Mammalian Genome 2: 135-137, 1992
9 Springer-VerlagNew York Inc. 1992
Chromosomal localizations of mouse Fgf2 and
Fgf5 genes
Marie-Genevieve Mattei, 1 Marie-Jos~phe P6busque, 2 and Daniel Birnbaum 2 1U.242 INSERM, H6pital d'Enfants de La Timone, 5 Bd. J. Moulin, 13385 Marseille; zU.119 INSERM, 27 Bd. Le~ Roure, 13009 Marseille, France Received June 3, 1991; accepted July 24, 1991
Abstract. The mouse Fgf2 and Fgf5 genes, two mem-
bers of the FGF family whose human homologs are on the long arm of chromosome (chr) 4, were localized by using in situ chromosomal hybridization, to mouse Chrs 3 and 5, respectively. The FGF gene family (historically, Fibroblast Growth Factor) presently comprises seven members: FGF1 to FGF7 (formerly known as oLFGF, ~FGF, INT2, HST/ KFGF, FGF5, FGF6 and KGF, respectively). These genes have sequence identities in the range of 35-70%, and encode factors presenting various activities. FGFs are involved in cell proliferation, angiogenesis, embryogenesis and possibly oncogenesis (for review see Goldfarb 1990). In human, the chromosomal localization is known for six of the FGF genes (Table 1). They are localized on different chromosomes, except for two pairs of genes: FGF3/INT2 and FGF4/HST, that are colocalized on chr 11, in band q13 and separated by less than 40 kb (Adelaide et al. 1988; Casey et al. 1986; Nguyen et al. 1988), and FGF2 and FGF5, that are both localized on chr 4, but on different bands. In mouse, Fgf3/Int2 and Fgf4/Hst are also closely linked (Peters et al. 1989) and are on Chr 7. Both genes can be activated by the nearby integration of the mouse mammary tumor provirus in some virally-induced mammary tumors (Dickson et al. 1984; Peters et al. 1989). The murine Fgf6 gene is located on Chr 6 (deLapeyriere et al. 1990). The close linkage (35 kb in human and 17 kb in mouse) of FGF3 and FGF4 is a feature of the FGF family. It may be of functional significance or indicate a particular scheme of evolution. It is of interest to determine whether a similar organization exists for other pairs of genes. With this question in mind, we previously determined the precise localization of hu-
Offprint requests to: D. Birnbaum
man FGF2 and FGF5 on chr 4 (Lafage et al. 1990; Nguyen et al. 1988). We now wanted to determine, in case Fgf2 and Fgf5 were also syntenic in mouse, whether they might be more closely linked than they are in human. We report here that Fgf2 and Fgf5 are not localized on the same chromosome. In situ hybridizations were used to map the two FGF genes. The probe for murine Fgf2 was derived from cosmid clone cos bF1. This cosmid was isolated from a mouse cosmid library screened with a human FGF2 probe (Prats et al. 1989). A 0.55 kb Alu I fragment of cosmid cos bF1, containing sequences from the third exon of Fgf2 (not shown), was subcloned into a Bluescript (Stratagene) vector to give the pbFAlu subclone. The probe for murine Fgf5 was pmFGF-5, a 2 kb EcoR I cDNA, subcloned into a Bluescript vector (Haub et al. 1990). In situ hybridizations were performed according to previously described procedures (Mattei et al. 1985; deLapeyriere et al. 1990). Briefly, experiments were carried out on metaphase spreads of concanavalin-A stimulated lymphocytes from WMP male mice, in which all the autosomes except 19 were in the form of metacentric Robertsonian translocations. The use of Robertsonian chromosomes facilitates the identification of individual mouse chromosomes. Plasmids pbFAlu and pmFGF-5 were tritium-labeled by nicktranslation to respective specific activities of 1 • 108 and 1.2 x 108 d.p.m, ixg- 1. They were hybridized at a probe concentration of 25 ng/ml. The result of the in situ hybridization using the Fgf2 probe pbFAlu is shown in Fig. 1A. In the 100 metaphase cells examined after in situ hybridization, 172 silver grains were associated with chromosomes, and 65 of these (37.7%) were located on Chr 3. The distribution of the grains was not random: 85% of them mapped in the A2-B region of Chr 3. This result allowed us to map the pbFAlu probe, and consequently the Fgf2/[~Fgf gene, to the 3A2-3B region of the murine genome. The result of the in situ hybridization using the Fgf5 probe is shown in Fig. lB. In the 100
136
M-G. Mattei et al.: Localizations of Fgf2 and Fgt5
Table 1. Chromosomal localization of genes of the FGF family in human and mouse. Gene
Chromosomal Localization in Human Mouse
FGF1 FGF2
5q31.3--33.2 4q26-27
18 3 A2-B
FGF3 FGF4 FGF5 FGF6
llq13 11q13.3 4q21 12p13
7 7 5 6
FGF7
?
?
Reference Jaye et al. 1986; Cox et al. 1991 Lafage-Pochitaloff et al. 1990; Cox et al. 1991; this work Casey et al. 1986; Peters et al. 1984 Adelaide et al. 1988; Peters et al. 1990 Nguyen et al. 1988; this work Marics et al. 1989; deLapeyriere et al. 1990
F F E1-F F3-G1
metaphase cells examined, there were 346 silver grains associated with chromosomes, and 70 of these (20%) were located on Chr 5. The distribution was not random: 57 out 70 grains (81.4%) mapped to the E1-F region of Chr 5. This result allowed us to map the Fgf5 gene to the chromosomal region 5E1-5F. In mouse, the Fgf2 and Fgf5 genes are not syntenic. The paralogous genes are both localized on the same chromosome in human, namely chr 4, but on
different bands. In both species the two genes are therefore separated. If Fgf2 and Fgf5 ever happened to be closely linked, their separation took place more than 80 million years ago. Thus, in mouse too, the tandem linkage of Fgf3 and Fgf4 remains unique among the F g f genes. Mouse Chrs 3 and 5 host several genes having homologous genes on human chr 4 (Davisson et al. 1990). The mouse Fgf2 and Fgf5 genes indeed belong to con-
A
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9
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Fig. 1. (A) Localization of murine Fgf2 to mouse Chr 3 by in situ hybridization. Left: two partial WMP mouse metaphases, showing the specific site of hybridization to Chr 3; top: arrowheads indicate silver grains on Giemsa-stained chromosomes after autoradiography; bottom: chromosomes with silver grains were subsequently identified by R-banding. Right: diagram of WMP mouse Rb(3 ;12) chromosome, indicating the distribution of labeled sites on Chr 3. (B) Localization of murine FgJ5 to mouse Chr 5 by in situ hybridization. Left: two partial WMP mouse metaphases, showing the specific site of hybridization to Chr 5 (top and bottom: same as in (A). R-banding allows to distinguish unambiguously between Chr 3 and Chr 5; this latter is heavily positive in its one-third terminal portion. Right: diagram of WMP mouse Rb(5;14) chromosome, indicating the distribution of labeled sites.
M-G. Mattei et al.: Localizations of Fgf2 and Fgf5
served syntenic groups (Nadeau 1989) located on the long arm of human chr 4 (Lyon and Kirby 1990). The localizations described here will help in the comparative mapping of the involved chromosomes. Acknowledgments. This work was supported by INSERM. We
thank Mitchell Goldfarb (Columbia University, New York, N.Y.) for the gift of the murine Fgf5 probe and Victor Pirisi and Nathalie Roeckel for technical help.
References Adelaide, J., Mattei', M.G., Marics, I., Raybaud, F., Planche, J., deLapeyriere, O., and Birnbaum, D.: Chromosomal localization of the hst oncogene and its co-amplification with the int.2 oncogene in a human melanoma. Oncogene 2: 413--416, 1988. Casey, G., Smith, R., McGillivray, D., Peters, G., and Dickson, C.: Characterization and chromosome assignment of the human homolog of int-2, a potential proto-oncogene. Mol Cell Biol 6: 502510, 1986. Cox, R., Copeland, N., Jenkins, N., and Lehrach, H.: Interspersed repetitive element polymerase chain reaction product mapping using a mouse interspecific backcross. Genomics 10: 375-384, 1991. Davisson, M., LaUey, P., Peters, J., Doolittle, D., Hillyard, A., and Searle, A.: Report of the comparative subcommittee for human and mouse homologies. Cytogenet Cell Genet 55: 434-456, 1990. deLapeyriere, O., Rosnet, O., Benharroch, D., Raybaud, F., Marchetto, S., Planche, J., Galland, F., Mattei, M.G., Copeland, N., Jenkins, N., Coulier, F., and Birnbaum, D.: Structure, chromosome mapping and expression of the murine Fgf-6 gene. Oncogene 5: 823-831, 1990. Dickson, C., Smith, R., Brookes, S., and Peters, G.: Tumorigenesis by mouse mammary tumor virus: Provira! activation of a cellular gene in the common integration region int-2. Cell 37: 52%536, 1984. Goldfarb, M.: The fibroblast growth factor family. Cell Growth Differ 1: 439-445, 1990.
137 Haub, O., Drucker, B., and Goldfarb, M.: Expression of the murine fibroblast growth factor 5 gene in the adult central nervous system. Proc Natl Acad Sci USA 87: 8022-8026, 1990. Jaye, M., Howk, R., Burgess, W., Ricca, G., Chiu, I.M., Ravera, M., O'Brien, S., Modi, W., Maciag, T., and Drohan, W.: Human endothelial cell growth factor: Cloning, nucleotide sequence, and chromosome localization. Science 233: 541-544, 1986. Lafage-Pochitaloff, M., Galland, F., Simonetti, J., Prats, H., Matt e l M.G., and Birnbaum, D.: The human basic fibroblast growth factor gene is located on the long arm of chromsome 4 at bands q26-q27. Oncogene Res 5: 241-244, 1990. Lyon, M. and Kirby, M.: Mouse chromosome atlas. Mouse News Lett 87: 31-33, 1990. Marics, I., Adelaide, J., Raybaud, F., Mattei', M.G., Coulier, F., Planche, J., deLapeyriere, O., and Birnbaum, D.: Characterization of the HST-related FGF.6 gene, a new member of the fibroblast growth factor gene family. Oncogene 4: 335-340, 1989. Mattei, M.G., Philip, N., Passage, E., Moisan, J.P., Mandel, J.L., and MatteL J.F.: DNA probe localization at 18p113 band by in situ hybridization and identification of a small supernumerary chromosome. Human Genet 69: 268-271, 1985. Nadeau, J.: Maps of linkage and synteny homologies between mouse and man. Trends Genet 5: 82-86, 1989. Nguyen, C., Roux, D., MatteL M.G., deLapeyriere, O., Goldfarb, M., Birnbaum, D., and Jordan, B.: The FGF-related oncogenes hst and int.2, and the bcl.1 locus are contained within one megabase in band q13 of chromosome 11, while the fgf.5 oncogene maps to 4q21. Oncogene 3: 703-708, 1988. Peters, G., Brookes, S., Smith, R., Placzek, M., and Dickson, C.: The mouse homolog of the hst/k-FGF gene is adjacent to int-2 and is activated by proviral insertion in some virally induced mammary tumors. Proc Natl Acad Sci USA 86: 5678-5682, 1989. Peters, G., Kozak, C., and Dickson0 C.: Mouse mammary tumor virus integration regions int-1 and int-2 map on different mouse chromosomes. Mol Cell Biol 4: 375-378, 1984. Prats, H., Kaghad, M., Prats, A.C., Klagsbrun, M., L61ias, J.M., Liauzun, P., Chalon, P., Tauber, J.P., Amalric, F., Smith, J., and Caput, D.: High molecular mass forms of basic fibroblast growth factor are initiated by alternative CUG codons. Proc Natl Acad Sci USA 86: 1836-1840, 1989.
9 Springer-VerlagNew York Inc. 1992
Chromosomal localizations of mouse Fgf2 and
Fgf5 genes
Marie-Genevieve Mattei, 1 Marie-Jos~phe P6busque, 2 and Daniel Birnbaum 2 1U.242 INSERM, H6pital d'Enfants de La Timone, 5 Bd. J. Moulin, 13385 Marseille; zU.119 INSERM, 27 Bd. Le~ Roure, 13009 Marseille, France Received June 3, 1991; accepted July 24, 1991
Abstract. The mouse Fgf2 and Fgf5 genes, two mem-
bers of the FGF family whose human homologs are on the long arm of chromosome (chr) 4, were localized by using in situ chromosomal hybridization, to mouse Chrs 3 and 5, respectively. The FGF gene family (historically, Fibroblast Growth Factor) presently comprises seven members: FGF1 to FGF7 (formerly known as oLFGF, ~FGF, INT2, HST/ KFGF, FGF5, FGF6 and KGF, respectively). These genes have sequence identities in the range of 35-70%, and encode factors presenting various activities. FGFs are involved in cell proliferation, angiogenesis, embryogenesis and possibly oncogenesis (for review see Goldfarb 1990). In human, the chromosomal localization is known for six of the FGF genes (Table 1). They are localized on different chromosomes, except for two pairs of genes: FGF3/INT2 and FGF4/HST, that are colocalized on chr 11, in band q13 and separated by less than 40 kb (Adelaide et al. 1988; Casey et al. 1986; Nguyen et al. 1988), and FGF2 and FGF5, that are both localized on chr 4, but on different bands. In mouse, Fgf3/Int2 and Fgf4/Hst are also closely linked (Peters et al. 1989) and are on Chr 7. Both genes can be activated by the nearby integration of the mouse mammary tumor provirus in some virally-induced mammary tumors (Dickson et al. 1984; Peters et al. 1989). The murine Fgf6 gene is located on Chr 6 (deLapeyriere et al. 1990). The close linkage (35 kb in human and 17 kb in mouse) of FGF3 and FGF4 is a feature of the FGF family. It may be of functional significance or indicate a particular scheme of evolution. It is of interest to determine whether a similar organization exists for other pairs of genes. With this question in mind, we previously determined the precise localization of hu-
Offprint requests to: D. Birnbaum
man FGF2 and FGF5 on chr 4 (Lafage et al. 1990; Nguyen et al. 1988). We now wanted to determine, in case Fgf2 and Fgf5 were also syntenic in mouse, whether they might be more closely linked than they are in human. We report here that Fgf2 and Fgf5 are not localized on the same chromosome. In situ hybridizations were used to map the two FGF genes. The probe for murine Fgf2 was derived from cosmid clone cos bF1. This cosmid was isolated from a mouse cosmid library screened with a human FGF2 probe (Prats et al. 1989). A 0.55 kb Alu I fragment of cosmid cos bF1, containing sequences from the third exon of Fgf2 (not shown), was subcloned into a Bluescript (Stratagene) vector to give the pbFAlu subclone. The probe for murine Fgf5 was pmFGF-5, a 2 kb EcoR I cDNA, subcloned into a Bluescript vector (Haub et al. 1990). In situ hybridizations were performed according to previously described procedures (Mattei et al. 1985; deLapeyriere et al. 1990). Briefly, experiments were carried out on metaphase spreads of concanavalin-A stimulated lymphocytes from WMP male mice, in which all the autosomes except 19 were in the form of metacentric Robertsonian translocations. The use of Robertsonian chromosomes facilitates the identification of individual mouse chromosomes. Plasmids pbFAlu and pmFGF-5 were tritium-labeled by nicktranslation to respective specific activities of 1 • 108 and 1.2 x 108 d.p.m, ixg- 1. They were hybridized at a probe concentration of 25 ng/ml. The result of the in situ hybridization using the Fgf2 probe pbFAlu is shown in Fig. 1A. In the 100 metaphase cells examined after in situ hybridization, 172 silver grains were associated with chromosomes, and 65 of these (37.7%) were located on Chr 3. The distribution of the grains was not random: 85% of them mapped in the A2-B region of Chr 3. This result allowed us to map the pbFAlu probe, and consequently the Fgf2/[~Fgf gene, to the 3A2-3B region of the murine genome. The result of the in situ hybridization using the Fgf5 probe is shown in Fig. lB. In the 100
136
M-G. Mattei et al.: Localizations of Fgf2 and Fgt5
Table 1. Chromosomal localization of genes of the FGF family in human and mouse. Gene
Chromosomal Localization in Human Mouse
FGF1 FGF2
5q31.3--33.2 4q26-27
18 3 A2-B
FGF3 FGF4 FGF5 FGF6
llq13 11q13.3 4q21 12p13
7 7 5 6
FGF7
?
?
Reference Jaye et al. 1986; Cox et al. 1991 Lafage-Pochitaloff et al. 1990; Cox et al. 1991; this work Casey et al. 1986; Peters et al. 1984 Adelaide et al. 1988; Peters et al. 1990 Nguyen et al. 1988; this work Marics et al. 1989; deLapeyriere et al. 1990
F F E1-F F3-G1
metaphase cells examined, there were 346 silver grains associated with chromosomes, and 70 of these (20%) were located on Chr 5. The distribution was not random: 57 out 70 grains (81.4%) mapped to the E1-F region of Chr 5. This result allowed us to map the Fgf5 gene to the chromosomal region 5E1-5F. In mouse, the Fgf2 and Fgf5 genes are not syntenic. The paralogous genes are both localized on the same chromosome in human, namely chr 4, but on
different bands. In both species the two genes are therefore separated. If Fgf2 and Fgf5 ever happened to be closely linked, their separation took place more than 80 million years ago. Thus, in mouse too, the tandem linkage of Fgf3 and Fgf4 remains unique among the F g f genes. Mouse Chrs 3 and 5 host several genes having homologous genes on human chr 4 (Davisson et al. 1990). The mouse Fgf2 and Fgf5 genes indeed belong to con-
A
t
: c...n...-
D
9
G .......
9
............. 2 .....
:
B
l ...............
14 ............... go
|lh..o..
eeeseseeeeeeeeeeeeeseseeeee eeeeeeeeeeeeeeQeeeeee iooo ee
Fig. 1. (A) Localization of murine Fgf2 to mouse Chr 3 by in situ hybridization. Left: two partial WMP mouse metaphases, showing the specific site of hybridization to Chr 3; top: arrowheads indicate silver grains on Giemsa-stained chromosomes after autoradiography; bottom: chromosomes with silver grains were subsequently identified by R-banding. Right: diagram of WMP mouse Rb(3 ;12) chromosome, indicating the distribution of labeled sites on Chr 3. (B) Localization of murine FgJ5 to mouse Chr 5 by in situ hybridization. Left: two partial WMP mouse metaphases, showing the specific site of hybridization to Chr 5 (top and bottom: same as in (A). R-banding allows to distinguish unambiguously between Chr 3 and Chr 5; this latter is heavily positive in its one-third terminal portion. Right: diagram of WMP mouse Rb(5;14) chromosome, indicating the distribution of labeled sites.
M-G. Mattei et al.: Localizations of Fgf2 and Fgf5
served syntenic groups (Nadeau 1989) located on the long arm of human chr 4 (Lyon and Kirby 1990). The localizations described here will help in the comparative mapping of the involved chromosomes. Acknowledgments. This work was supported by INSERM. We
thank Mitchell Goldfarb (Columbia University, New York, N.Y.) for the gift of the murine Fgf5 probe and Victor Pirisi and Nathalie Roeckel for technical help.
References Adelaide, J., Mattei', M.G., Marics, I., Raybaud, F., Planche, J., deLapeyriere, O., and Birnbaum, D.: Chromosomal localization of the hst oncogene and its co-amplification with the int.2 oncogene in a human melanoma. Oncogene 2: 413--416, 1988. Casey, G., Smith, R., McGillivray, D., Peters, G., and Dickson, C.: Characterization and chromosome assignment of the human homolog of int-2, a potential proto-oncogene. Mol Cell Biol 6: 502510, 1986. Cox, R., Copeland, N., Jenkins, N., and Lehrach, H.: Interspersed repetitive element polymerase chain reaction product mapping using a mouse interspecific backcross. Genomics 10: 375-384, 1991. Davisson, M., LaUey, P., Peters, J., Doolittle, D., Hillyard, A., and Searle, A.: Report of the comparative subcommittee for human and mouse homologies. Cytogenet Cell Genet 55: 434-456, 1990. deLapeyriere, O., Rosnet, O., Benharroch, D., Raybaud, F., Marchetto, S., Planche, J., Galland, F., Mattei, M.G., Copeland, N., Jenkins, N., Coulier, F., and Birnbaum, D.: Structure, chromosome mapping and expression of the murine Fgf-6 gene. Oncogene 5: 823-831, 1990. Dickson, C., Smith, R., Brookes, S., and Peters, G.: Tumorigenesis by mouse mammary tumor virus: Provira! activation of a cellular gene in the common integration region int-2. Cell 37: 52%536, 1984. Goldfarb, M.: The fibroblast growth factor family. Cell Growth Differ 1: 439-445, 1990.
137 Haub, O., Drucker, B., and Goldfarb, M.: Expression of the murine fibroblast growth factor 5 gene in the adult central nervous system. Proc Natl Acad Sci USA 87: 8022-8026, 1990. Jaye, M., Howk, R., Burgess, W., Ricca, G., Chiu, I.M., Ravera, M., O'Brien, S., Modi, W., Maciag, T., and Drohan, W.: Human endothelial cell growth factor: Cloning, nucleotide sequence, and chromosome localization. Science 233: 541-544, 1986. Lafage-Pochitaloff, M., Galland, F., Simonetti, J., Prats, H., Matt e l M.G., and Birnbaum, D.: The human basic fibroblast growth factor gene is located on the long arm of chromsome 4 at bands q26-q27. Oncogene Res 5: 241-244, 1990. Lyon, M. and Kirby, M.: Mouse chromosome atlas. Mouse News Lett 87: 31-33, 1990. Marics, I., Adelaide, J., Raybaud, F., Mattei', M.G., Coulier, F., Planche, J., deLapeyriere, O., and Birnbaum, D.: Characterization of the HST-related FGF.6 gene, a new member of the fibroblast growth factor gene family. Oncogene 4: 335-340, 1989. Mattei, M.G., Philip, N., Passage, E., Moisan, J.P., Mandel, J.L., and MatteL J.F.: DNA probe localization at 18p113 band by in situ hybridization and identification of a small supernumerary chromosome. Human Genet 69: 268-271, 1985. Nadeau, J.: Maps of linkage and synteny homologies between mouse and man. Trends Genet 5: 82-86, 1989. Nguyen, C., Roux, D., MatteL M.G., deLapeyriere, O., Goldfarb, M., Birnbaum, D., and Jordan, B.: The FGF-related oncogenes hst and int.2, and the bcl.1 locus are contained within one megabase in band q13 of chromosome 11, while the fgf.5 oncogene maps to 4q21. Oncogene 3: 703-708, 1988. Peters, G., Brookes, S., Smith, R., Placzek, M., and Dickson, C.: The mouse homolog of the hst/k-FGF gene is adjacent to int-2 and is activated by proviral insertion in some virally induced mammary tumors. Proc Natl Acad Sci USA 86: 5678-5682, 1989. Peters, G., Kozak, C., and Dickson0 C.: Mouse mammary tumor virus integration regions int-1 and int-2 map on different mouse chromosomes. Mol Cell Biol 4: 375-378, 1984. Prats, H., Kaghad, M., Prats, A.C., Klagsbrun, M., L61ias, J.M., Liauzun, P., Chalon, P., Tauber, J.P., Amalric, F., Smith, J., and Caput, D.: High molecular mass forms of basic fibroblast growth factor are initiated by alternative CUG codons. Proc Natl Acad Sci USA 86: 1836-1840, 1989.
