and 5'GCC AGA CTG TTG GAA ACA GCC 3'(anti-sense). Those used for PCR of the YAC screening were 5' AGG CTG CAA GAC CAG CCT 3'(sense strand) and 5' TGT CTT GTT CCA CCG CGA AGG AGA 3' (anti-sense). Preparation and trypsin-Giemsa banding of chromosomes was performed as described by Davisson and Akeson (1987), with minor modifications for obtaining mitotic chromosomes from bone marrow in vivo. The localization of the A 2 M gene by isotopic in situ hybridization with a PCR-generated probe and the nonisotopic hybridization with a YAC clone were performed on metaphase chromosomes from both the NMRI and Robertsonian translocation Rb(6;15)lAld strains (The Jackson Laboratory, Bar Harbor, Me.). Isotopic labeling and the subsequent hybridization were performed according to the procedure of Harper and Saunders (1981) with the staining procedure described by Cannizzarro and Emanuel (1984). Slides were developed after 5-10 days of exposure. The biotinylation of the YAC probe by nick translation and the nonisotopic hybridization to Robertsonian translo-
470
C. Hilliker et al.: The mouse c~-2-macroglobulin gene
cation metaphases was performed according to the procedure of Wada and co-workers (1990). A partial histogram summarizing the results of the isotopic in situ analysis to NMRI and Rb(6; 15) 1Aid chromosomes is presented in Fig. 1 (a,b). Analysis of 85 metaphases from the NMRI strain yielded 173 grains (approximately 2.0 grains/metaphase). Significant hybridization was found on Chromosome (Chr) 6 (34/173 grains or 19.6%), with grains randomly distributed over the other chromosomes. For the Robertsonian translocation Rb(6;15)lAld, 75 metaphases were scored for 162 grains (approximately 2.2 grains/metaphase). Again the hybridization signal was predominantly localized on Chr 6 (35/162 grains or 21%), with the rest of the grains being randomly distributed over all the chromosomes. The assignment to Chr 6 is evidenced by 64.7% (22/34; Fig. la) of the grains localizing to the region 6 F1-G3 in the NMRI strain and 72% (25/35) (Fig. lb) of the grains in the same region for the Robertsonian translocation Rb(6.15)lAid. In addition, fluorescent in situ hybridization (FISH) with the YAC clone, containing the A2M gene, showed hybridization signal in the telomeric region of mouse Chr 6 in the reported area of localization (Fig. 2), with about 85% of the metaphases displaying the yellow fluorescent signal on
'6
oo
O0
00000 000 000000000 0000000001 a
0000 b
Fig. 1 (a-b). Partial histogram (Evans 1990) of NMRI and (a) Robertsonian translocation Rb(6;15)lAld; (b) chromosomes hybridized with the A2M PCR-generated probe.
Fig. 2. Nonisotopic in situ hybridization with the YAC clone containing the A2M gene to Rb(6;15)lAld metaphase spreads.
both chromatids of the two Chr 6 homologs. These results therefore support the localization of the mouse A2M gene to Chr 6 F1-G3. The human A2M gene was originally assigned to human Chr 12 based on the analysis of its segregation in panels of human-rodent somatic cell hybrids (Bell et al. 1985; Kan et al. 1985). Its localization was further refined to the chromosome region 12p12.3-p13.3 by in situ hybridization (Fukushima et al. 1988; Devriendt et al. 1989). A conserved linkage group could be present distally on mouse Chr 6 and on human Chr 12p. To date 11 genes have been mapped to human Chr 12p and mouse Chr 6. The genes involved are (mouse locus in parenthesis) CD4(Ly-4),TNFRI(Tnfr-1),S YBI(SybI ) ,PRH1 (Prp), FGF6 (Fgf-6), GAPD ( Gap d), TPI1 (Tp i1),LDHB(Ldh-2), PTHLH(Pthlh), and KRAS2(Kras-2) (Davisson et al. 1991). Cahilly and George (1985) mapped the cKi-ras proto-oncogene to mouse Chr 6F3-G3 by in situ hybridization, de Lapeyriere and colleagues (1990) also used in situ hybridization to map the Fgf-6 gene to the region 6F1-G3. Both genes map to the same region, to which we have mapped the mouse A2M gene. We preferred to use in situ hybridization as the method for mapping the mouse A2M gene because a gene located near the end of a chromosome would be especially prone to being separated from the rest of its linkage group in a translocation, which might occur in a somatic cell hybrid, that could be cytologically undetectable. Comparison of the cDNA sequences of complement components and of A2M strongly suggest an evolutionary relationship (Sottrup-Jensen et al., 1985). Human and mouse A2M, C3, and C4 contain domains with high sequence identity, with C3 and C4 more related to each other than to A2M. Members of this superfamily have in common the presence of an internal thiol ester and the mechanism of action. Activation of the thioester occurs by proteotysis at the bait region in
C. Hilliker et al.: The mouse a-2-macroglobulin gene
A2M or at the equivalently positioned arginine residue in complement components, allowing for a major conformational change, for an eventual covalent reaction at the thioester and for the exposure of a receptor recognition site. The chromosomal localization for the complement components C3, C4, and C5 are on three different chromosomes in humans: C3 on 19p13.3-13.2 (Whitehead et al. 1982; Davies et al. 1983), C4 on 6p21.3 (Carroll et al. 1984), and C5 on 9q22-q33 (Jeremiah et al. 1988). In the mouse, C5 is on Chr 2 (D'Eustachio et al. 1986), while C3 and C4 are located on Chr 17 (da Silva et al. 1978; White et al. 1984). These positions are different from the one identified here for the mouse A 2 M gene. It remains to be shown that, as in humans, other members of the A2M family of proteinase inhibitors are clustered on mouse Chr 6. This work is currently in progress, both by in situ hybridization and by analyzing the mouse genomic YAC clone.
Acknowledgments. The authors wish to thank M.T. Davisson and
E. Akeson of The Jackson Laboratory for help with the karyotype. These investigations have been supported by grant 3.0069.89 from the National Science Foundation of Belgium, by a grant "Geconcerteerde Acties" from the Belgian Government, by the "Interuniversity Network" for Fundamental Research sponsored by the Belgian Government (1987-1991), by the NFWO-Levenslijn Fund, and by the VLAB program for Biotechnology.
References Bell, G.I., Rail, L.B., Sanchez-Pescador, R., Merryweather, J.P., Scott, J., Eddy, R.L., and Shows, T.B.: Human alpha-2macroglobulin gene is located on chromosome 12. Somatic Cell Mol Genet 11: 285-289, 1985. Cahilly, L.A. and George, D.L.: Regional mapping of cKi-ras protooncogene on mouse chromosome 6 by in situ hybridization. Cytogenet Cell Genet 39: 140-144, 1985. Cannizzarro, L.A. and Emanuel, B.S.: An improved method for G-banding chromosomes after in situ hybridization. Cytogenet Cell Genet 38: 308-309, 1984. Carroll, M.C., Campbell, R.D., Bentley, D.R., and Porter, R.R.: A molecular map of the human major histocompatibility complex class III region linking complement genes C4, C2 and factor B. Nature 307: 237-241, 1984. da Silva, F.P., Hoecker, G.F., Day, N.K., Vienne, K., and Rubinstein, P.: Murine complement component 3: genetic variation and linkage to H-2. Proc Natl Acad Sei USA 75: 963-965, 1978. Davies, K.E., Jackson, J., Williamson, R., Harper, P.S., Ball, S., Sarfarazi, M., Meridith, L., and Fey, G.: Linkage analysis of myotonic dystrophy and sequences on chromosome 19 using a cloned complement 3 gene probe. J Med Genet 20: 259-263, 1983. Davisson, M.T. and Akeson, E.C.: An improved method for preparing G-banded chromosomes from mouse peripheral blood. Cytogenet Cell Genet 45: 70-74, 1987. Davisson, M.T., Lalley, P.A., Peters, J., Doolittle, D.P., Hillyard, A.L., and Searle, A.G.: Report of the comparative committee for human, mouse and other rodents. Cytogenet Cell Genet 58:11521189, 1991. de Lapeyriere, O., Rosnet, O., Benharroch, D., Raybaund, F., Marchetto, S., Planche, J., Galland, F., Mattei, M.-G., Copeland, N., Jenkins, N., Coulier, F., and Birnbaum, D.: Structure, chromosome mapping and expression in the murine Fgf-6 gene. Oncogene 5: 823-831, 1990.
471 D'Eustachio, P., Kristensen, T., Wetsel, R.A., Riblet, R., Taylor, B.A., and Tack, B.F.: Chromosomal localization of the genes encoding complement component C5 and factor H in the mouse. J Immunol 137: 3390-3395, 1986. Devriendt, K., Zhang, J., Van Leuven, F., Van den Berghe, H., Cassiman, J.-J., and Marynen, P.: A cluster of alpha-2macroglobulin-related genes (A2M) on human chromosome 12p: cloning of the pregnancy-zone protein gene and a A 2 M pseudogene. Gene 81: 325-334, 1989. Evans, E.P.: Standard normal chromosomes. In M.F. Lyon and A.G. Searle (eds.); Genetic Variants and Strains o f the Laboratory Mouse, pp. 576-578, Oxford University Press, New York, 1990. Fukushima, Y., Bell, G.I., and Shows, T.B.: The polymorphic human alpha-2-macroglobulin gene (A2M) is located on chromosome 12 in the region 12p12.3-p13.3. Cytogenet Cell Genet 43: 58-59, 1988. Gauthier, F. and Mouray, H.: Rat alpha-2 acute phase macroglobulin. Biochem J 159: 661-665, 1976. Harper, M.E. and Saunders, G.F.: Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization. Chromosoma 83: 431-439, 1981. Hilliker, C.E., Darville, M.I., Aly, M.S., Chikri, M., Szpirer, C., Marynen, P., Rousseau, G.-G., and Cassiman, J.-J.: Human and rat chromosomal localization of two genes for 6-phosphofructo2-kinase/fructose 2,6-biphosphatase by analysis of somatic cell hybrids and in situ hybridization. Genomics I0: 867-873, 1991. James, K.: Alpha-2-macroglobulin and its possible importance in immune systems. Trends Biochem Sci 5: 43-47, 1980. Jeremiah, S.J., West, L.F., Davis, M., Povey, S., Carritt, B., and Fey, G.: The assignment of the gene coding for complement C5 to chromosome 9q22-q33. Ann Hum Genet 52: 111-116, 1988. Kan, C.C., Solomon, E., Belt, K.T., chain, A.C., Hiorns, L.R., and Fey, G.: Nucleotide sequence of cDNA encoding human alpha2-macroglobulin and assignment of the chromosomal locus. Proc Natl Acad Sci USA 82: 2282-2286, 1985. Overbergh, L. and Van Leuven, F.: Molecular cloning and sequencing of murinogtobulin cDNA's. Hoppe-Seylers Z Physiol Chem 372: 144, 1991. Overbergh, L., Torrekens, S., and Van Leuven, F.: Molecular characterization of the proteinase inhibitors of the alpha-2macroglobulin family in the mouse. Arch Int Physiol Biochim 99: B40, 1991a. Overbergh, L., Torrekens, S., Van Leuven, F., and Van den Berghe, H.: Molecular characterization of the murinoglobulins. J Biol Chem 266: 16903-16910, 1991b. Sottrup-Jensen, L.: Alpha macroglobulins: structure, shape, and mechanism of proteinase complex formation. J Biol Chem 264: 11539-11542, 1989. Sottrup-Jensen, L., Stepanik, T.M., Kristensen, T., LCnblad, P.B., Jones, C.M., Weirzbicki, D.M., Magnusson, S., Domdey, H., Wetsel, R.A., Lundwall, .A., Tack, B.F., and Fey, G.H.: Common evolutionary origin of alpha-2-macroglobulin and complement components C3 and C4. Proc Natl Acad Sci USA 82: 9-13, 1985. Van Leuven, F.: Human alpha-2-macroglobulin: structure and function. Trends Biochem Sci 7: 185-187, 1982. Van Leuven, F. and Overbergh, L.: Murine alpha-2-macroglobulin. Hoppe-Seylers Z Physiol Chem 372: 147, 1991. Wada, M., Little, R.D., Abidi, F., Porta, G., Labella, T., Cooper, T., Della VaUe, G., D'Urso, M., and Schlessinger, D.: Human Xq24-q28: approaches to mapping with yeast artificial chromosomes. Am J Hum Genet 46: 95-106, 1990. White, P.C., Chaplin, D.D., Weis, J.H., Dupont, B., New, M.I., and Seidman, J.G.: Two steroid 21-hydroxylase genes are located in the mouse s region. Nature 312: 465-467, 1984. Whitehead, A.S., Solomon, E., Chambers, S., Bodmer, W.F., Povey, S., and Fey, G.: Assignment of the structural gene for the third component of human complement to chromosome 19. Proc Natl Acad Sci USA 79: 5021-5025, 1982.
9 Springer-VerlagNew York Inc. 1992
Assignment of mouse c band F1-G3
-2-macroglobulin gene to Chromosome 6
Carl Hilliker, Lut Overbergh, Paul Petit, Fred Van Leuven, and Herman Van den Berghe Center for Human Genetics, Campus Gasthuisberg O&N, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium Received March 6, 1992; accepted April 10, 1992
a-2-macroglobulin (A2M) is a major proteinase inhibitor and scavenger in the circulation of humans (Van Leuven 1982; Sottrup-Jensen 1989). Homologous pro, teins were shown to be present in all other vertebrates examined. Despite its in-depth molecular characterization as a proteinase inhibitor, the physiological function in vivo of A2M has yet to be determined. That A2M is part of a defense system in the body is supported only by circumstantial evidence (James 1980; Van Leuven 1982; Sottrup-Jensen 1989). It is suggested by the fact that A2M is the only known plasma inhibitor of the four different classes of proteinases originating from endogenous sources as well as from parasites and pathogens. Also, A2Ms are "acute phase" proteins in some species, such as the rat (Gauthier and Mouray 1976). Furthermore, no quantitative or qualitative deficiencies have been noted in humans. In an effort to elucidate the physiology of A2M in vivo, we are characterizing the family of the macroglobulin and murinoglobulin proteinase inhibitors in the mouse. Cloning of cDNA and of the corresponding genes will eventually result in the production of transgenic mice. The results reported here are part of this effort. Structurally, proteinase inhibitors of the eL macroglobulin family (AM) are characterized by a subunit structure containing about 1500 amino acids, with three specific structural features: a bait region, an internal thioester, and a receptor-binding domain (Van Leuven 1982; Sottrup-Jensen 1989). The bait region is the actual sequence of about 60 amino acids, located in the middle of the subunit, which is typical for each type of A2M. Its sequence determines the specificity of possible proteolytic activation and thereby determines the proteinases that can be bound by each A2M type. We chose to include the bait region because it represents the most specific sequence that distinOffprint requests to: F. Van Leuven
guishes it from other members of the murinoglobulin family (Overbergh et al. 1991a,b). The 1.7-kb probe used for the isotopic in situ hybridization was generated by means of the polymerase chain reaction (PCR) essentially as described previously (Hilliker et al. 1991). The PCR product encompasses position 1741 in exon 14 to position 3517 in exon 27 of mouse A2M cDNA, including the bait region (Overbergh et al. 1991a; Overbergh and Van Leuven 1991; Van Leuven and Overbergh 1991). The nonisotopic in situ hybridization was performed with a yeast artificial chromosome (YAC) clone of 245 kb, which contains the entire A 2 M gene. The YAC clone was obtained by screening the Princeton Mouse YAC Library by PCR with specific primers. The sequences of the primers used to generate the isotropic probe are 5' TCA GCA CAG AGC CTG CCA 3'(sense strand) and 5'GCC AGA CTG TTG GAA ACA GCC 3'(anti-sense). Those used for PCR of the YAC screening were 5' AGG CTG CAA GAC CAG CCT 3'(sense strand) and 5' TGT CTT GTT CCA CCG CGA AGG AGA 3' (anti-sense). Preparation and trypsin-Giemsa banding of chromosomes was performed as described by Davisson and Akeson (1987), with minor modifications for obtaining mitotic chromosomes from bone marrow in vivo. The localization of the A 2 M gene by isotopic in situ hybridization with a PCR-generated probe and the nonisotopic hybridization with a YAC clone were performed on metaphase chromosomes from both the NMRI and Robertsonian translocation Rb(6;15)lAld strains (The Jackson Laboratory, Bar Harbor, Me.). Isotopic labeling and the subsequent hybridization were performed according to the procedure of Harper and Saunders (1981) with the staining procedure described by Cannizzarro and Emanuel (1984). Slides were developed after 5-10 days of exposure. The biotinylation of the YAC probe by nick translation and the nonisotopic hybridization to Robertsonian translo-
470
C. Hilliker et al.: The mouse c~-2-macroglobulin gene
cation metaphases was performed according to the procedure of Wada and co-workers (1990). A partial histogram summarizing the results of the isotopic in situ analysis to NMRI and Rb(6; 15) 1Aid chromosomes is presented in Fig. 1 (a,b). Analysis of 85 metaphases from the NMRI strain yielded 173 grains (approximately 2.0 grains/metaphase). Significant hybridization was found on Chromosome (Chr) 6 (34/173 grains or 19.6%), with grains randomly distributed over the other chromosomes. For the Robertsonian translocation Rb(6;15)lAld, 75 metaphases were scored for 162 grains (approximately 2.2 grains/metaphase). Again the hybridization signal was predominantly localized on Chr 6 (35/162 grains or 21%), with the rest of the grains being randomly distributed over all the chromosomes. The assignment to Chr 6 is evidenced by 64.7% (22/34; Fig. la) of the grains localizing to the region 6 F1-G3 in the NMRI strain and 72% (25/35) (Fig. lb) of the grains in the same region for the Robertsonian translocation Rb(6.15)lAid. In addition, fluorescent in situ hybridization (FISH) with the YAC clone, containing the A2M gene, showed hybridization signal in the telomeric region of mouse Chr 6 in the reported area of localization (Fig. 2), with about 85% of the metaphases displaying the yellow fluorescent signal on
'6
oo
O0
00000 000 000000000 0000000001 a
0000 b
Fig. 1 (a-b). Partial histogram (Evans 1990) of NMRI and (a) Robertsonian translocation Rb(6;15)lAld; (b) chromosomes hybridized with the A2M PCR-generated probe.
Fig. 2. Nonisotopic in situ hybridization with the YAC clone containing the A2M gene to Rb(6;15)lAld metaphase spreads.
both chromatids of the two Chr 6 homologs. These results therefore support the localization of the mouse A2M gene to Chr 6 F1-G3. The human A2M gene was originally assigned to human Chr 12 based on the analysis of its segregation in panels of human-rodent somatic cell hybrids (Bell et al. 1985; Kan et al. 1985). Its localization was further refined to the chromosome region 12p12.3-p13.3 by in situ hybridization (Fukushima et al. 1988; Devriendt et al. 1989). A conserved linkage group could be present distally on mouse Chr 6 and on human Chr 12p. To date 11 genes have been mapped to human Chr 12p and mouse Chr 6. The genes involved are (mouse locus in parenthesis) CD4(Ly-4),TNFRI(Tnfr-1),S YBI(SybI ) ,PRH1 (Prp), FGF6 (Fgf-6), GAPD ( Gap d), TPI1 (Tp i1),LDHB(Ldh-2), PTHLH(Pthlh), and KRAS2(Kras-2) (Davisson et al. 1991). Cahilly and George (1985) mapped the cKi-ras proto-oncogene to mouse Chr 6F3-G3 by in situ hybridization, de Lapeyriere and colleagues (1990) also used in situ hybridization to map the Fgf-6 gene to the region 6F1-G3. Both genes map to the same region, to which we have mapped the mouse A2M gene. We preferred to use in situ hybridization as the method for mapping the mouse A2M gene because a gene located near the end of a chromosome would be especially prone to being separated from the rest of its linkage group in a translocation, which might occur in a somatic cell hybrid, that could be cytologically undetectable. Comparison of the cDNA sequences of complement components and of A2M strongly suggest an evolutionary relationship (Sottrup-Jensen et al., 1985). Human and mouse A2M, C3, and C4 contain domains with high sequence identity, with C3 and C4 more related to each other than to A2M. Members of this superfamily have in common the presence of an internal thiol ester and the mechanism of action. Activation of the thioester occurs by proteotysis at the bait region in
C. Hilliker et al.: The mouse a-2-macroglobulin gene
A2M or at the equivalently positioned arginine residue in complement components, allowing for a major conformational change, for an eventual covalent reaction at the thioester and for the exposure of a receptor recognition site. The chromosomal localization for the complement components C3, C4, and C5 are on three different chromosomes in humans: C3 on 19p13.3-13.2 (Whitehead et al. 1982; Davies et al. 1983), C4 on 6p21.3 (Carroll et al. 1984), and C5 on 9q22-q33 (Jeremiah et al. 1988). In the mouse, C5 is on Chr 2 (D'Eustachio et al. 1986), while C3 and C4 are located on Chr 17 (da Silva et al. 1978; White et al. 1984). These positions are different from the one identified here for the mouse A 2 M gene. It remains to be shown that, as in humans, other members of the A2M family of proteinase inhibitors are clustered on mouse Chr 6. This work is currently in progress, both by in situ hybridization and by analyzing the mouse genomic YAC clone.
Acknowledgments. The authors wish to thank M.T. Davisson and
E. Akeson of The Jackson Laboratory for help with the karyotype. These investigations have been supported by grant 3.0069.89 from the National Science Foundation of Belgium, by a grant "Geconcerteerde Acties" from the Belgian Government, by the "Interuniversity Network" for Fundamental Research sponsored by the Belgian Government (1987-1991), by the NFWO-Levenslijn Fund, and by the VLAB program for Biotechnology.
References Bell, G.I., Rail, L.B., Sanchez-Pescador, R., Merryweather, J.P., Scott, J., Eddy, R.L., and Shows, T.B.: Human alpha-2macroglobulin gene is located on chromosome 12. Somatic Cell Mol Genet 11: 285-289, 1985. Cahilly, L.A. and George, D.L.: Regional mapping of cKi-ras protooncogene on mouse chromosome 6 by in situ hybridization. Cytogenet Cell Genet 39: 140-144, 1985. Cannizzarro, L.A. and Emanuel, B.S.: An improved method for G-banding chromosomes after in situ hybridization. Cytogenet Cell Genet 38: 308-309, 1984. Carroll, M.C., Campbell, R.D., Bentley, D.R., and Porter, R.R.: A molecular map of the human major histocompatibility complex class III region linking complement genes C4, C2 and factor B. Nature 307: 237-241, 1984. da Silva, F.P., Hoecker, G.F., Day, N.K., Vienne, K., and Rubinstein, P.: Murine complement component 3: genetic variation and linkage to H-2. Proc Natl Acad Sei USA 75: 963-965, 1978. Davies, K.E., Jackson, J., Williamson, R., Harper, P.S., Ball, S., Sarfarazi, M., Meridith, L., and Fey, G.: Linkage analysis of myotonic dystrophy and sequences on chromosome 19 using a cloned complement 3 gene probe. J Med Genet 20: 259-263, 1983. Davisson, M.T. and Akeson, E.C.: An improved method for preparing G-banded chromosomes from mouse peripheral blood. Cytogenet Cell Genet 45: 70-74, 1987. Davisson, M.T., Lalley, P.A., Peters, J., Doolittle, D.P., Hillyard, A.L., and Searle, A.G.: Report of the comparative committee for human, mouse and other rodents. Cytogenet Cell Genet 58:11521189, 1991. de Lapeyriere, O., Rosnet, O., Benharroch, D., Raybaund, F., Marchetto, S., Planche, J., Galland, F., Mattei, M.-G., Copeland, N., Jenkins, N., Coulier, F., and Birnbaum, D.: Structure, chromosome mapping and expression in the murine Fgf-6 gene. Oncogene 5: 823-831, 1990.
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