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. HIMMELBAUER II, 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 A human-hamster cell hybrid carrying a ABSTRACT single copy of chromosome 16 as the only human genetic material was irradiated with a single dose of -rays (7000 rads; 1 rad = 0.01 Gy) and then fused with a thymidine kinasedeficient hamster cell line (RJKM) to generate radiation hybrids retaining unselected fragments of this human chromosome. In two experiments, 223 hybrids were isolated in hypoxanthine/aminopterine/thymidine (HAT) medium and screened with 38 DNA probes, corresponding to anonymous DNA or gene sequences localized on chromosome 16. The most likely order and location of the 38 DNA sequences were established by multiple pairwise analysis and scaled to estimate physical distance in megabases. The order and the distances thus obtained are mostly consistent with available data on genetic and physical mapping of these markers, illustrating the usefulness of radiation hybrids for mapping.
found to be mostly consistent with currently available physical and genetic linkage data.
MATERIALS AND METHODS RJ83.1FT is a hamster-human hybrid cell line containing a single chromosome 16 (as the only human genetic material), which is retained in about 95% of cells in absence of deliberate selection. This hybrid originated from the fusion of an HPRT-deficient hamster cell line (RJK88) with human lymphocytes. RJKM, a Chinese hamster cell line deficient in thymidine kinase activity, was obtained from T. Mohandas (Harbor General Hospital, University of California, Los Angeles). Cells were grown at 370C in RPMI 1640 medium supplemented with 18% (vol/vol) fetal calf serum, penicillin (100 units/ml), and streptomycin (100 ttg/ml). Prior to irradiation, RJKM cells were grown in the presence of 50 nM 5-bromodeoxyuridine (BrdUrd) for 4 days and in its absence for 2 days prior to the experiment to minimize both thymidine kinase-sufficient revertants and the intracellular content of BrdUrd. On the day of fusion, 1 x 107 RJ83.1FT cells were treated with trypsin, washed, and resuspended in 10 ml of serum-free RPMI 1640 medium. This cell suspension was y-irradiated at 0C using a Gammacell 1000 apparatus (Atomic Energy, Ottawa) at a rate of 437 rads/min (1 rad = 0.01 Gy) for about 16 min (exposure, 7000 rads). An equal proportion of irradiated RJ83.1FT cells and unirradiated RJKM cells were mixed and centrifuged, and 0.5 ml of 50% (wt/vol) polyethylene glycol Mr 1000 in RPMI 1640 medium supplemented with 10% (vol/vol) dimethyl sulfoxide, was added to the cell pellet, following a protocol essentially identical to a published procedure (13). After fusion, cells were plated in 100-mm plastic dishes and incubated at 370C for 2-3 weeks. Hypoxanthine/aminopterin/thymidine (HAT) medium was added 2 days after fusion and replaced every 3-4 days thereafter. HAT-resistant colonies, visible at 10-14 days, were isolated with cloning cylinders. Only one colony per dish was picked and expanded for analysis. No colonies were observed in control dishes of irradiated RJ83.1FT cells plated in HAT medium. Revertant RJKM colonies were observed at a frequency of 5 x 10-6. The cloned DNA sequences used as probes in this study are listed in Table 1. Probe 16.2.4 was isolated by B. Wirth at the Imperial Cancer Research Fund. DNA (10 ,ug) prepared from the hybrids and the parental cell lines was digested with Taq I, HindIII, Msp I, or Pst I, electrophoresed in 0.8% agarose, transferred to a nylon membrane (Hybond, Amersham), and hybridized to each probe by following established conditions (31). Each filter was reused up to 10 times.
Somatic cell hybrids represent a powerful approach for mapping of human DNA sequences and a useful reagent for cloning. In recent years various procedures have been developed for the transfer of small fragments of the human genome into a rodent-cell background. One of these methods is represented by the irradiation and fusion gene transfer technique, first described by Goss and Harris (1). Irradiationreduced cell hybrids have been generated for selected or unselected portions of the human genome to introduce DNA fragments carrying genes responsible for inherited human diseases into a rodent background (2-4). These hybrids have also proved to be useful for mapping purposes (5-9). In particular, maps of the proximal and distal long arm of chromosome 21 have been constructed by analyzing the cosegregation of chromosome 21-specific DNA sequences in human-hamster radiation hybrids that were not subjected to deliberate selection for any particular chromosome 21 gene (10, 11). By using such radiation hybrids, it has been possible to order human DNA sequences independently of any other information and to estimate distances between loci on the basis of the principle that the probability of cotransference of a pair of loci decreases with the distance between them. We have followed this approach to construct a radiation map of chromosome 16 starting from a human-hamster hybrid retaining human chromosome 16 as its only human genetic component. Retention frequencies for each of 38 markers and cotransference frequencies for each pair of markers were submitted to multiple pairwise analysis. The best order of markers was sought and distances were scaled to the estimated physical lengths of the p and q arms (12). This map was The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: Mb, megabase(s); lod, logarithm of odds; cR, centi-
ray(s). tTo whom reprint requests should be addressed.
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Table 1. Radiation hybrid map of chromosome 16
Locus (5'HVR) D16S85 D16S21
D16S84 D16S139 D16S125 D16S94 D16S138 D16S63 D16S80 D16S144 D16S45 D16S82 D16S81 D16S143 D16S119 D16S3 D16S79 D16S96 D16S120 D16S101 D16S36 cen D16Z3
Probe 5'HVR 3'HVR FR3.42 CMM65 N54 26.6 VK5 N2 CRI0327 24.1 LOM2B CRI090 41.1 3.15
16/116 2.36 ACH92 36.1 VK20B 1.57 VK22
16/12
Flanking marker D16S85 D16S21 D16S84 D16S139 D16S125 D16S94 D16S138 D16S63 D16S80 D16S144 D16S45 D16S82 D16S81 D16S143 D16S119 D16S3 D16S79 D16S% D16S120 D16S101 D16S36 cen D16Z3 (16.2.4) D16S123 D16S124 D16S107 D16S179 D16S10 D16S177
Support 1.5 2.7 -0.2 1.1 0.0 0.0 1.5 0.0 4.1 0.0 0.0 0.0 0.0 0.0 10.8 1.3 1.3 3.6 18.9 3.4 5.4 9.3 0.1 2.5 14.7 13.4 8.2 0.7 0.1 0.0 0.9 0.6 12.1 0.2 0.3 5.1 2.3
Informative hybrids, no. 30 94 94 29
96 30 30 30 30 30 30 96 30 30 30 30 30 30
96 30
Isolated reactions
Retention frequency 1 0.30 0.30 0.27 0.27 0.27
0.27 0.23 0.23 0.27 0.27 0.27 0.27 0.27 0.24 0.20 0.23 0.27 0.33 0.37 0.33 0.67
-
2 0.26 0.23
+
0.25 0.20 0.24 0.24
0.02
0.18
0.01
0.01
0.24
0.02
0.02
0.47
0.09 0.10
0.04 0.03
Ref. for locus 14 14 15 16 17 18 19 20 14 21 22 14 18 18 23 24 25 18 26 24 26 23
27 0.04 0.23 0.64 0.83 30 HUR195 0.07 0.07 0.70 30 16.2.4 (16.2.4) 24 0.16 0.03 0.40 30 2.46 D16S123 24 0.03 0.05 0.20 0.43 95 1.99 D16S124 26 0.03 0.32 30 VK26.F13 D16S107 23 0.30 30 D16S179 16/67 28 0.29 30 ACHF3.A3 D16S10 23 0.27 30 Uvo D16S177 16/63 29 0.02 0.01 0.08 0.23 90 D16S4 V%1 Uvo 25 30 0.03 0.27 TAT D16S4 ACH207 30 0.02 0.24 0.27 D16S14 TAT BSO.9 25 30 0.23 D16S162 D16S14 ACH202 23 30 0.27 D16S15 D16S162 16/08 25 30 0.20 D16S176 ACH208 D16S15 23 30 0.23 D16S5 4.3 D16S176 16/60 96 25 0.12 0.17 D16S5 ACH224 1, First experiment; 2, second experiment; +, positive isolated reactions; -, negative isolated reactions. Local support is log1o(L1, L2), where L1 is the likelihood under the favored order and L2 is the likelihood under transposition with the flanking locus (32).
For each hybrid a record was created with a field for each of the probes, coded 0, 1, or 8 for negative, positive, or not done, respectively. There were two experiments. In the first 37 probes were used on 58 hybrids. In the second 14 probes were used on 165 hybrids. No hybrid was positive for all tested probes. However, 28 hybrids in the first experiment and 99 hybrids in the second experiment were negative with all probes used and so were uninformative with respect to differential retention and cotransference. These hybrids were excluded, leaving a maximum of 30 observations on each probe in the first experiment and 66 in the second (Table 1). Retention and cotransference frequencies were converted to estimates of association (6) and corresponding logarithm of odds (lod scores) (Z) and submitted to multiple pairwise analysis by the MAP program (32, 33) as described by Lawrence et al. (12). In a given analysis only paracentric loci (in the same chromosome arm) were mapped, to satisfy the assumption that maximal retention is terminal.
RESULTS Experiments 1 and 2 were analyzed separately and found to give consistent locus orders. Therefore, the data were pooled
to construct the map. The order shown in Table 1 maximized the likelihood on the assumption of independent lod scores (except for the order of D16S84 and D16S21, which is based on previous long-range restriction mapping) (15). The radiation hybrids give trivial support for the alternative order of markers (Z = 0.2, odds = 1.7:1). A lod testing the preferred order against an inversion of a pair of probes is called local support. Local support is weak also for the four clusters
(D16S139, D16S125, D16S94), (D16S138, D16S63), (D16S80, D16S144, D16S45, D16S82, D16S81, D16S143), and (D16S10, D16S177) of the short arm, and for the two clusters (D16S107, D16S179, D16S10, D16S177, UVO) and (TAT, D16S14) of the long arm. For other markers, local support >2 (corresponding to odds of 100:1) is strong support for order. Many loci, especially near the centromere, give isolated reactions discordant with flanking markers. D16Z3 and D16S123 show a remarkably high frequency of positive (0.23) and negative (0.16) isolated reactions, respectively. The order and support shown in Table 1 were based on separate estimates of the asymptote L for each experiment and chromosome arm (Table 2). L denotes the conditional probability that a locus separated from its centromere be
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Table 2. x2tests (E = 0, p = 1, K = 1) X2 value p arm q arm Hypothesis 1 2 2 1 L= P 331.00 133.48 221.88 61.22 L = Pmin 216.42 125.82 116.28 37.07 L 211.18 87.69 115.58 21.91 Value of L 0.178 0.170 0.160 0.160 1, First experiment; 2, second experiment.
Total 747.58 495.59 436.36
retained, whereas Pi is the marginal probability that locus i is retained. The hypothesis of equal retention frequencies (L = P) is strongly rejected (x2 = 311.22) as is the hypothesis that L is the minimum value of Pi (X2 = 59.23). As described by Lawrence et al. (12), these tests were based on the assumptions of no interference (p = 1) and consistent maps for conditional retention and loss (K = 1). In the pooled data, residual x2 is acceptable for the p arm but surprisingly small for the q arm (Table 3). This probably reflects cotransference frequencies less than L, which give a lod score of zero and an expected lod score near zero, reducing the degrees of freedom from its conventional value of n - k, where n is the number of observed lod scores and k is the number of parameters estimated. For constructing the map and testing hypotheses this hypovariability is inconsequential. It is absent from the p arm since several loci with zero distance were pooled into megaloci, whereas small but nonzero intervals in the q arm were not pooled. We did not attempt error filtration because the etiology of isolated reactions is complex and not attributable solely to error (12). Estimates of map length in centirays (cR) are dosagedependent and were, therefore, scaled to megabases (Mb) under the plausible but unproven assumption that radiation breakage is uniform on the physical map. The distance between 5'-HVR and the centromere was estimated by MAP to be 152 cR, which we take as the length of the p arm corresponding to 39 Mb (34). The distance between the centromere and D16S5 was estimated as 235 cR. The distal marker is proximal to band q24.2, leaving unspanned the subtelomeric 18% of the q arm (35). From a physical estimate of 59 Mb for 16q (34), we calculate that the map spans 48 Mb of the q arm. The cR estimates are in reasonable agreement, although they suggest a larger map for the q arm than one might expect from the physical length. Information about this map is given in Table 4.
DISCUSSION The analysis of the data obtained with radiation hybrids of chromosome 16 suggests some conclusions concerning the use of this mapping approach that are of general interest. Clusters of probes whose relative order is weakly supported by our radiation hybrid data have been identified in different regions of the chromosome 16 (Table 1). In particular no resolution was achieved for the distal part of 16p where the numerous sequences, which have been ordered by other means, are very close to each other (15, 16, 19); since not all of these sequences have been separated by radiation events, we could only order clusters within which the relative order of the individual sequences cannot be determined. The Table 3. Arm lengths with megaloci Arm Mb cR cR scaled 39 152 39 p 59* 235 48 q df, Degrees of freedom. *Including region distal to D16S5.
Ratio
x2
0.26 0.20
257.19 154.83
df 256 238
relative order of the probes within the clusters (D16S107, D16S179, D16S10, D16S177, UVO) and (TAT, D16S14) on 16q is also weakly supported by our data. A higher radiation dose or a larger sample should provide order within these clusters. The decrease of the retention frequencies of the probes tested (Table 1), observed in both arms of the chromosome from the centromere to the telomere, suggests a preferential retention in our radiation hybrids of some DNA sequences near the centromere or else selection against subtelomeric sequences. This observation is in keeping with a previous report (6) and with a theoretic model that predicts the preferential retention of both centromere and telomeres in radiation hybrids (12). However, the slight increase of the presence of human fragments containing the p-telomeric locus D16S85 is too weak to confirm the expected preferential retention of the telomere of the short arm, while we could not test the retention of the q-telomere because our most distal probe D16S5 is 18% of the long arm away from its telomere. A possible radiosensitivity of the centromeric region might account for the positive and negative isolated reactions that have been observed clustered around the centromere and decreased toward the telomeres (Table 1). Since isolated positive and negative reactions cannot be explained only in term of the postulated preferential retention of the centromere, a different mechanism (perhaps a single ionization) must be invoked that is able to cut out short DNA fragments: once generated they might then be preferentially retained at some locations (e.g., high frequency of positive reactions at locus D16Z3) or lost at other locations (high frequency of negative reactions at locus D16S123). Radiosensitivity and/or preferential retention of the centromeric heterochromatin, which is proximal in the long arm, might also play a role in the apparent increase of the length of 16q when it is estimated in radiation units (cR), as observed above. The multiple pairwise analysis enabled us to estimate distances between pairs of adjacent loci in addition to their most likely order. The reliability of the map thus obtained has been assessed by comparing the location of the 38 probes considered, as suggested by the present work, with that known by previous genetic, physical, and cytogenetic assignment, as shown in Table 4. The most likely order of the 38 human DNA sequences is, with few exceptions, in agreement with the order of markers deduced from linkage data (16, 37, 38), from somatic cell hybrids carrying rearranged portions of this chromosome (36, 39), and from physical pulsed-field gel electrophoresis data (15, 39, 40). One of the exceptions is the order pter-D16S21-D16S84-cen, which is based on longrange restriction mapping (15) but is weakly contradicted by our radiation hybrid data (Z = 0.2 and odds = 1.7:1 for the alternative order). Other apparent inconsistencies found in the order of some loci, as predicted by our radiation map of the long arm with respect to the map recently obtained using a panel of somatic cell hybrids carrying rearranged portions of 16q (36), may be due to their weak local support, as discussed above. The discrepant location of both D16S124 and D16S36, which our radiation data mapped with strong support more proximal, respectively, in the long and in the short arms with respect to the rearranged somatic cell hybrids map, may be due to partial hybridization on another location as suggested from loci duplications reported for chromosome 16 (41). The physical distances between some probes, obtained through radiation hybrid data, are in agreement with pulsedfield gel electrophoresis data available for the same intervals of the short arm. In particular, as shown in Table 4, the physical distance between D16S84 and D16S94 has been estimated to be <1 Mb (39), which fits with our estimate of 1 Mb. Similarly D16S45 is -2 Mb away from D16S63 and within 200 kilobases of D16S144 (39). On the other hand the
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Table 4. Location database for chromosome 16
Radiation hybrid map, Locus Region Male Female Mb pter 0.0 (5'HVR) A p13.3 0.0 D16S85 A p13.3 0.0 0.0 0.0 0.35 0.8 D16S21 B p13.3 3.0 0.5 1.7 1.10 D16S84 D 7.0 1.0 1.7 2.00 p D16S139 E p13 2.2 D16S125 E p13 2.2 D16S94 E p13.3 14.6 <3.00 2.7 D16S138 p13 3.5 D16S63 F p13 14.0 2.0 3.5 D16S80 F pl3.3-pl3.13 4.8 D16S144 F pter-pl3.1 4.8 D16S45 F pter-p13 17.5-19.9* 2.5-3.2* 4.8 D16S81 F p13.3 4.8 D16S82 F p13.3 4.8 D16S143 F pter-pl3.1 4.8 D16S119 F pl3.3-pl3.13 7.9 D16S3 F pl3.3-pl3.13 9.1 D16S79 G pl3.13-pl3.11 38.2 10.5 D16S96 G pl3.13-pl3.11 13.2 D16S120 K pl3.11-pll.2 20.0 K D16S101 pl3.11-pll.2 24.3 D16S36 G pter-p13 32.0 cen cen 39.0 D16Z3 M qll.2 43.1 (16.2.4) 46.6 D16S123 NO 54.4 ql2-q13 D16S124 T ql3-q21 61.7 D16S107 ql3-q21 65.8 Q D16S179 p 66.6 q D16S10 57.2 ql3-q22.1 67.1 Q D16S177 67.3 Q q Uvo T q22.1 68.1 D16S4 S q22.1 60.0 68.8 x TAT q22.1 60.0 72.1 w D16S14 q22.1 72.9 w D16S162 ql2-qter 73.8 D16S15 VWXY q22-q24 77.3 x D16S176 81.2 q x D16S5 q23.1-q24 87.0 qter qter 98.0 Regions (A-Z) have been defined through the breakpoints present in somatic cell hybrids carrying rearranged portions of the short (25) and the long (36) arm of chromosome 16, as follows: pter(A)JS(B)PK32(C)CY14(D)NOH1(E)-
Cytogenetic assignment pter
Genetic map, cM Both sexes
Physical map, Mb 0.0
23HA(F)CY19(G)SMI/FRA16A(H)PK30/PAR(I)CY13(J)CY15(K)CY12(L)centromere(M)CY8(N)CY135(0)CY7(P)CY13OP(Q)CY125P/FRA16B(R)CY13OD(S)CY4(T)CY6/CY125D(U)CY5(V)CY170(W)CY124(X)CY120(Y)CY2/ CY3(Z)qter. Cytogenetic assignment and genetic and physical estimates of distance have been reported (15, 16, 19, 36-39). The physical map distance was from pulsed-field gel electrophoresis data. Estimates of distance of the radiation hybrids map in centirays were scaled to megabases according to the total length of chromosome 16, as shown in Table 3. *The two values represent different estimates of the same interval, as reported in the literature.
apparent overestimate of the distances between the most distal probes in 16p and the p-telomere might be due to the
mentioned preferential retention of the telomeric sequences, which accounts then for the apparent increase in the length of this region. In spite of some inconsistencies, which still need to be reconciled, possibly by a different mapping approach, radiation hybrids have been confirmed to be a powerful tool to obtain order and distance between pair of markers. The integration of the map obtained through radiation hybrids with available cytogenetic, genetic, and physical data may then represent an useful method to construct location databases for human chromosomes, as shown in Table 4 for chromosome 16 and reported for other human chromosomes (12, 32).
We thank S. Castagnola, S. Giambarrasi, A. De Lapi, G. Caridi, and G. Panzica for their technical assistance; Drs. R. K. Moyzis, G. Scherer, R. Kemler, B. Wirth, and V. J. Hyland for providing probes for the characterization of RH; Dr. M. Rocchi for providing the initial monochromosomal hybrid; and Prof. I. Barrai and Dr. V. J. Hyland for helpful discussion. This work was supported by "Progetto Finalizzato Ingegneria Genetica", Consiglio Nazionale delle Ricerche (Rome). 1. Goss, S. J. & Harris, H. (1975) Nature (London) 255, 14451458. 2. Cox, D. R., Pritchard, C. A., Uglum, E., Casher, D., Kobori, J. & Myers, R. M. (1989) Genomics 4, 397-407. 3. Glaser, T., Rose, E., Morse, H., Housman, D. & Jones, C. (1990) Genomics 6, 48-64. 4. Goodfellow, P. J., Povey, S., Nevanlinna, H. A. & Goodfellow, P. N. (1990) Somatic Cell Mol. Genet. 16, 163-171.
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