Cytogenet. Cell Genet. 19: 44—48 (1977)
BRIEF REPORT
Somatic cell genetic evidence for the presence of a gene for citrullinemia on human chromosome 9 B. C arritt Institute of Genetics, University of Glasgow, Glasgow
In an earlier report (C arritt et al., 1977) we showed that the Chinese hamster cell line Don and its TK derivative a23 (W esti rvi i .d et al., 1971) lack detectable activity of the enzyme argininosuccinate synthetase (ASS) and that in hybrids formed between a23 and HGPRT human fibroblasts, ASS activity segregated concordantly with human chromo some 9 and AKi. It was therefore concluded that chromosome 9 carries a human gene responsible for ASS expression by these hybrids. However, since the parental type of ASS in the hybrids could not be determined, we were unable to distinguish between two possibilities: (1) that human chromosome 9 carries the structural gene for ASS and (2) that human chromosome 9 carries a positive control gene capable of activating a Chinese hamster ASS structural gene. As an approach to this question, I have made use of a human cell line (GM-63) which has an ASS with an altered Km toward citrulline (T edesco and M eeeman , 1967), the effect of which is to depress ASS activity to 20-30 % of wild-type levels under certain conditions of assay ( T edesco and M ellman , 1967, and table I).
Supported by a grant from the Cancer Research Campaign. Request reprints from: Dr. Ben C arritt, Institute of Genetics, University of Glasgow, Church Street, Glasgow G il 5JS (Scotland).
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Cell lines a23 and GM-63 were fused in suspension using 1000 HAU UV-inactivated Sendai virus, and hybrid colonies were isolated in Ham’s F12 + HAT + 2.5 iim ouabain (Baker et al., 1974). A total of 17 independent primary hybrid clones (DOCI clones) were screened at an early stage for the possession of human AKt, a marker for chromosome 9 (P ovey et al., 1976; W estervuld et al., 1976); 7 clones were positive for the marker. These 7 clones were then grown to a larger population size and, after confirming that they had retained the AKj marker, were assayed for ASS activity.
Brief Report
45
As shown in table I, ASS activities were in all cases lower than that in the human citrullinémie parental cell (GM-63); in two clones activities svere about one-half that in GM-63. This compares with activities which are generally in the range of 4-8 units/mg for Don X human hybrids pos sessing human chromosome 9 contributed by a human parent with wildtype (16 units/mg) levels of ASS activity, although hybrid clones with full parental levels of ASS were sometimes found (C arritt et al., 1977). This result is consistent with the idea that the defective allele in the citrullinémie cell (i.e., the structural gene for ASS) is linked to AKt. However, other interpretations must be considered: 1. It is possible that the failure to detect significant levels of ASS activity in most clones of the present series of hybrids that express human AKi is due to a disruption of the ASS/AK| linkage on chromosome 9 by chromosome rearrangement, either in the human parent or in the hybrids. This possibility was excluded by examination of a minimum of
Table I. Argininosuccinate synthetase (ASS) activities in cell lines and in Chinese hamster-human citrullinémie cell hybrids containing human chromosome 9. Cells
Human fibroblast (Col)1' Human HGPRT fibroblast (GM-29)'' Human citrullinémie fibroblast (GM-63) Chinese hamster T K - (a23) DOC1 hybrids 2b 3b 6a 7a 8 lib 15a
ASS activity (units-'/mg protein) 16.7 ±1.3 (3) 15.810.5 (8) 4.3 ±0.6 (4) Data from C arritt et al. (1977).
Brief Report
46
25 G-banded metaphases of each clone: a normal chromosome 9 was evident in more than 50 "/» of cells in clones expressing AK|. 2. It was apparent from our previous study (C arritt et a!., 1977) that clonal heterogeneity with respect to chromosome 9 resulted in low levels of ASS activity in hybrids without affecting the ability to detect human AKt. Analysis of subclones revealed a direct relationship between the level of ASS activity in a primary clone and the proportion of AKr positive subclones obtained from it (C arro t et al., 1977). These AKrpositive subclones also had higher levels of ASS activity than their parental pri mary clone. A total of 15 subclones were isolated from three primary DOCI clones. AKi-positive subclones were obtained at a frequency higher than 70 % from these primary clones, suggesting that the original clones were rel atively homogeneous with respect to chromosome 9. As shown in table II. although ASS activities in the AK,-positive subcloncs were in general
Table II. Human AK, and ASS activity in subclones of Chinese hamster-human citrullinémie cell hybrids. DOCI subclone
AK,->
8.1 8.2 8.3 8.4
+
llb .l llb.3 llb.4 lib .5
+
15a.2 15a.3 15a.4 15a.5 15a.6 15a.8 15a.9
+ -
+ -
+ +
+ + + + -f* +
ASS activity (units/mg) 1.4 1.1 20 > 20
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« Cells were sonicated briefly in 0.05 M tris HCI, pH 8.3, and centrifuged at 100,000Xg for 60 min. The supernatant, containing 6-15 mg/nil protein, was as sayed for ASS activity at 5 mM aspartate and 0.3-4 m.M citrulline as described pre viously (C arritt et al., 1977).
48
Brief Report
I thank my colleagues, Drs. M.L. H ooper , P.S. Goldfarb, and C. M acD onald, for helpful discussions and Professors J.A. P ateman and J.H. Subak-S harpe for their interest.
Baker, R.M.; B runette , D.M.; M ankowitz , R.: T hompson , L.H.: W hitmore . G.F.; StMlNOViTCH, L., and T ill, J.E.: Ouabain-resistant mutants of mouse and hamster
cells in culture. Cell 1: 9-21 (1974). C arritt , B.; G oldfarb, P.S.G.; H ooper , M.L., and Slack, C.: Chromosome assign
ment of a human gene for argininosuccinatc synthetase expression in Chinese hamster X human hybrids. Expl Cell Res. 106: 71-78 (1977). P ovey , S.; S laughter, C.A.; W ilson , D.E.; G ormley , I.P.; Buckton, K.E.; P erry. P.. and Bobrow , M.: Evidence for the assignment of the loci AK,. AIÇ, and ACONs to chromosome 9 in man. Ann. hum. Genet. 39: 413-422 (1976). R atner, S.: Argininosuccinases and adenylsuccinases. In P.D. Boyer, ed.: The enzymes, Vol. 7, pp. 167-197 (Academic Press. New York 1972). T edesco , T.A. and M ellman, W.J.: Argininosuccinate synthetase activity and citrulline metabolism in cells cultured from a citrullinémie subject. Proc. natn. Acad. Sci. USA 57: 829-834 (1967). W esterveld , A.; J ongsma, A.P.M.; M eera K han, P.; Someren , H. van, and Bootsma , D.: Assignment of the AK,:Np:ABO linkage group to human chromo some 9. Proc. natn. Acad. Sci. USA 73: 895-899 (1976). W esterveld , A.; V isser , R.P.L.S.; M eera K han. P., and Bootsma. D.: Loss of human genetic markers in man X Chinese hamster somatic cell hybrids. Nature new Biol. 234: 20-24 (1971).
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 8:55:50 AM
Manuscript received 7 February 1977; accepted for publication 10 May 1977.
BRIEF REPORT
Somatic cell genetic evidence for the presence of a gene for citrullinemia on human chromosome 9 B. C arritt Institute of Genetics, University of Glasgow, Glasgow
In an earlier report (C arritt et al., 1977) we showed that the Chinese hamster cell line Don and its TK derivative a23 (W esti rvi i .d et al., 1971) lack detectable activity of the enzyme argininosuccinate synthetase (ASS) and that in hybrids formed between a23 and HGPRT human fibroblasts, ASS activity segregated concordantly with human chromo some 9 and AKi. It was therefore concluded that chromosome 9 carries a human gene responsible for ASS expression by these hybrids. However, since the parental type of ASS in the hybrids could not be determined, we were unable to distinguish between two possibilities: (1) that human chromosome 9 carries the structural gene for ASS and (2) that human chromosome 9 carries a positive control gene capable of activating a Chinese hamster ASS structural gene. As an approach to this question, I have made use of a human cell line (GM-63) which has an ASS with an altered Km toward citrulline (T edesco and M eeeman , 1967), the effect of which is to depress ASS activity to 20-30 % of wild-type levels under certain conditions of assay ( T edesco and M ellman , 1967, and table I).
Supported by a grant from the Cancer Research Campaign. Request reprints from: Dr. Ben C arritt, Institute of Genetics, University of Glasgow, Church Street, Glasgow G il 5JS (Scotland).
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 8:55:50 AM
Cell lines a23 and GM-63 were fused in suspension using 1000 HAU UV-inactivated Sendai virus, and hybrid colonies were isolated in Ham’s F12 + HAT + 2.5 iim ouabain (Baker et al., 1974). A total of 17 independent primary hybrid clones (DOCI clones) were screened at an early stage for the possession of human AKt, a marker for chromosome 9 (P ovey et al., 1976; W estervuld et al., 1976); 7 clones were positive for the marker. These 7 clones were then grown to a larger population size and, after confirming that they had retained the AKj marker, were assayed for ASS activity.
Brief Report
45
As shown in table I, ASS activities were in all cases lower than that in the human citrullinémie parental cell (GM-63); in two clones activities svere about one-half that in GM-63. This compares with activities which are generally in the range of 4-8 units/mg for Don X human hybrids pos sessing human chromosome 9 contributed by a human parent with wildtype (16 units/mg) levels of ASS activity, although hybrid clones with full parental levels of ASS were sometimes found (C arritt et al., 1977). This result is consistent with the idea that the defective allele in the citrullinémie cell (i.e., the structural gene for ASS) is linked to AKt. However, other interpretations must be considered: 1. It is possible that the failure to detect significant levels of ASS activity in most clones of the present series of hybrids that express human AKi is due to a disruption of the ASS/AK| linkage on chromosome 9 by chromosome rearrangement, either in the human parent or in the hybrids. This possibility was excluded by examination of a minimum of
Table I. Argininosuccinate synthetase (ASS) activities in cell lines and in Chinese hamster-human citrullinémie cell hybrids containing human chromosome 9. Cells
Human fibroblast (Col)1' Human HGPRT fibroblast (GM-29)'' Human citrullinémie fibroblast (GM-63) Chinese hamster T K - (a23) DOC1 hybrids 2b 3b 6a 7a 8 lib 15a
ASS activity (units-'/mg protein) 16.7 ±1.3 (3) 15.810.5 (8) 4.3 ±0.6 (4) Data from C arritt et al. (1977).
Brief Report
46
25 G-banded metaphases of each clone: a normal chromosome 9 was evident in more than 50 "/» of cells in clones expressing AK|. 2. It was apparent from our previous study (C arritt et a!., 1977) that clonal heterogeneity with respect to chromosome 9 resulted in low levels of ASS activity in hybrids without affecting the ability to detect human AKt. Analysis of subclones revealed a direct relationship between the level of ASS activity in a primary clone and the proportion of AKr positive subclones obtained from it (C arro t et al., 1977). These AKrpositive subclones also had higher levels of ASS activity than their parental pri mary clone. A total of 15 subclones were isolated from three primary DOCI clones. AKi-positive subclones were obtained at a frequency higher than 70 % from these primary clones, suggesting that the original clones were rel atively homogeneous with respect to chromosome 9. As shown in table II. although ASS activities in the AK,-positive subcloncs were in general
Table II. Human AK, and ASS activity in subclones of Chinese hamster-human citrullinémie cell hybrids. DOCI subclone
AK,->
8.1 8.2 8.3 8.4
+
llb .l llb.3 llb.4 lib .5
+
15a.2 15a.3 15a.4 15a.5 15a.6 15a.8 15a.9
+ -
+ -
+ +
+ + + + -f* +
ASS activity (units/mg) 1.4 1.1 20 > 20
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 8:55:50 AM
« Cells were sonicated briefly in 0.05 M tris HCI, pH 8.3, and centrifuged at 100,000Xg for 60 min. The supernatant, containing 6-15 mg/nil protein, was as sayed for ASS activity at 5 mM aspartate and 0.3-4 m.M citrulline as described pre viously (C arritt et al., 1977).
48
Brief Report
I thank my colleagues, Drs. M.L. H ooper , P.S. Goldfarb, and C. M acD onald, for helpful discussions and Professors J.A. P ateman and J.H. Subak-S harpe for their interest.
Baker, R.M.; B runette , D.M.; M ankowitz , R.: T hompson , L.H.: W hitmore . G.F.; StMlNOViTCH, L., and T ill, J.E.: Ouabain-resistant mutants of mouse and hamster
cells in culture. Cell 1: 9-21 (1974). C arritt , B.; G oldfarb, P.S.G.; H ooper , M.L., and Slack, C.: Chromosome assign
ment of a human gene for argininosuccinatc synthetase expression in Chinese hamster X human hybrids. Expl Cell Res. 106: 71-78 (1977). P ovey , S.; S laughter, C.A.; W ilson , D.E.; G ormley , I.P.; Buckton, K.E.; P erry. P.. and Bobrow , M.: Evidence for the assignment of the loci AK,. AIÇ, and ACONs to chromosome 9 in man. Ann. hum. Genet. 39: 413-422 (1976). R atner, S.: Argininosuccinases and adenylsuccinases. In P.D. Boyer, ed.: The enzymes, Vol. 7, pp. 167-197 (Academic Press. New York 1972). T edesco , T.A. and M ellman, W.J.: Argininosuccinate synthetase activity and citrulline metabolism in cells cultured from a citrullinémie subject. Proc. natn. Acad. Sci. USA 57: 829-834 (1967). W esterveld , A.; J ongsma, A.P.M.; M eera K han, P.; Someren , H. van, and Bootsma , D.: Assignment of the AK,:Np:ABO linkage group to human chromo some 9. Proc. natn. Acad. Sci. USA 73: 895-899 (1976). W esterveld , A.; V isser , R.P.L.S.; M eera K han. P., and Bootsma. D.: Loss of human genetic markers in man X Chinese hamster somatic cell hybrids. Nature new Biol. 234: 20-24 (1971).
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 8:55:50 AM
Manuscript received 7 February 1977; accepted for publication 10 May 1977.
