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Sensitive Mouse Cells by Mouse-Human Cell Hybridization

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Sensitive Mouse Cells by Mouse-Human Cell Hybridization Proc. Nati. Acad. Sci. USA Vol. 80, pp. 5655-5659, September 1983 Genetics Human chromosome 13 compensates a DNA repair defect in UV- sensitive mouse cells by mouse-human cell hybridization (human gene mapping/UV sensitivity/somatic cell hybrids) TADA-AKI HoRi, TADAHIRO SHIOMI, AND KOKI SATO Division of Genetics, National Institute of Radiological Sciences, 4-Anagawa, Chiba 260, Japan Communicated by Hitoshi Kihara, May 23, 1983 ABSTRACT A human chromosome responsible for excision of an UV-sensitive mouse mutation, and we report the suc- repair of UV-induced DNA damage has been identified by study- cessful human chromosome mapping of a gene responsible for ing somatic cell hybrids between an UV-sensitive mutant of mouse UV-sensitivity in mammalian cells. lymphoma L5178Y cells and normal human lymphocytes. An au- tosomal recessive mutant, Q31, of complementation group I is de- MATERIALS AND METHODS ficient in excision repair of UV-induced DNA damage. Somatic cell hybrids between Q31 and human lymphocytes exhibited the Cells and Culture Conditions. Mouse lymphoma L5178Y cell same UV resistance as did parental L5178Y cells. In addition, both line and its UV-sensitive mutant Q31 were cultured in Fischer's the levels of UV-induced unscheduled DNA synthesis and chro- medium (Nissui Seiyaku, Tokyo) supplemented with 10% calf mosomal sensitivity were recovered from the UV-resistant hybrid serum (Flow Laboratories) at 370C in a CO2 incubator (5% CO2/ clones. Segregation of the hybrid cells gave rise to UV-sensitive 95% air). Peripheral lymphocytes of a healthy male adult were clones. The segregation of UV sensitivity was not correlated with cultured in Eagle's minimal essential medium (Nissui Seiyaku) the loss of human X chromosome. Karyotype analysis of the segre- supplemented with 15% fetal calf serum (GIBCO) and 2% phy- gants gave evidence that a gene on human chromosome 13 com- tohemagglutinin (Wellcome). pensates for UV hypersensitivity of Q31 mutant. Cell Fusion. Q31TG3, a 6-thioguanine-resistant clone of Q31, was fused with phytohemagglutinin-stimulated human lym- DNA repair has been implicated as modulating mutagenesis phocytes in polyethylene glycol 4000. Cells were mixed in a 1:1 and carcinogenesis of certain human genetic diseases. For ex- ratio (5-10 x 105 cells) in a test tube and washed with serum- ample, somatic cells derived from a xeroderma pigmentosum free minimal essential medium by centrifugation. The pellet of (XP) patient who has a genetic predisposition to sunlight-in- mixed cells was treated with 0.25 ml of polyethylene glycol 4000 duced skin cancer are known to be hypersensitive to the killing for 2 min. Immediately after cell fusion, cells (2 x 105) were effects of UV (1) and significantly more sensitive to UV-induced plated in 60-mm plastic Petri dishes containing 5 ml of growth mutagenesis than are cells from normal individuals (2). Most XP medium. After 24 hr of incubation, the cells were tranferred cells are defective in the first step of excision repair of UV-in- into HAT medium (100 uM hypoxanthine/10 uM, amethop- duced DNA damage and fall into seven complementation groups terin/40 ,uM thymidine) from which the hybrid cells were se- (3-5). However, the genetic basis of phenotypic complemen- lected. tation is not yet clear, and human enzymes responsible for DNA Assays for UV Sensitivity. Qualitative assay. The cell sus- repair in vivo remain to be identified. pension spotting method (8) was used for isolating UV-sensitive The chromosome assignment of human gene(s) that function and UV-resistant clones of mouse-human hybrid cells. In brief, in the DNA repair process is an important approach to under- cell suspensions from individual colonies developed on agar plates standing the genetic basis of complementation and may provide were spotted onto two sets of 0.5% soft agar plates, one of which clues for identifying gene product from specific human chro- was irradiated with UV at a dose of 4 J/m2. After 4-6 days of mosomal DNA. Many human genes have been mapped by con- incubation of both sets of plates, UV-sensitive and UV-resistant cordant segregation of specific human chromosomes and spe- clones were isolated from the nonirradiated plates. cific gene markers in rodent-human cell hybrids. UV-sensitive Quantitative assay. Cells (100-2,000) suspended in 0.2 ml of mutants with DNA repair defects are now available in rodent saline G (11) containing 0.2% calf serum were spread onto the cell lines (6, 7), and mutants from mouse lymphoma L5178Y central part of each plastic dish (60 mm; Falcon) and were ir- cells fall into four complementation groups (8). An autosomal radiated with UV at a dose rate of 0.4 J/m2/sec. These cells recessive mutant, Q31, of complementation group I (8, 9) is were incubated in soft agar medium to determine the surviving completely deficient in excision repair of UV-induced DNA fraction of the irradiated cells. damage (10). In an attempt to understand the genetic basis of Assay of Unscheduled DNA Synthesis (UDS). Cells were complementation, we have initiated a program of mapping hu- exposed to UV (30 J/m2) at a dose rate of 0.4 J/m2/sec and man chromosomes that carry gene(s) which compensate the DNA incubated for 3 hr in minimal essential medium containing [3H]- repair defects of UV-sensitive mouse mutants in mouse-human thymidine (12.5 ,uCi/ml, 20 Ci/mmol; 1 Ci = 3.7 X 10"° Bq). cell hybrids. The cells were washed with saline G, treated with hypotonic In the present study, we examined somatic cell hybrids pro- solution (1% sodium citrate) for 15 min, and fixed with meth- duced from the fusion between UV-sensitive Q31 cells and nor- anol/acetic acid mixture, 3:1 (vol/vol). The fixed cells were mal human lymphocytes. We mapped a gene on human chro- spread into glass slides and treated with 5% trichloroacetic acid mosome 13 that appears to compensate for the DNA repair defect at 4°C. After the slides were washed with water and ethanol, they were dried, dipped into 1: 1-diluted NRM2 nuclear emul- The publication costs of this article were defrayed in part bypage charge payment. This article must therefore be hereby marked "advertise- Abbreviations: XP, xeroderma pigmentosum; HAT, hypoxanthine/ ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. amethopterin/thymidine; UDS, unscheduled DNA synthesis. 5655 Downloaded by guest on October 1, 2021 5656 Genetics: Hori et al. Proc. Natl. Acad. Sci. USA 80 (1983) Table 1. Segregation tests for UV sensitivity identified by their G-banding patterns. The alkaline Giemsa staining method (13), with slight modifications, was used to No. of No. of confirm the presence of human chromosomes. By this method, clones HAT or TG segregantst human chromosomes stain faint blue and mouse chromosomes Period Cells tested resistance* Uvr UV stain dark purple. The constitutive heterochromatin of mouse I HLQ3-1 60 HATr 58 2 chromosomes stains a light blue, whereas that of some of the 57 TGr 56 1 human chromosomes stain purple. HLQ3-2 60 HATr 35 25 79 TGr 40 39 II HLQ3-1-1 150 TGr 25 125 RESULTS HLQ3-2-1 120 r ~r 47 73 Subcloning of Hybrid Cells and Segregation Tests for UV in 1R3 132 TGr 127 5 Sensitivity. Cells that survived after the first HAT selection were 1R5 260 TGr 258 2 plated onto two sets of agar plates containing either HAT or 6- 2R5 180 TGr 178 2 thioguanine (5 tug/ml) to determine the efficiency of HAT se- IV iR5-R1 240 TGr 239 1 lection. The plating efficiency on the HAT plate was low, i.e., * HATr, HAT resistant; TGr, 6-thioguanine resistant. 3%, indicating that the initial HAT selection was inefficient. Of tUW, UV resistant; UV8, UV sensitive. 25 HAT-resistant clones tested for UV sensitivity, 23 clones were UV resistant. Two UV-resistant clones, HLQ3-1 and HLQ3-2, sion (Sakura, Tokyo), and exposed for 5-7 days at 4°C. The slides were isolated. They exhibited essentially the same UV-resistant were developed, fixed, and stained with 3% Giemsa solution dose-response curves as did the parental L5178Y cells (data not for 3 min. shown). These hybrid clones contained 7-16 human chromo- To assay the activity of UDS, we scored the numbers of sil- somes and nearly two sets of mouse genomes. These results ver grains on lightly labeled non-S-phase nuclei. In these con- indicated that some human chromosomes can compensate the ditions, cells in the S phase were heavily labeled and clearly UV sensitivity of Q31 in mouse-human cell hybrids. None of distinguishable from non-S-phase cells. For control and UV- the 6-thioguanine-resistant clones tested (125 clones) were UV treated cells, 50 and 100 non-S-phase nuclei were examined, resistant. These cells might be either unfused or self-fused Q31 respectively. cells that survived in inefficient HAT selection. Assays for Chromosomal Sensitivity. Exponentially growing From the initial two hybrid clones HLQ3-1 and HLQ3-2, cells were exposed to UV at a dose of 6 J/m2, incubated for 6 subclones were isolated at various intervals, and segregation of hr. and treated with colchicine (0.2 jig/ml) for an additional 3 UV-sensitive clones was tested by the cell suspension spotting hr. The harvested cells were treated with 1% sodium citrate for method. The results are shown in Table 1. In the period I (8 15 min and fixed with the 3:1 methanol/acetic acid mixture. days after the first cloning), the majority of HLQ3-1 and HLQ3- Chromosome spreads were made by the air-drying method and 2 cells were still HAT resistant and segregation of 6-thiogua- stained with 3% Giemsa solution for 10 min.
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