Hum. Genet. 33, 109--112 (1976) © by Springer-Verlag 1976
Regional Mapping of Human Chromosome 10: Assignment of the Gene for Cytoplasraic Glutamate-Oxaloacetate Transaminase to 10q24 -~ qter B. Hellkuhl and K.-tt. Grzeschik Institut ffir Humangenetik der Universit~t Mfinster/Westf. Received December 1, 1975
Summary. Segregation analysis of human-mouse somatic cell hybrids involving fibroblasts of a 10/17 translocation carrier as human partner allowed to assign the gene for human soluble GOT to 10q24 -~qter.
Introduction H u m a n gene m a p p i n g b y the use of h u m a n - r o d e n t somatic cell hybrids has proven to be at present the most powerful m e t h o d on the w a y t o w a r d a detailed gene m a p of man. B e y o n d the assignment of genes to a particular chromosome it allows chromosome submapping if h u m a n diploid cells carrying a reciprocal translocation are used as h u m a n fusion partners (Grzeschik, 1973). Genes coding for h u m a n cytoplasmic glntamate-oxaloacetate transaminase (GOT-S, EC 2.6.1.1) and hexokinase (HK, EC 2.7.1.1) have been assigned to chromosome 10 (Creagan et al., 1973; Shows, 1973; MeA1pine et al., 1974). We now localize one of these genes to a particular region of this chromosome based on simultaneous chromosome analysis and enzyme electrophoresis on cell populations of human-mouse hybrid lines in which part of a broken chromosome 10 was present. Material and Methods Cell Culture. The hybrids were produced by fusion of mouse RAG cells, deficient in hypoxanthine-guanine-phosphoribosyltransferase (Klebe et al., 1970) and diploid fibroblasts of a translocation carrier (G-M216, 46X¥, t(10; 17)(q24;p13) (Fig. 1), obtained from the Human Genetic Mutant Cell Repository, Institute for Medical l~esearch, Camden, New Jersey). Selection of hybrid colonies and processing of hybrid clones for segregation analysis have been described previously (Grzeschik, 1973). Chromosome Analysis. ttuman chromosomes in hybrid metaphases as a rule were identified using the Q-banding technique (Caspersson eta]., 1970), since we consider this method to be more consistent and powerful than G-banding. At least 30 metaphases of each hybrid clone were analyzed for the presence of chromosomes 10, 17, 10q--, and 17p-~-.
110
B. Hellkuhl and K.-H. Grzeschik t (10;17) (q24;p13)
10
lOq-
17
17p*
Fig. 1. Diagrammatic representation of 10/17 chromosome transloeation in GM 216 fibroblasts. Breaks presumably occurred in 10@4 and 17p13
Electrophoresis. The electrophoretie separation and identification of GOT from cell lysates was achieved on Cellogel (Chemetron, Milan). Conditions of electrophoresis: citrate-PO~ buffer 0.01M, ptI 7.0, 2 h, 200 V, room temperature. Reaction mixture: tris-HC1 buffer 0.5M, pit 7.8, 2 ml; a-ketoglutarate 15 mg/ml tI~O, pH 7.0, 0.2 ml; pyridoxal-5-phosphate, 0.1 mg/ml H20, 0.3 ml; fast blue-BB salt 4rag; modified from K6mpf et al. (1971). Results and Discussion I n the electrophoretic system used here the human cytoplasmic GOT could be separated from the enzyme of mouse, Syrian, and Chinese hamster (Fig. 2a). I n human-mouse somatic cell hybrids two types of patterns were observed: A single b a n d - - m o s t probably the mouse b a n d - - a n d a three bands pattern conraining a heteropolymeric band in between both parental bands (Fig. 2b). The heteropolymeric band migrated in the middle between the human and mouse band and not, as observed by Shows (1973) on starch gel, closer to the mouse band. Since the translocation involved a human chromosome 17, a thymidine kinasedeficient animal cell line could have been used for fusion. However, as selection for the presence of human T K could also have favored the selection of hybrids containing fragments or rearrangement products of the 17p@ chromosome, selective pressure on chromosome 17 was avoided. Out of three primary hybrid colonies two contained both pieces of chromosome 10 in addition to an intact No. 10. From one of these cultures 30 subclones were isolated and scored for the presence of human chromosomes 10, 17, 17p~-, and 10q-- (Fig. 1).
Human GOT-S to 10q24 ->qter
111
Fig. 2a and b. Electropherogram of soluble GOT from cultured cells on Cellogel. (a) 1 Chinese hamster Wg 3-h. 2 Syrian hamster TG-2.3 Mouse A9. d Human AM. (b) 1 Mouse A9.2 Human AM. 3 - - 6 Human-mouse somatic cell hybrids containing no part of chromosome i0 (3 and 5) or no part of chromosome 10 but q24 ~qter (4 and 6) Table 1. Segregation of human chromosomes 10, 10q--, 17p-~, and of human GOT-S in secondary hybrid clones :No. of
10
10q--
17p-~
GOT-S
+ __ --
± -L --
~: -F ÷
÷ -~ ÷
clones 14 11 2
Since we did not succeed in separating h u m a n H K clearly from the mouse H K on starch gel and Cellogel we could not localize its gone to one of the parts of chromosome 10. Correlation data on the presence in the hybrids of GOT-S, chromosome 10, and both chromosomal fragments are given in Table 1. The m a j o r i t y of the clones retained art intact chromosome No. 10 in addition to one or the other translocation products or b o t h the 1 0 q - - and 17p~- together. However, the segregation patterns in the clones retaining only 1 0 q - - or 17p~showed a clear correlation of the presence of the 17p~- chromosome and GOT-S. Therefore we can assign the gone for h u m a n GOT-S to the distal p a r t (10q24 -~ qter) of the long arm of chromosome 10. These results were briefly reported at the Baltimore Gone Mapping Conference (i975). T h e y are corroborated b y data
112
B. Hellkuhl and K.-H. Grzeschik
of Chern et al. (1975) p r e s e n t e d a t t h e same meeting. Using h u m a n cells c a r r y i n g t h e s a m e translocation, these a u t h o r s p r o d u c e d h y b r i d s w i t h a T K - n e g a t i v e mouse cell line. The segregation d a t a t h e y o b t a i n e d agree w i t h our conclusion. Supported by the Deutsche Forschungsgemeinschaft, GR 373/6.
References Caspersson, T., Zech, L., Johansson, C., Modest, E. J. : Identification of human chromosomes by DNA-binding fluorescent agents. Chromosoma (Berl.) 30, 215--227 (1970) Chern, C. J., Mellman, W. J., Croce, C. M. : Assignment of the gene for cytoplasmic glutamicoxaloacetic transaminase to the regions q24--qter of human chromosome 10. Human Gene Mapping Conference, Baltimore, Md. (1975) Creagan, R., Tischfield, J., McMorris, F. A., Chen, S., Hirschi, M., Chen, T. R., Ricciuti, F., Ruddle, F. H. : Assignment of the genes for human peptidase A to chromosome 18 and cytoplasmic glutamic oxaloacetate transaminase to chromosome 10 using somatic cell hybrids. Cytogenet. Cell Genet. 12, 187 (1973) Grzeschik, K.-H. : Syrian hamster-human somatic cell hybrids: isolaiion and characterization. Humangenetik 20, 211--218 (1973) Klebe, R. J., Chen, T. R., Ruddle, F. H. : Controlled production of proliferating somatic cell hybrids. J. Cell Biol. 45, 74--78 (1970) KSmpf, J., Ritter, H., Schmitt, J.: Transspecific variability of glutamie oxaloacetic transaminase (EC 2.6.1.1) in primates. Humangenetik 13, 72--74 (1971) McAlpine, P. J., Mohandas, T., Hamerton, J. L.: Isozyme analysis of somatic cell hybrids: assignment of the phosphoglucomutase 2 (PGM2) gene locus to chromosome 4 in man with data on the molecular structure and human chromosome assignments of six additional markers. Proc. 3rd Int. Conf. Isozymes, :New Haven, April, 1974 Shows, T. B. : Synteny of human genes for glutamic oxaloaeetic transaminase and hexokinase in somatic cell hybrids. Cytogenet. Cell Genet. 13, 143--/45 (1974) B. Hellkuhl K.-H. Grzeschik Institut fiir Humangenetik der Universit~t Vesaliusweg 12--14 D-4400 M/inster Federal Republic of Germany
Regional Mapping of Human Chromosome 10: Assignment of the Gene for Cytoplasraic Glutamate-Oxaloacetate Transaminase to 10q24 -~ qter B. Hellkuhl and K.-tt. Grzeschik Institut ffir Humangenetik der Universit~t Mfinster/Westf. Received December 1, 1975
Summary. Segregation analysis of human-mouse somatic cell hybrids involving fibroblasts of a 10/17 translocation carrier as human partner allowed to assign the gene for human soluble GOT to 10q24 -~qter.
Introduction H u m a n gene m a p p i n g b y the use of h u m a n - r o d e n t somatic cell hybrids has proven to be at present the most powerful m e t h o d on the w a y t o w a r d a detailed gene m a p of man. B e y o n d the assignment of genes to a particular chromosome it allows chromosome submapping if h u m a n diploid cells carrying a reciprocal translocation are used as h u m a n fusion partners (Grzeschik, 1973). Genes coding for h u m a n cytoplasmic glntamate-oxaloacetate transaminase (GOT-S, EC 2.6.1.1) and hexokinase (HK, EC 2.7.1.1) have been assigned to chromosome 10 (Creagan et al., 1973; Shows, 1973; MeA1pine et al., 1974). We now localize one of these genes to a particular region of this chromosome based on simultaneous chromosome analysis and enzyme electrophoresis on cell populations of human-mouse hybrid lines in which part of a broken chromosome 10 was present. Material and Methods Cell Culture. The hybrids were produced by fusion of mouse RAG cells, deficient in hypoxanthine-guanine-phosphoribosyltransferase (Klebe et al., 1970) and diploid fibroblasts of a translocation carrier (G-M216, 46X¥, t(10; 17)(q24;p13) (Fig. 1), obtained from the Human Genetic Mutant Cell Repository, Institute for Medical l~esearch, Camden, New Jersey). Selection of hybrid colonies and processing of hybrid clones for segregation analysis have been described previously (Grzeschik, 1973). Chromosome Analysis. ttuman chromosomes in hybrid metaphases as a rule were identified using the Q-banding technique (Caspersson eta]., 1970), since we consider this method to be more consistent and powerful than G-banding. At least 30 metaphases of each hybrid clone were analyzed for the presence of chromosomes 10, 17, 10q--, and 17p-~-.
110
B. Hellkuhl and K.-H. Grzeschik t (10;17) (q24;p13)
10
lOq-
17
17p*
Fig. 1. Diagrammatic representation of 10/17 chromosome transloeation in GM 216 fibroblasts. Breaks presumably occurred in 10@4 and 17p13
Electrophoresis. The electrophoretie separation and identification of GOT from cell lysates was achieved on Cellogel (Chemetron, Milan). Conditions of electrophoresis: citrate-PO~ buffer 0.01M, ptI 7.0, 2 h, 200 V, room temperature. Reaction mixture: tris-HC1 buffer 0.5M, pit 7.8, 2 ml; a-ketoglutarate 15 mg/ml tI~O, pH 7.0, 0.2 ml; pyridoxal-5-phosphate, 0.1 mg/ml H20, 0.3 ml; fast blue-BB salt 4rag; modified from K6mpf et al. (1971). Results and Discussion I n the electrophoretic system used here the human cytoplasmic GOT could be separated from the enzyme of mouse, Syrian, and Chinese hamster (Fig. 2a). I n human-mouse somatic cell hybrids two types of patterns were observed: A single b a n d - - m o s t probably the mouse b a n d - - a n d a three bands pattern conraining a heteropolymeric band in between both parental bands (Fig. 2b). The heteropolymeric band migrated in the middle between the human and mouse band and not, as observed by Shows (1973) on starch gel, closer to the mouse band. Since the translocation involved a human chromosome 17, a thymidine kinasedeficient animal cell line could have been used for fusion. However, as selection for the presence of human T K could also have favored the selection of hybrids containing fragments or rearrangement products of the 17p@ chromosome, selective pressure on chromosome 17 was avoided. Out of three primary hybrid colonies two contained both pieces of chromosome 10 in addition to an intact No. 10. From one of these cultures 30 subclones were isolated and scored for the presence of human chromosomes 10, 17, 17p~-, and 10q-- (Fig. 1).
Human GOT-S to 10q24 ->qter
111
Fig. 2a and b. Electropherogram of soluble GOT from cultured cells on Cellogel. (a) 1 Chinese hamster Wg 3-h. 2 Syrian hamster TG-2.3 Mouse A9. d Human AM. (b) 1 Mouse A9.2 Human AM. 3 - - 6 Human-mouse somatic cell hybrids containing no part of chromosome i0 (3 and 5) or no part of chromosome 10 but q24 ~qter (4 and 6) Table 1. Segregation of human chromosomes 10, 10q--, 17p-~, and of human GOT-S in secondary hybrid clones :No. of
10
10q--
17p-~
GOT-S
+ __ --
± -L --
~: -F ÷
÷ -~ ÷
clones 14 11 2
Since we did not succeed in separating h u m a n H K clearly from the mouse H K on starch gel and Cellogel we could not localize its gone to one of the parts of chromosome 10. Correlation data on the presence in the hybrids of GOT-S, chromosome 10, and both chromosomal fragments are given in Table 1. The m a j o r i t y of the clones retained art intact chromosome No. 10 in addition to one or the other translocation products or b o t h the 1 0 q - - and 17p~- together. However, the segregation patterns in the clones retaining only 1 0 q - - or 17p~showed a clear correlation of the presence of the 17p~- chromosome and GOT-S. Therefore we can assign the gone for h u m a n GOT-S to the distal p a r t (10q24 -~ qter) of the long arm of chromosome 10. These results were briefly reported at the Baltimore Gone Mapping Conference (i975). T h e y are corroborated b y data
112
B. Hellkuhl and K.-H. Grzeschik
of Chern et al. (1975) p r e s e n t e d a t t h e same meeting. Using h u m a n cells c a r r y i n g t h e s a m e translocation, these a u t h o r s p r o d u c e d h y b r i d s w i t h a T K - n e g a t i v e mouse cell line. The segregation d a t a t h e y o b t a i n e d agree w i t h our conclusion. Supported by the Deutsche Forschungsgemeinschaft, GR 373/6.
References Caspersson, T., Zech, L., Johansson, C., Modest, E. J. : Identification of human chromosomes by DNA-binding fluorescent agents. Chromosoma (Berl.) 30, 215--227 (1970) Chern, C. J., Mellman, W. J., Croce, C. M. : Assignment of the gene for cytoplasmic glutamicoxaloacetic transaminase to the regions q24--qter of human chromosome 10. Human Gene Mapping Conference, Baltimore, Md. (1975) Creagan, R., Tischfield, J., McMorris, F. A., Chen, S., Hirschi, M., Chen, T. R., Ricciuti, F., Ruddle, F. H. : Assignment of the genes for human peptidase A to chromosome 18 and cytoplasmic glutamic oxaloacetate transaminase to chromosome 10 using somatic cell hybrids. Cytogenet. Cell Genet. 12, 187 (1973) Grzeschik, K.-H. : Syrian hamster-human somatic cell hybrids: isolaiion and characterization. Humangenetik 20, 211--218 (1973) Klebe, R. J., Chen, T. R., Ruddle, F. H. : Controlled production of proliferating somatic cell hybrids. J. Cell Biol. 45, 74--78 (1970) KSmpf, J., Ritter, H., Schmitt, J.: Transspecific variability of glutamie oxaloacetic transaminase (EC 2.6.1.1) in primates. Humangenetik 13, 72--74 (1971) McAlpine, P. J., Mohandas, T., Hamerton, J. L.: Isozyme analysis of somatic cell hybrids: assignment of the phosphoglucomutase 2 (PGM2) gene locus to chromosome 4 in man with data on the molecular structure and human chromosome assignments of six additional markers. Proc. 3rd Int. Conf. Isozymes, :New Haven, April, 1974 Shows, T. B. : Synteny of human genes for glutamic oxaloaeetic transaminase and hexokinase in somatic cell hybrids. Cytogenet. Cell Genet. 13, 143--/45 (1974) B. Hellkuhl K.-H. Grzeschik Institut fiir Humangenetik der Universit~t Vesaliusweg 12--14 D-4400 M/inster Federal Republic of Germany
