Cytogenet. Cell Genet. 19: 1-6 (1977)
Assignment of the gene for enolase to mouse chromosome 4 using somatic cell hybrids G.G. D'A ncona and C.M. Croce The Wistar Institute of Anatomy and Biology, Philadelphia, Pa.
A bslracl Somatic cell hybrids between mouse peritoneal macrophages (MPM) and human fibrosarcoma cells (HT-1080) or Chinese hamster cells (A3) were analyzed by starchgel electrophoresis for the expression of murine enolase. Data obtained indicate that the locus for enolase is on chromosome 4 in the mouse.
Supported, in part, by USPHS research grants CA-16685 and CA-10815 from the National Cancer Institute, GM-20700 from the National Institute of General Medical Studies, and VC-220 from the American Cancer Society. C.M.C. is the recipient of a Basil O'Connor Starter Grant from The National Foundation—March of Dimes and a Career Development Award (CA-00143) from the National Cancer Institute. Request reprints from: Dr. C.M. C roce, The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, PA 19104 (USA).
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 6:00:18 AM
Somatic cell hybrids have been recently used to determine homologies in the rodent and primate gene maps. The most striking example of such a homology between chromosomes of different species is the syntenic relationship of the genes for glucose-6-phosphate dehydrogenase (G6PD), hypoxanthine phosphoribosyltransferase (HPRT), phosphoglycerate kinase (PGK), «-galactosidase {uGAL), and phospliorylase kinase (PHK) on both the human and the mouse X chromosomes (E pstein , 1969, 1972; H uijing et al., 1973; Kozak, et ah, 1974, 1975; Shows et ah, 1976). Synteny of genes for these isoenzymes has also been established in other mammalian species (see reviews by Warburton and P earson, 1976). The close linkage between the structural genes for the murine isozymes 6-phosphogluconate dehydrogenase (Pgd) and phosphoglucomutase-2 (Pgm-2) on mouse chromosome 4 (Chapman, 1975) is paralleled by the
2
D ’A ncona, C roce
Assignment of ENO to mouse chrom osom e 4
close linkage of the two homologous human genes (6PGD and PGM\) on the short arm of human chromosome 1. Since it has been shown that 6PGD and PGM\ are syntenic with the locus for cnolase 1 (ENO\) on chromosome 1 in the chimpanzee (de G rouchy et al., 1976) and on the short arm of chromosome 1 in man (H amerton, 1976), we have investi gated somatic cell hybrids in which mouse chromosomes segregate to determine whether the structural locus for enolase is also syntenic with Pgd and Pgm-2 in the mouse. For this purpose we have used two types of somatic cell hybrids that segregate mouse chromosomes (C roce et al., 1972; Croce, 1976): (1) hybrids between human fibrosarcoma (HT-1080-6TG) cells and mouse peritoneal macrophages (MPM) (HT-1080 X MPM) and (2) hybrids between Chinese hamster (A3) cells and MPM (A3 X MPM).
Materials and methods Cells. Hybrids between HT-1080-6TG cells and MPM of BALB/c, C57BL/6J, 129, and AKR mouse strains were obtained as described (C roce, 1976). These hybrids were studied for the expression of mouse enolase and the presence of mouse chromosomes. In addition, A3 cells derived from a Chinese hamster cell line deficient in thymidine kinase were fused with MPM from either BALB/c or AKR mice, and hybrids were selected in HAT medium (Littlefield, 1964; C roce et al., 1972). The A3 X MPM hybrids were studied for the expression of mouse enolase and Pgd. Isoenzyme analysis. The procedure for starch-gel electrophoresis and isozyme visualization of enolase and PGD has been described elsewhere (O menn and C ohen , 1971; R uddle and N ichols, 1971). Enolase assays were performed after electro phoresis at 6 V/cm for the hybrids between human and mouse cells and at 3.5 V/cni for the hybrids between Chinese hamster and mouse cells.
The results of the karyologic and enzyme analyses for enolase of the human-mouse hybrids are summarized in table I. Whereas mouse enolase was expressed only in those clones that retained mouse chromosome 4, no enolase activity was detected in clones in which this chromosome was missing (table I and fig. 1). One particular hybrid. 55-54F2, retained at least one copy of all the mouse chromosomes except mouse chromo somes 4 and 11 (M iller et al., 1976), and did not express mouse enolase.
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 6:00:18 AM
Results
D'A ncona, C roce Assignment of UNO to mouse chromosome 4
3
Table /. Concordant segregation of the expression of mouse enolase and the presence of mouse chromosome 4 in mouse-human hybrids segregating mouse chromosomes. Mouse chromosome 4
Mouse enolase
1
2
3
4
5
11
0
0
4
6
7
8
Fig. 1. Two zymograms (lanes 1-4 and lanes 5-8) of enolase on starch gel. Human cells (lane 3); mouse cells (lane 2); HT-1080-6TG X MPM hybrid clone with positive expression of mouse enolase (lane 1); HT-1080-6TG X MPM hybrid clone with negative expression of mouse enolase (lane 4). Mouse and Chinese hamster cells are in lanes 7 and 6, respectively. A hybrid clone with negative expression of mouse enolase is in lane 5. A hybrid clone expressing a heteropolymer of mouse and Chinese hamster enolase is in lane 8.
Since electrophoretic separation of mouse PGD and human 6PGD isoenzymes is not possible, we studied Chinese hamster X mouse cell hybrids in order to establish concordant segregation of the genes for mouse enolase and the Pgd gene that has been assigned to mouse chro mosome 4 (C hapman, 1975). The results of this analysis (table III) indicate that expression of the mouse enolase locus segregates concordantly with the expression of mouse Pgd in 30 independent Chinese hamster-mouse somatic cell hybrids. The zymograms of enolase and 6PGD are shown in figs. 1 and 2. One heteropolymeric band is expressed in those hybrids that retain the gene for mouse enolase and Pgd (fig. 1, channels 1 and 8, and fig. 2, channels 1 and 8).
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 6:00:18 AM
Asynteny between the expression of mouse enolase and galactokinase. which has been assigned to mouse chromosome 11 (M cBrf.pn et al., 1977), was observed in these hybrid clones (table II).
4
D'A ncona, C roce Assignment of ENO to mouse chromosome 4
Table II. Discordant segregation of the expression of mouse enolase and of mouse galactokinase in hybrids segregating mouse chromosomes. Mouse galactokinase 9 15
Mouse enolase
4 7
Table III. Concordant segregation between mouse enolase and 6-phosphogluconate dehydrogenase (Pgd) in mouse-Chinese hamster hybrids segregating mouse chro mosomes. Mouse enolase
+ Mouse PGD
+
0
26
Fig. 2. Zymogram of PGD on starch gel. A3 X MPM hybrid clones expressing mouse PGD and a heteropolymer (lanes 1 and 2); mouse cells (lane 3); Chinese hamster cells (lane 4); A3 X MPM hybrid clones with no expression of mouse PGD (lanes 5 and 6).
The data presented in this report indicate that the structural locus for enolase is on chromosome 4 in the mouse and that the syntenic relation ships of the genes that code for 6PGD, PGM, and ENO have been conserved during evolution. In order to further investigate the homology
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 6:00:18 AM
Discussion
D 'A ncona, C roce
Assignment of ENO to mouse chrom osom e 4
5
between the short arm of human chromosome 1 and mouse chromosome 4, it is of interest to know whether other chromosome markers assigned to the short arm of human chromosome 1, such as adenylate kinase 2 and uridine monophosphate kinase, are also present on mouse chromosome 4.
References
linkage with metabolically related enzyme loci. Biochem. Genet. 13: 844-856 (1975). C roce, C.M.: Loss of mouse chromosomes in somatic cell hybrids between HT-1080 human fibrosarcoma cells and mouse peritoneal macrophages. Proc. natn. Acad. Sci. USA 73: 3248-3252 (1976). C roce, C.M.; Koprow ski , H., and E agle, H.: Effect of environmental pH on the efficiency of cellular hybridization. Proc. natn. Acad. Sci. USA 69: 1953-1956 (1972). G rouchy, 1. d e ; F inaz, C.; C ochet , C.; Van Cong, N.; R ebourcet, R., and F rezal, J.: Chimpanzee gene mapping. In Baltimore Conference (1975): Third international workshop on human gene mapping. Birth Defects: Original Article Series, Vol. 12, No. 7, pp. 416-419 (The National Foundation, New York 1976). E pstein , C.T.: Mammalian oocytes: X chromosome activity. Science 163: 1078-1079 (1969). E pstein , C.T.: Expression of the mammalian X chromosome before and after fertilization. Science 175: 1467-1468 (1972). H amerton, J.L.: Report of the committee on the genetic constitution of chromo somes 1 and 2. In Baltimore C onference (1975): Third international workshop on human gene mapping. Birth Defects: Original Article Scries, Vol. 12, No. 7, pp. 7-23 (The National Foundation, New York 1976). H uijing , F.; E icher , E.M., and C oleman , D.L.: Localization of phosphorylase kinase (Phk) in the mouse X chromosome. Biochem. Genet. 9: 193-196 (1973). Kozak, L.P.: M cL ean, G.K., and E icher, E.M.: X linkage of phosphoglycerate kinase in the mouse. Biochem. Genet. 11: 41-47 (1974). Kozak, C.; N ichols , E.A., and R uddle , E.H.: Gene linkage analysis in the mouse by somatic ceil hybridization: assignment of adenine phosphoribosyltransferase to chromosome 8 and a-galactosidase to the X chromosome. Somat. Cell Genet. 1: 60-70 (1975). L ittlefield , J.W.: Selecting of hybrids from mating of fibroblasts in vitro and their presumed recombinants. Science 145: 709-710 (1964). M c Breen , P.; O rkw iszew ski , K.G.; C hern , C.J.; M ellman, W.J., and C roce, C.M.: Synteny of the genes for thymidine kinase and galactokinase in the mouse and their assignment to mouse chromosome 11. Cvtogenet. Cell Genet. 19: 7-13 (1977). M iller , O.J.; M iller , D.A.; D ev , V.G.; T antravahi, R., and C roce, C.M.: Expres
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 6:00:18 AM
C hapman, V.M.: 6-Phosphogluconate dehydrogenase (PGD) genetics in the mouse:
6
D’A ncona, C roce
Assignment of ENO to mouse chromosome 4
sion of human and suppression of mouse nucleolus organizer activity in mousehuman somatic cell hybrids. Proc. natn. Acad. Sci. USA 73: 4531-4535 (1976). O menn , G.S. and C ohen , P.T.W.: Electrophoretic methods for differentiating gly colytic enzymes of mouse and human origin. In Vitro 7: 132-139 (1971). R uddle , F.H . and N ichols , E.A.: Starch gel electrophoretic phenotypes of mouse X human somatic cell hybrids and mouse isozyme polymorphisms. In Vitro 7: 120-131 (1971). S how s , T.B.; B rown , J.A., and C hapman, V.M.: Comparative gene mapping of HPRT, G6PD. and PGK in man, mouse and muntjac deer. In B altimore C onference (1975): Third international workshop on human gene mapping. Birth Defects: Original Article Series, Vol. 12, No. 7, pp. 436-439 (The National Foundation, New York 1976). W arburton, D. and P earson, P.L.: Report of the committee on comparative map ping. In Baltimore C onference (1975): Third iniernational workshop on human gene mapping. Birth Defects: Original Article Series, Vol. 12, No. 7, pp. 75-82 (The National Foundation, New York 1976).
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 6:00:18 AM
Manuscript received 5 April 1977; accepted for publication 2 May 1977.
Assignment of the gene for enolase to mouse chromosome 4 using somatic cell hybrids G.G. D'A ncona and C.M. Croce The Wistar Institute of Anatomy and Biology, Philadelphia, Pa.
A bslracl Somatic cell hybrids between mouse peritoneal macrophages (MPM) and human fibrosarcoma cells (HT-1080) or Chinese hamster cells (A3) were analyzed by starchgel electrophoresis for the expression of murine enolase. Data obtained indicate that the locus for enolase is on chromosome 4 in the mouse.
Supported, in part, by USPHS research grants CA-16685 and CA-10815 from the National Cancer Institute, GM-20700 from the National Institute of General Medical Studies, and VC-220 from the American Cancer Society. C.M.C. is the recipient of a Basil O'Connor Starter Grant from The National Foundation—March of Dimes and a Career Development Award (CA-00143) from the National Cancer Institute. Request reprints from: Dr. C.M. C roce, The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, PA 19104 (USA).
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 6:00:18 AM
Somatic cell hybrids have been recently used to determine homologies in the rodent and primate gene maps. The most striking example of such a homology between chromosomes of different species is the syntenic relationship of the genes for glucose-6-phosphate dehydrogenase (G6PD), hypoxanthine phosphoribosyltransferase (HPRT), phosphoglycerate kinase (PGK), «-galactosidase {uGAL), and phospliorylase kinase (PHK) on both the human and the mouse X chromosomes (E pstein , 1969, 1972; H uijing et al., 1973; Kozak, et ah, 1974, 1975; Shows et ah, 1976). Synteny of genes for these isoenzymes has also been established in other mammalian species (see reviews by Warburton and P earson, 1976). The close linkage between the structural genes for the murine isozymes 6-phosphogluconate dehydrogenase (Pgd) and phosphoglucomutase-2 (Pgm-2) on mouse chromosome 4 (Chapman, 1975) is paralleled by the
2
D ’A ncona, C roce
Assignment of ENO to mouse chrom osom e 4
close linkage of the two homologous human genes (6PGD and PGM\) on the short arm of human chromosome 1. Since it has been shown that 6PGD and PGM\ are syntenic with the locus for cnolase 1 (ENO\) on chromosome 1 in the chimpanzee (de G rouchy et al., 1976) and on the short arm of chromosome 1 in man (H amerton, 1976), we have investi gated somatic cell hybrids in which mouse chromosomes segregate to determine whether the structural locus for enolase is also syntenic with Pgd and Pgm-2 in the mouse. For this purpose we have used two types of somatic cell hybrids that segregate mouse chromosomes (C roce et al., 1972; Croce, 1976): (1) hybrids between human fibrosarcoma (HT-1080-6TG) cells and mouse peritoneal macrophages (MPM) (HT-1080 X MPM) and (2) hybrids between Chinese hamster (A3) cells and MPM (A3 X MPM).
Materials and methods Cells. Hybrids between HT-1080-6TG cells and MPM of BALB/c, C57BL/6J, 129, and AKR mouse strains were obtained as described (C roce, 1976). These hybrids were studied for the expression of mouse enolase and the presence of mouse chromosomes. In addition, A3 cells derived from a Chinese hamster cell line deficient in thymidine kinase were fused with MPM from either BALB/c or AKR mice, and hybrids were selected in HAT medium (Littlefield, 1964; C roce et al., 1972). The A3 X MPM hybrids were studied for the expression of mouse enolase and Pgd. Isoenzyme analysis. The procedure for starch-gel electrophoresis and isozyme visualization of enolase and PGD has been described elsewhere (O menn and C ohen , 1971; R uddle and N ichols, 1971). Enolase assays were performed after electro phoresis at 6 V/cm for the hybrids between human and mouse cells and at 3.5 V/cni for the hybrids between Chinese hamster and mouse cells.
The results of the karyologic and enzyme analyses for enolase of the human-mouse hybrids are summarized in table I. Whereas mouse enolase was expressed only in those clones that retained mouse chromosome 4, no enolase activity was detected in clones in which this chromosome was missing (table I and fig. 1). One particular hybrid. 55-54F2, retained at least one copy of all the mouse chromosomes except mouse chromo somes 4 and 11 (M iller et al., 1976), and did not express mouse enolase.
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 6:00:18 AM
Results
D'A ncona, C roce Assignment of UNO to mouse chromosome 4
3
Table /. Concordant segregation of the expression of mouse enolase and the presence of mouse chromosome 4 in mouse-human hybrids segregating mouse chromosomes. Mouse chromosome 4
Mouse enolase
1
2
3
4
5
11
0
0
4
6
7
8
Fig. 1. Two zymograms (lanes 1-4 and lanes 5-8) of enolase on starch gel. Human cells (lane 3); mouse cells (lane 2); HT-1080-6TG X MPM hybrid clone with positive expression of mouse enolase (lane 1); HT-1080-6TG X MPM hybrid clone with negative expression of mouse enolase (lane 4). Mouse and Chinese hamster cells are in lanes 7 and 6, respectively. A hybrid clone with negative expression of mouse enolase is in lane 5. A hybrid clone expressing a heteropolymer of mouse and Chinese hamster enolase is in lane 8.
Since electrophoretic separation of mouse PGD and human 6PGD isoenzymes is not possible, we studied Chinese hamster X mouse cell hybrids in order to establish concordant segregation of the genes for mouse enolase and the Pgd gene that has been assigned to mouse chro mosome 4 (C hapman, 1975). The results of this analysis (table III) indicate that expression of the mouse enolase locus segregates concordantly with the expression of mouse Pgd in 30 independent Chinese hamster-mouse somatic cell hybrids. The zymograms of enolase and 6PGD are shown in figs. 1 and 2. One heteropolymeric band is expressed in those hybrids that retain the gene for mouse enolase and Pgd (fig. 1, channels 1 and 8, and fig. 2, channels 1 and 8).
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 6:00:18 AM
Asynteny between the expression of mouse enolase and galactokinase. which has been assigned to mouse chromosome 11 (M cBrf.pn et al., 1977), was observed in these hybrid clones (table II).
4
D'A ncona, C roce Assignment of ENO to mouse chromosome 4
Table II. Discordant segregation of the expression of mouse enolase and of mouse galactokinase in hybrids segregating mouse chromosomes. Mouse galactokinase 9 15
Mouse enolase
4 7
Table III. Concordant segregation between mouse enolase and 6-phosphogluconate dehydrogenase (Pgd) in mouse-Chinese hamster hybrids segregating mouse chro mosomes. Mouse enolase
+ Mouse PGD
+
0
26
Fig. 2. Zymogram of PGD on starch gel. A3 X MPM hybrid clones expressing mouse PGD and a heteropolymer (lanes 1 and 2); mouse cells (lane 3); Chinese hamster cells (lane 4); A3 X MPM hybrid clones with no expression of mouse PGD (lanes 5 and 6).
The data presented in this report indicate that the structural locus for enolase is on chromosome 4 in the mouse and that the syntenic relation ships of the genes that code for 6PGD, PGM, and ENO have been conserved during evolution. In order to further investigate the homology
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 6:00:18 AM
Discussion
D 'A ncona, C roce
Assignment of ENO to mouse chrom osom e 4
5
between the short arm of human chromosome 1 and mouse chromosome 4, it is of interest to know whether other chromosome markers assigned to the short arm of human chromosome 1, such as adenylate kinase 2 and uridine monophosphate kinase, are also present on mouse chromosome 4.
References
linkage with metabolically related enzyme loci. Biochem. Genet. 13: 844-856 (1975). C roce, C.M.: Loss of mouse chromosomes in somatic cell hybrids between HT-1080 human fibrosarcoma cells and mouse peritoneal macrophages. Proc. natn. Acad. Sci. USA 73: 3248-3252 (1976). C roce, C.M.; Koprow ski , H., and E agle, H.: Effect of environmental pH on the efficiency of cellular hybridization. Proc. natn. Acad. Sci. USA 69: 1953-1956 (1972). G rouchy, 1. d e ; F inaz, C.; C ochet , C.; Van Cong, N.; R ebourcet, R., and F rezal, J.: Chimpanzee gene mapping. In Baltimore Conference (1975): Third international workshop on human gene mapping. Birth Defects: Original Article Series, Vol. 12, No. 7, pp. 416-419 (The National Foundation, New York 1976). E pstein , C.T.: Mammalian oocytes: X chromosome activity. Science 163: 1078-1079 (1969). E pstein , C.T.: Expression of the mammalian X chromosome before and after fertilization. Science 175: 1467-1468 (1972). H amerton, J.L.: Report of the committee on the genetic constitution of chromo somes 1 and 2. In Baltimore C onference (1975): Third international workshop on human gene mapping. Birth Defects: Original Article Scries, Vol. 12, No. 7, pp. 7-23 (The National Foundation, New York 1976). H uijing , F.; E icher , E.M., and C oleman , D.L.: Localization of phosphorylase kinase (Phk) in the mouse X chromosome. Biochem. Genet. 9: 193-196 (1973). Kozak, L.P.: M cL ean, G.K., and E icher, E.M.: X linkage of phosphoglycerate kinase in the mouse. Biochem. Genet. 11: 41-47 (1974). Kozak, C.; N ichols , E.A., and R uddle , E.H.: Gene linkage analysis in the mouse by somatic ceil hybridization: assignment of adenine phosphoribosyltransferase to chromosome 8 and a-galactosidase to the X chromosome. Somat. Cell Genet. 1: 60-70 (1975). L ittlefield , J.W.: Selecting of hybrids from mating of fibroblasts in vitro and their presumed recombinants. Science 145: 709-710 (1964). M c Breen , P.; O rkw iszew ski , K.G.; C hern , C.J.; M ellman, W.J., and C roce, C.M.: Synteny of the genes for thymidine kinase and galactokinase in the mouse and their assignment to mouse chromosome 11. Cvtogenet. Cell Genet. 19: 7-13 (1977). M iller , O.J.; M iller , D.A.; D ev , V.G.; T antravahi, R., and C roce, C.M.: Expres
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 6:00:18 AM
C hapman, V.M.: 6-Phosphogluconate dehydrogenase (PGD) genetics in the mouse:
6
D’A ncona, C roce
Assignment of ENO to mouse chromosome 4
sion of human and suppression of mouse nucleolus organizer activity in mousehuman somatic cell hybrids. Proc. natn. Acad. Sci. USA 73: 4531-4535 (1976). O menn , G.S. and C ohen , P.T.W.: Electrophoretic methods for differentiating gly colytic enzymes of mouse and human origin. In Vitro 7: 132-139 (1971). R uddle , F.H . and N ichols , E.A.: Starch gel electrophoretic phenotypes of mouse X human somatic cell hybrids and mouse isozyme polymorphisms. In Vitro 7: 120-131 (1971). S how s , T.B.; B rown , J.A., and C hapman, V.M.: Comparative gene mapping of HPRT, G6PD. and PGK in man, mouse and muntjac deer. In B altimore C onference (1975): Third international workshop on human gene mapping. Birth Defects: Original Article Series, Vol. 12, No. 7, pp. 436-439 (The National Foundation, New York 1976). W arburton, D. and P earson, P.L.: Report of the committee on comparative map ping. In Baltimore C onference (1975): Third iniernational workshop on human gene mapping. Birth Defects: Original Article Series, Vol. 12, No. 7, pp. 75-82 (The National Foundation, New York 1976).
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 6:00:18 AM
Manuscript received 5 April 1977; accepted for publication 2 May 1977.
