260 (430)
Show s , Brown
Segregation of Es-D and APRT
Segregation of esterase-D and adenine phosphoribosyl transferase in somatic cell hybrids: gene linkage or chromosome association ? T.B. Shows1 and J.A. Brown2 'Department of Experimental Biology, Roswell Park Memorial Institute, New York State Department of Health, and d ep a rtm en t of Pediatrics, Children’s Hospital, State University of New York at Buffalo, Buffalo, N.Y.
Supported by NIH grants GM 20454, HD 05196, and HD-GM 06321, by National Science Foundation grant GB-39273, and by HEW Maternal and Child Health Services, Project 417.
Downloaded by: King's College London 137.73.144.138 - 1/17/2019 2:39:34 AM
Concordant segregation of enzymes and chromosomes predicts the assignment of genes to specific chromosomes in somatic cell hybrids. It is conceivable that nonrandom human chromosome loss may occur in cell hybrids which would nullify apparent gene linkages or chromosome assignments.1 The question of gene linkage or chromosome association has emerged when analyzing the segregation characteristics of esterase-D (Es-D), adenine phosphoribosyl transferase (APRT), and chromosome 16 in man-mouse somatic cell hybrids. APRT has been assigned to chromosome 16 in man with somatic cell hybrids.2’3 Genetic variants of (Es-D) were found in human populations by H opkinson et al.,1 but there are conflicting results with regard to the assignment of the Es-D gene to a specific chromosome.5 The segregation of Es-D and enzymes coded by genes assigned to 15 chromosomes was investigated in 82 primary man-mouse somatic cell hybrid clones. The clones were isolated from six hybridization experiments which employed four different human cell lines and three different mouse cell lines (table I). Concordant segregation of APRT and Es-D was observed for 73 of the 82 primary clones. Nine clones (10.9%) were discordant. This value is somewhat elevated for close linkage to be considered, since it has been our experience, using the same cell hybrids for gene linkage studies, that 4-6 °/o of primary clones are discordant for confirmed linked genes not under selective pressure.® This discordancy has been due to chromosome
Shows, Brown
261 (431)
Segregation of Es-D and APRT
Table I. Segregation of adenine phosphoribosyl transferase and estcrase-D in primary cell hybrid clones.“ Hybrid sets APRT+/Es-D +
APRT+/E s-D -
APRT-/ES-D+
WIL REW JVR TSR TSA REX
4 8 7 6 0 28
1 1 0 0 0 1
0 2 1 1 0 2
53
3
6
APRT-/ES-D* 8 5 3 0 2 2 20/82
“ Parental cells, fusion techniques, isolation of primary hybrid clones and hybrid sets have been described.7-9 Arabic numbers represent number of primary clones expressing the enzyme combinations. APRT and Es-D were determined by starch-gel electrophoresis as described previously.4-10 + = Enzyme activity; — = no activity.
Table II. Es-D, APRT, and chromosome 16 distribution in cell hybrids.“ Hybrid clones
JWR-22h JWR-26C JVR-18 JVR-22 ALR-1 BSH 6 ALR-1 BSH 5 ALR-1 BSAg 9 ALR-1 BSAg 10 ALA-1 BSH 8
Chromosome 16
Es-D
APRT
per cells examined
percentage of cells
0/20 8/20 11/25 1/22 10/29 16/32 3/27
___
___
—
40% 44% 5% 34% 50% 11%
+
+
3/32 1/10
9% 10%
+
+
+
—
-
+
+
+ —
+ very weak —
breakage. Linkage of ES-D to all other enzymes tested could be dis counted. Chromosome and enzyme analyses in nine hybrid clones were consistent with the joint segregation of APRT, Es-D, and chromosome 16 (table II).
Downloaded by: King's College London 137.73.144.138 - 1/17/2019 2:39:34 AM
“ Hybrid clones have been described.11-12 Human chromosomes were identified by G-banding and C-banding techniques.13-14
262 (432)
Show s , B rown
Segregation of Es-D and APRT
The two enzymes could not be detected by our assay system in clones that retained chromosome 16 in less than 10% of the cells examined. There was one exception (ALA-1 BSH 8) tested in which only 10% of the cells possessed an intact chromosome 16 yet Es-D but not APRT was expressed. Chromosome breakage could have occurred in this clone, or perhaps Es-D is located on a different chromosome. We were not able to unequivocally identify chromosomes 13, 14, or 15 in these cell hybrids. Chromosome and enzyme analyses could rule out assignment of Es-D to all chromosomes except Nos. 13, 15, and 16. If the Es-D gene is not located on chromosome 16, then it is located on either chromosome 13 or 15, which indicates that one of these two chromosomes segregates nonrandomly with chromosome 16 in 90 % of our cell hybrids. In fact, van H f.yningen et al. contend that Es-D is located on chromosome 13.5 Such nonrandom segregation may represent a selective advantage or dependence of chromosome 16 on either chromosome 13 or 15 in our cell hybrids. References 1 N orum, R.A. and M igeon, B.R.: Non-random loss of human markers from manmouse somatic cell hybrids. Nature, Lond. (1974, in press). 2 Tischfield, J.A. and Ruddle, F.H.: Assignment of the gene for adenine phosphoribosyltransferase to human chromosome 16 by mouse-human somatic cell hybridization. Proc. natn. Acad. Sci. USA 71: 45-49 (1974). 3 Kahan, B.; H eld, K.R. and D eM ars, R.: Genes for human adenine phospho ribosyltransferase on chromosome no. 16. Genetics (1974, in press). 4 H opkinson, D.A.; M estriner, M.A.; Cortner, J. and H arris, H.: Esterase D:
a new human polymorphism. Ann. hum. Genet. 37: 119-137 (1973).
dehydrogenase to chromosome 7, for mannose phosphate isomerase and pyruvate kinase to chromosome 15, and, probably, for human esterase-D to chromosome 13 using man-mouse hybrids. This conference (1974). 6 S hows, T.B.: Somatic cell genetics of enzyme markers associated with three human linkage groups, pp. 15-25. In R.L. D avidson and F. de la Cruz, eds.: Somatic cell hybridization (Raven Press, New York 1974). 7 Shows, T.B.: Genetics of human-mouse somatic cell hybrids: linkage of human genes for lactate dehydrogenase-A and esterase-A4. Proc. natn. Acad. Sci. USA 69: 348-352 (1972). 8 L alley, P.A.; Rattazzi, M.C. and S hows, T.B.: Human /?-D-N-acety 1-hexosami nidase A and B: expression and linkage relationships in somatic cell hybrids. Proc. natn. Acad. Sci. USA 71: 1569-1573 (1974).
Downloaded by: King's College London 137.73.144.138 - 1/17/2019 2:39:34 AM
5 H eyningen, V. van ; Bobrow, M.; Bodmer, W.F.; P ovey, S.; G ardiner, S.E. and H opkinson, D.A.: Assignment of the genes for human mitochondrial malate
S hows, Brown
Segregation of Es-D and APRT
263 (433)
9 S how s , T.B. and Brown , J.A.: Localization of genes coding for PGK, HPRT, and G6PD on the long arm of the X chromosome in somatic cell hybrids. This conference (1974). 10 M owbray, S.: W atson, B. and H arris, H.: A search for electrophoretic variants of human adenine phosphoribosyl transferase. Ann. hum. Genet. 36: 153-162 (1972). 11 S hows, T.B. and Brown, J.A.: Mapping chromosomes I and 2 employing a 1/2 translocation in somatic cell hybrids. This conference (1974). 12 S how s , T.B. and Brown , J.A.: An (X q-;9p+ ) translocation suggests the assign
Downloaded by: King's College London 137.73.144.138 - 1/17/2019 2:39:34 AM
ment of G6PD, HPRT, and PGK to the long arm of the X chromosome in somatic cell hybrids, pp. 146-149. In N ew H aven Conference (1973): First inter national workshop on human gene mapping. Birth Defects: Original Article Series, Vol. 10. No. 3 (The National Foundation, New York 1974). 13 P ath,, S.; M errick , S. and L ubs , H.A.: Identification of each human chromo some with a modified Giemsa stain. Science 173: 821-822 (1971). 14 D ev , G.V.; M iller , D.A.; A lderdice , P.W. and M iller , O.I.: Method for locating the centromeres of mouse meiotic chromosomes and its application to T163H and T70H translocations. Expl Cell Res. 73: 259-262 (1972).
Show s , Brown
Segregation of Es-D and APRT
Segregation of esterase-D and adenine phosphoribosyl transferase in somatic cell hybrids: gene linkage or chromosome association ? T.B. Shows1 and J.A. Brown2 'Department of Experimental Biology, Roswell Park Memorial Institute, New York State Department of Health, and d ep a rtm en t of Pediatrics, Children’s Hospital, State University of New York at Buffalo, Buffalo, N.Y.
Supported by NIH grants GM 20454, HD 05196, and HD-GM 06321, by National Science Foundation grant GB-39273, and by HEW Maternal and Child Health Services, Project 417.
Downloaded by: King's College London 137.73.144.138 - 1/17/2019 2:39:34 AM
Concordant segregation of enzymes and chromosomes predicts the assignment of genes to specific chromosomes in somatic cell hybrids. It is conceivable that nonrandom human chromosome loss may occur in cell hybrids which would nullify apparent gene linkages or chromosome assignments.1 The question of gene linkage or chromosome association has emerged when analyzing the segregation characteristics of esterase-D (Es-D), adenine phosphoribosyl transferase (APRT), and chromosome 16 in man-mouse somatic cell hybrids. APRT has been assigned to chromosome 16 in man with somatic cell hybrids.2’3 Genetic variants of (Es-D) were found in human populations by H opkinson et al.,1 but there are conflicting results with regard to the assignment of the Es-D gene to a specific chromosome.5 The segregation of Es-D and enzymes coded by genes assigned to 15 chromosomes was investigated in 82 primary man-mouse somatic cell hybrid clones. The clones were isolated from six hybridization experiments which employed four different human cell lines and three different mouse cell lines (table I). Concordant segregation of APRT and Es-D was observed for 73 of the 82 primary clones. Nine clones (10.9%) were discordant. This value is somewhat elevated for close linkage to be considered, since it has been our experience, using the same cell hybrids for gene linkage studies, that 4-6 °/o of primary clones are discordant for confirmed linked genes not under selective pressure.® This discordancy has been due to chromosome
Shows, Brown
261 (431)
Segregation of Es-D and APRT
Table I. Segregation of adenine phosphoribosyl transferase and estcrase-D in primary cell hybrid clones.“ Hybrid sets APRT+/Es-D +
APRT+/E s-D -
APRT-/ES-D+
WIL REW JVR TSR TSA REX
4 8 7 6 0 28
1 1 0 0 0 1
0 2 1 1 0 2
53
3
6
APRT-/ES-D* 8 5 3 0 2 2 20/82
“ Parental cells, fusion techniques, isolation of primary hybrid clones and hybrid sets have been described.7-9 Arabic numbers represent number of primary clones expressing the enzyme combinations. APRT and Es-D were determined by starch-gel electrophoresis as described previously.4-10 + = Enzyme activity; — = no activity.
Table II. Es-D, APRT, and chromosome 16 distribution in cell hybrids.“ Hybrid clones
JWR-22h JWR-26C JVR-18 JVR-22 ALR-1 BSH 6 ALR-1 BSH 5 ALR-1 BSAg 9 ALR-1 BSAg 10 ALA-1 BSH 8
Chromosome 16
Es-D
APRT
per cells examined
percentage of cells
0/20 8/20 11/25 1/22 10/29 16/32 3/27
___
___
—
40% 44% 5% 34% 50% 11%
+
+
3/32 1/10
9% 10%
+
+
+
—
-
+
+
+ —
+ very weak —
breakage. Linkage of ES-D to all other enzymes tested could be dis counted. Chromosome and enzyme analyses in nine hybrid clones were consistent with the joint segregation of APRT, Es-D, and chromosome 16 (table II).
Downloaded by: King's College London 137.73.144.138 - 1/17/2019 2:39:34 AM
“ Hybrid clones have been described.11-12 Human chromosomes were identified by G-banding and C-banding techniques.13-14
262 (432)
Show s , B rown
Segregation of Es-D and APRT
The two enzymes could not be detected by our assay system in clones that retained chromosome 16 in less than 10% of the cells examined. There was one exception (ALA-1 BSH 8) tested in which only 10% of the cells possessed an intact chromosome 16 yet Es-D but not APRT was expressed. Chromosome breakage could have occurred in this clone, or perhaps Es-D is located on a different chromosome. We were not able to unequivocally identify chromosomes 13, 14, or 15 in these cell hybrids. Chromosome and enzyme analyses could rule out assignment of Es-D to all chromosomes except Nos. 13, 15, and 16. If the Es-D gene is not located on chromosome 16, then it is located on either chromosome 13 or 15, which indicates that one of these two chromosomes segregates nonrandomly with chromosome 16 in 90 % of our cell hybrids. In fact, van H f.yningen et al. contend that Es-D is located on chromosome 13.5 Such nonrandom segregation may represent a selective advantage or dependence of chromosome 16 on either chromosome 13 or 15 in our cell hybrids. References 1 N orum, R.A. and M igeon, B.R.: Non-random loss of human markers from manmouse somatic cell hybrids. Nature, Lond. (1974, in press). 2 Tischfield, J.A. and Ruddle, F.H.: Assignment of the gene for adenine phosphoribosyltransferase to human chromosome 16 by mouse-human somatic cell hybridization. Proc. natn. Acad. Sci. USA 71: 45-49 (1974). 3 Kahan, B.; H eld, K.R. and D eM ars, R.: Genes for human adenine phospho ribosyltransferase on chromosome no. 16. Genetics (1974, in press). 4 H opkinson, D.A.; M estriner, M.A.; Cortner, J. and H arris, H.: Esterase D:
a new human polymorphism. Ann. hum. Genet. 37: 119-137 (1973).
dehydrogenase to chromosome 7, for mannose phosphate isomerase and pyruvate kinase to chromosome 15, and, probably, for human esterase-D to chromosome 13 using man-mouse hybrids. This conference (1974). 6 S hows, T.B.: Somatic cell genetics of enzyme markers associated with three human linkage groups, pp. 15-25. In R.L. D avidson and F. de la Cruz, eds.: Somatic cell hybridization (Raven Press, New York 1974). 7 Shows, T.B.: Genetics of human-mouse somatic cell hybrids: linkage of human genes for lactate dehydrogenase-A and esterase-A4. Proc. natn. Acad. Sci. USA 69: 348-352 (1972). 8 L alley, P.A.; Rattazzi, M.C. and S hows, T.B.: Human /?-D-N-acety 1-hexosami nidase A and B: expression and linkage relationships in somatic cell hybrids. Proc. natn. Acad. Sci. USA 71: 1569-1573 (1974).
Downloaded by: King's College London 137.73.144.138 - 1/17/2019 2:39:34 AM
5 H eyningen, V. van ; Bobrow, M.; Bodmer, W.F.; P ovey, S.; G ardiner, S.E. and H opkinson, D.A.: Assignment of the genes for human mitochondrial malate
S hows, Brown
Segregation of Es-D and APRT
263 (433)
9 S how s , T.B. and Brown , J.A.: Localization of genes coding for PGK, HPRT, and G6PD on the long arm of the X chromosome in somatic cell hybrids. This conference (1974). 10 M owbray, S.: W atson, B. and H arris, H.: A search for electrophoretic variants of human adenine phosphoribosyl transferase. Ann. hum. Genet. 36: 153-162 (1972). 11 S hows, T.B. and Brown, J.A.: Mapping chromosomes I and 2 employing a 1/2 translocation in somatic cell hybrids. This conference (1974). 12 S how s , T.B. and Brown , J.A.: An (X q-;9p+ ) translocation suggests the assign
Downloaded by: King's College London 137.73.144.138 - 1/17/2019 2:39:34 AM
ment of G6PD, HPRT, and PGK to the long arm of the X chromosome in somatic cell hybrids, pp. 146-149. In N ew H aven Conference (1973): First inter national workshop on human gene mapping. Birth Defects: Original Article Series, Vol. 10. No. 3 (The National Foundation, New York 1974). 13 P ath,, S.; M errick , S. and L ubs , H.A.: Identification of each human chromo some with a modified Giemsa stain. Science 173: 821-822 (1971). 14 D ev , G.V.; M iller , D.A.; A lderdice , P.W. and M iller , O.I.: Method for locating the centromeres of mouse meiotic chromosomes and its application to T163H and T70H translocations. Expl Cell Res. 73: 259-262 (1972).
