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Chromosome Rearrangements in Human-Chinese Hamster Somatic Cell Hybrids J

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Chromosome Rearrangements in Human-Chinese Hamster Somatic Cell Hybrids J Am J Hum Genet 27:595-608, 1975 Localization of Human Gene Loci Using Spontaneous Chromosome Rearrangements in Human-Chinese Hamster Somatic Cell Hybrids J. L. HAMERTON,' T. MOHANDAS,' PHYLLIS J. MCALPINE,' AND G. R. DOUGLAS1"2 INTRODUCTION Human-rodent somatic cell hybrids have been utilized extensively for human gene assignments (see [1, 2] for review). The use of hybrid lines originating from the fusion of human cells carrying chromosome structural changes with rodent cells has allowed the assignment of gene loci to specific chromosome regions [3-5]. Regional localization of gene loci can also be achieved using spontaneously derived or induced structural alterations in the human chromosomes in human-rodent hybrid cell lines [6-9]. The use of methods similar to those used for mapping Drosophila polytene chromosomes (see [10] for review) allows hybrid cell lines carrying chromosome rearrangements to be used to assign gene loci to specific chromosome bands. We report here the regional and subregional localization of gene loci on human chromosomes 2, 12, and X using structural rearrangements of the human chromosomes which originated spontaneously in human-Chinese ham- ster somatic cell hybrids. These rearrangements include deletion, intraspecific translocation, and interspecific translocation. MATERIALS AND METHODS Hybrid line 1610 was derived from fusion between a mutant Chinese hamster cell line (CHW-1102) deficient in hypoxanthine guanine phosphoribosyltransferase (HPRT) [11] and a strain of human diploid fibroblasts (DCF) derived from a male fetus. Lines 1705 and 4105 were derived from fusion of CHW-1102 with leukocytes from two dif- ferent human donors. Methods for the production, propagation, cloning, and cytological analysis of hybrid lines have already been described [7, 12, 13]. The cytological and biochemical analyses of each hybrid line and clone were performed on cells at the same passage level or the closest passage levels possible. Cytological analyses were carried Received October 29, 1974; revised February 21, 1975. This work was supported by grant MA-4061 from the Medical Research Council of Canada and by the Children's Hospital Research Foundation, Winnipeg. T. Mohandas and G. R. Douglas were the recipients of postdoctoral fellowships from the Medical Research Council. 1 Division of Genetics, Department of Paediatrics, University of Manitoba, Winnipeg, Mani- toba. Address reprint requests to J. L. Hamerton, Department of Genetics, Health Sciences Centre-Children's Centre, 685 Bannatyne Avenue, Winnipeg, Manitoba, R3E OW1, Canada. 2 Present address: Human Development Division, Department of Health and Welfare, Environmental Health Centre, Tunney's Pasture, Ottawa, Ontario. o 1975 by the American Society of Human Genetics. All rights reserved. 595 596 HAMERTON ET AL. out by T. Mohandas and G. R. Douglas and enzyme analyses by P. J. McAlpine. After both sets of results were available, the data were pooled and analyzed for gene assign- ments. Lysates of the lines and clones were examined electrophoretically for the follow- ing enzyme markers by the methods indicated: red cell acid phosphatase (AcP1) [14]; cytoplasmic isocitrate dehydrogenase (IDH1) [15]; peptidase B (Pep B) [16]; glucose- 6-phosphate dehydrogenase (G6PD) [17]; phosphoglycerate kinase (PGK) [18]; a ga- lactosidase (a-gal) [19]; hypoxanthine guanine phosphoribosyltransferase (HPRT) [20]; and peptidase A (Pep A) [16]. Cellogel (Chemetron, Italy) with 0.1 M Tris- citrate pH 8.6 buffer was used for the electrophoretic examination of lactate dehydro- genase (LDH) and NAD-dependent cytoplasmic malate dehydrogenase (MDH1). Iso- zymes were visualized with slight modifications of previously published methods [21, 22]. The nomenclature used to describe chromosome rearrangements is a modification of that proposed by the Paris Conference [23]. The prefixes Hs and Cg placed before the chromosome number indicate human and Chinese hamster chromosomes, respectively. RESULTS Cytological and Biochemical Analyses on Hybrids Carrying Rearrangements In- volving Chromosome 2 An interspecific presumptive reciprocal translocation between human chromo- some 2 and Chinese hamster chromosome 9, t(Hs2;Cg9)(qi1;?), subsequently referred to as t(a), was identified in a secondary clone (1610-09-51) of hybrid line 1610. This rearrangement had presumably arisen after cloning, since cells were observed with a normal intact chromosome 2, with t(a), and with a deleted form of t(a), del[t(a)(Hs2q22)], referred to subsequently as delt(a)l (fig. 1). Sixty-three tertiary clones derived from 1610-09-51 could be separated into four phenotypic classes (table 1) on the basis of presence or absence of the genetic markers AcP1, MDH1, and IDH1 previously assigned to chromosome 2 [24-29]. Twenty-two clones representing each of the four classes were analyzed cyto- logically and the results correlated with their biochemical phenotypes (table 2). In addition to an intact chromosome 2, t(a), and delt(a) , a second deleted form of the translocation, del [t(a) (Hs2q24)], referred to subsequently as delt(a)2, was identified in tertiary clone 51-68. One tertiary clone (51-02) carried both products of the translocation, t(a) and del(Hs2)(ql1), and thus appeared to carry all the chromosome 2 material (fig. 1). Another tertiary clone (51-19) car- ried a 2p deletion, del(Hs2)(p23), and a translocation of this deleted segment to Cg9, resulting in a dicentric product tdic(Hs2;Cg9)(p23;?) referred to sub- sequently as tdic(b) (fig. 2). The data presented in table 2 show that in the presence of an intact human chromosome 2, the human forms of the genetic markers AcP1, IDH1, and MDH1 were expressed. In the absence of an intact human chromosome 2 or when a re- arranged chromosome 2 of type t(a), delt(a) , or delt(a)2 was present, the human form of these three markers was not expressed. Tertiary clone 51-02, which car- ried both t(a) and del(Hs2) (ql 1) and thus, within the limits of resolution, was balanced with respect to chromosome 2 material, expressed the human MDH1 and AcPj gene loci but not the IDH1 locus. Tertiary clone 51-19, which carried IL. a-_ r -, e to" ~ I- Norma~~~~ -o a {e '-w1ida- < - - I- '. ; I-, 597 598 HAMERTON ET AL. TABLE 1 BIOCHEMICAL ANALYSIS OF TERTIARY CLONES DERIVED FROM SECONDARY CLONE 1610-09-51 HUMAN MARKERS PHENOTYPIC CLASS I DH1 MDH1 AcP1 No. SUBCLONES I............. + + + 52 I............. - - - 9 III............. + + 1 IV ............. + - + 1 NOTE.-+ = presence of human marker; -=absence. del(Hs2) (p23) and tdic(b) and was therefore deficient for the 2pter->2p23 region, expressed the human loci IDH1 and AcP1 but not MDH1. Cytological and Biochemical Analyses on Hybrids Carrying Rearrangements In- volving Chromosomes 12 and X Hybrid line 1705 initially had human chromosomes 5, 12, 18, and X and ex- pressed the human chromosome 12 markers LDH B and Pep B, the chromosome 18 marker Pep A, and the human X markers a-gal, PGK, HPRT, and G6PD. When this line was cloned, clone 1705-40 carried a translocation between the human X chromosome and human chromosome 12, t(X;12)(q24;q21) (fig. 3). Twenty-four secondary clones from 1705-40 could be separated into two pheno- typic classes with respect to the human enzyme markers (table 3). Six secondary clones, three from each phenotypic class, were analyzed cytologically and all (table 4) carried the derivative chromosome Xpter->Xq24::12q21- >12qter. These clones had lost both the human chromosome 12 markers LDH B and Pep B and the human X chromosome marker G6PD. The remaining X chromosome loci (a-gal, PGK, and HPRT) were expressed in all secondary clones. A deleted human chromosome 12, del(I 2) (q2 1) (fig. 3), with a breakpoint in the same band as the breakpoint in the t(X;12)(q24;q21) was identified in hybrid line 4105. This line was thus deficient for the 12q2l1->12qter region of chromosome 12 and expressed the human LDH B locus but not the human Pep B locus. Hybrid line 4105 also carried a deleted human X chromosome, del(X) (p21), but expressed the human chromosome markers a-gal, PGK, HPRT, and G6PD. Three secondary clones derived from 1705-40 which expressed human Pep A had human chromosome 18, while the three that did not express human Pep A did not have human chromosome 18. DISCUSSION Localization of Markers on Chromosome 2 The IDH1 and MDH1 human gene loci have been shown to be syntenic [24] and have been assigned to chromosome 2 [26-29]. The AcP1 locus has also been ++++++++++++IllI P4 Ca I ++++++++++++IIIIIIIII+I z ++++++++++++IIIIIIIIII+ .0 00 C1 z 0 0 I-IO I I IIICO, 0 CO, C11+ 0 Cd I8 8IIIII1 8I1Io8I 1 0o U) I1118888 18 1 1 1 11 118 1 .Ca ¢1 0E CZ Ca Z ¢ 0 0 -4 0 Z VI 0 b-Ca li U) w Ei 0 0 IN E _0 I aI .0 -Z oIt- r, lao 6C1CE5 0 0 CaOA ooC8- )C 100 0 u 50S 11 tCto Itt_' UW 0 h tQ 41;¢ C,4 NC, C-4rq C4 Ca C,4 er) rqC~q t4 eCa '4 4 Q 0, U U 6 o b w Z U U z *^ bo 0 bo *-; q U W &' V C/) U- 0 C%-a d-C4e i r - Ni0 00 Cn)e4 0', 1.- 4-+m 0o 00000- O \o o Do _- C \0o o o \o 000 599 600 HAMERTON ET AL. 3 3 3 a b c FIG. 2.-Diagrams and photomicrographs of interspecific chromosome rearrangement in tertiary clone 1610-09-51-19. General description as in fig. 1. a, Normal human chromosome 2; arrow indicates breakpoint. b, Normal Chinese hamster chromosome 9. c, Deleted human chro- mosome 2, del(Hs2)(p23). d, Translocation between deleted human chromosome 2 and Chinese hamster chromosome 9, tdic(b) = t(Hs2 ;Cg9) (Cg9qter-- Cg9pter?: :Hs2p23-oHs2qter).
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