Proc. Nati. Acad. Sci. USA Vol. 89, pp. 104-108, January 1992 Medical Sciences Construction of a map of chromosome 16 by using radiation hybrids (somatic cell hybrids/physical maps/multiple pairwise analysis) I. CECCHERINI*t, G. ROMEO*, S. LAWRENCEt, M. H. BREUNING§, P. C. HARRIS¶, H. HIMMELBAUERII, A. M. FRISCHAUFII, G. R. SUTHERLAND**, G. G. GERMINOtt, S. T. REEDERS#t, AND N. E. MORTONt *Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy; tDepartment of Community Medicine, University of Southampton, Southampton General Hospital, Southampton S09 4XY, United Kingdom; §Department of Human Genetics, State University of Leiden, Wassenaarseweg 72-2333 AL Leiden, The Netherlands; 1Medical Research Council Molecular Haematology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom; IIImperial Cancer Research Fund, P.O. Box 123, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom; **Department of Cytogenetics and Molecular Genetics, Adelaide Children's Hospital, North Adelaide 5006, Australia; ttDepartment of Nephrology, School of Medicine, P.O. Box 3333, Yale University, New Haven, CT 06510; and tHoward Hughes Medical Institute, Yale University, New Haven, CT 06520 Contributed by N. E. Morton, September 9, 1991 ABSTRACT A human-hamster cell hybrid carrying a found to be mostly consistent with currently available phys- single copy of chromosome 16 as the only human genetic ical and genetic linkage data. material was irradiated with a single dose of -rays (7000 rads; 1 rad = 0.01 Gy) and then fused with a thymidine kinase- MATERIALS AND METHODS deficient hamster cell line (RJKM) to generate radiation hy- RJ83.1FT is a hamster-human hybrid cell line containing a brids retaining unselected fragments of this human chromo- single chromosome 16 (as the only human genetic material), some. In two experiments, 223 hybrids were isolated in hypo- which is retained in about 95% of cells in absence of delib- xanthine/aminopterine/thymidine (HAT) medium and erate selection. This hybrid originated from the fusion of an screened with 38 DNA probes, corresponding to anonymous HPRT-deficient hamster cell line (RJK88) with human lym- DNA or gene sequences localized on chromosome 16. The most phocytes. RJKM, a Chinese hamster cell line deficient in likely order and location of the 38 DNA sequences were thymidine kinase activity, was obtained from T. Mohandas established by multiple pairwise analysis and scaled to estimate (Harbor General Hospital, University of California, Los physical distance in megabases. The order and the distances Angeles). Cells were grown at 370C in RPMI 1640 medium thus obtained are mostly consistent with available data on supplemented with 18% (vol/vol) fetal calf serum, penicillin genetic and physical mapping ofthese markers, illustrating the (100 units/ml), and streptomycin (100 ttg/ml). usefulness of radiation hybrids for mapping. Prior to irradiation, RJKM cells were grown in the pres- ence of 50 nM 5-bromodeoxyuridine (BrdUrd) for 4 days and Somatic cell hybrids represent a powerful approach for in its absence for 2 days prior to the experiment to minimize mapping of human DNA sequences and a useful reagent for both thymidine kinase-sufficient revertants and the intracel- cloning. In recent years various procedures have been de- lular content of BrdUrd. On the day of fusion, 1 x 107 veloped for the transfer of small fragments of the human RJ83.1FT cells were treated with trypsin, washed, and re- genome into a rodent-cell background. One ofthese methods suspended in 10 ml of serum-free RPMI 1640 medium. This is represented by the irradiation and fusion gene transfer cell suspension was y-irradiated at 0C using a Gammacell technique, first described by Goss and Harris (1). Irradiation- 1000 apparatus (Atomic Energy, Ottawa) at a rate of 437 reduced cell hybrids have been generated for selected or rads/min (1 rad = 0.01 Gy) for about 16 min (exposure, 7000 unselected portions of the human genome to introduce DNA rads). An equal proportion of irradiated RJ83.1FT cells and fragments carrying genes responsible for inherited human unirradiated RJKM cells were mixed and centrifuged, and 0.5 diseases into a rodent background (2-4). These hybrids have ml of50% (wt/vol) polyethylene glycol Mr 1000 in RPMI 1640 also proved to be useful for mapping purposes (5-9). In medium supplemented with 10% (vol/vol) dimethyl sulfox- particular, maps of the proximal and distal long arm of ide, was added to the cell pellet, following a protocol essen- the tially identical to a published procedure (13). After fusion, chromosome 21 have been constructed by analyzing cells were plated in 100-mm plastic dishes and incubated at cosegregation of chromosome 21-specific DNA sequences in 370C for 2-3 weeks. Hypoxanthine/aminopterin/thymidine human-hamster radiation hybrids that were not subjected to (HAT) medium was added 2 days after fusion and replaced deliberate selection for any particular chromosome 21 gene every 3-4 days thereafter. HAT-resistant colonies, visible at (10, 11). By using such radiation hybrids, it has been possible 10-14 days, were isolated with cloning cylinders. Only one to order human DNA sequences independently of any other colony per dish was picked and expanded for analysis. No information and to estimate distances between loci on the colonies were observed in control dishes of irradiated basis of the principle that the probability ofcotransference of RJ83.1FT cells plated in HAT medium. Revertant RJKM a pair of loci decreases with the distance between them. We colonies were observed at a frequency of 5 x 10-6. have followed this approach to construct a radiation map of The cloned DNA sequences used as probes in this study are chromosome 16 starting from a human-hamster hybrid re- listed in Table 1. Probe 16.2.4 was isolated by B. Wirth at the taining human chromosome 16 as its only human genetic Imperial Cancer Research Fund. DNA (10 ,ug) prepared from component. Retention frequencies for each of38 markers and the hybrids and the parental cell lines was digested with Taq cotransference frequencies for each pair of markers were I, HindIII, Msp I, or Pst I, electrophoresed in 0.8% agarose, submitted to multiple pairwise analysis. The best order of transferred to a nylon membrane (Hybond, Amersham), and markers was sought and distances were scaled to the esti- hybridized to each probe by following established conditions mated physical lengths ofthe p and q arms (12). This map was (31). Each filter was reused up to 10 times. The publication costs of this article were defrayed in part by page charge Abbreviations: Mb, megabase(s); lod, logarithm of odds; cR, centi- payment. This article must therefore be hereby marked "advertisement" ray(s). in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 104 Downloaded by guest on October 1, 2021 Medical Sciences: Ceccherini et al. Proc. Natl. Acad. Sci. USA 89 (1992) 105 Table 1. Radiation hybrid map of chromosome 16 Informative Retention Isolated Ref. Flanking hybrids, frequency reactions for Locus Probe marker Support no. 1 2 + - locus (5'HVR) 5'HVR D16S85 1.5 0.30 0.26 14 D16S85 3'HVR D16S21 2.7 0.30 0.23 14 D16S21 FR3.42 D16S84 -0.2 30 0.27 15 D16S84 CMM65 D16S139 1.1 94 0.27 0.25 0.02 16 D16S139 N54 D16S125 0.0 94 0.27 0.20 17 D16S125 26.6 D16S94 0.0 29 0.24 18 D16S94 VK5 D16S138 1.5 96 0.27 0.24 19 D16S138 N2 D16S63 0.0 30 0.23 20 D16S63 CRI0327 D16S80 4.1 30 0.23 14 D16S80 24.1 D16S144 0.0 30 0.27 21 D16S144 LOM2B D16S45 0.0 30 0.27 22 D16S45 CRI090 D16S82 0.0 30 0.27 14 D16S82 41.1 D16S81 0.0 30 0.27 18 D16S81 3.15 D16S143 0.0 96 0.27 0.18 0.01 0.01 18 D16S143 16/116 D16S119 10.8 30 0.24 23 D16S119 2.36 D16S3 1.3 30 0.20 24 D16S3 ACH92 D16S79 1.3 30 0.23 25 D16S79 36.1 D16S% 3.6 30 0.27 18 D16S96 VK20B D16S120 18.9 30 0.33 0.24 0.02 0.02 26 D16S120 1.57 D16S101 3.4 30 0.37 24 D16S101 VK22 D16S36 5.4 96 0.33 0.47 0.09 0.04 26 D16S36 16/12 cen 9.3 30 0.67 0.10 0.03 23 cen D16Z3 0.1 D16Z3 HUR195 (16.2.4) 2.5 30 0.83 0.64 0.23 0.04 27 (16.2.4) 16.2.4 D16S123 14.7 30 0.70 0.07 0.07 D16S123 2.46 D16S124 13.4 30 0.40 0.03 0.16 24 D16S124 1.99 D16S107 8.2 95 0.43 0.20 0.05 0.03 24 D16S107 VK26.F13 D16S179 0.7 30 0.32 0.03 26 D16S179 16/67 D16S10 0.1 30 0.30 23 D16S10 ACHF3.A3 D16S177 0.0 30 0.29 28 D16S177 16/63 Uvo 0.9 30 0.27 23 Uvo V%1 D16S4 0.6 90 0.23 0.08 0.01 0.02 29 D16S4 ACH207 TAT 12.1 30 0.27 0.03 25 TAT BSO.9 D16S14 0.2 0.27 0.24 0.02 30 D16S14 ACH202 D16S162 0.3 30 0.23 25 D16S162 16/08 D16S15 5.1 30 0.27 23 D16S15 ACH208 D16S176 2.3 30 0.20 25 D16S176 16/60 D16S5 4.3 30 0.23 23 D16S5 ACH224 96 0.17 0.12 25 1, First experiment; 2, second experiment; +, positive isolated reactions; -, negative isolated reactions.