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

ABSTRACT A human-hamster cell hybrid carrying a
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 kinase-
deficient hamster cell line (RJKM) to generate radiation hy-
brids retaining unselected fragments of this human chromo-
some. In two experiments, 223 hybrids were isolated in hypo-
xanthine/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.
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 de-
veloped 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). Irradiation-
reduced 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 re-
taining 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 esti-
mated physical lengths of the p and q arms (12). This map was
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104
found to be mostly consistent with currently available phys-
ical 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 delib-
erate selection. This hybrid originated from the fusion of an
HPRT-deficient hamster cell line (RJK88) with human lym-
phocytes. 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 pres-
ence 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 intracel-
lular content of BrdUrd. On the day of fusion, 1 x 107
RJ83.1FT cells were treated with trypsin, washed, and re-
suspended 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 sulfox-
ide, was added to the cell pellet, following a protocol essen-
tially 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.
Abbreviations: Mb, megabase(s); lod, logarithm of odds; cR, centi-
ray(s).
tTo whom reprint requests should be addressed.
 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. 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 to construct the map. The order shown in Table 1 maximized
of the probes, coded 0, 1, or 8 for negative, positive, or not the likelihood on the assumption of independent lod scores
done, respectively. There were two experiments. In the first (except for the order of D16S84 and D16S21, which is based
37 probes were used on 58 hybrids. In the second 14 probes on previous long-range restriction mapping) (15). The radia-
were used on 165 hybrids. No hybrid was positive for all tion hybrids give trivial support for the alternative order of
tested probes. However, 28 hybrids in the first experiment markers (Z = 0.2, odds = 1.7:1). A lod testing the preferred
and 99 hybrids in the second experiment were negative with order against an inversion of a pair of probes is called local
all probes used and so were uninformative with respect to support. Local support is weak also for the four clusters
differential retention and cotransference. These hybrids were (D16S139, D16S125, D16S94), (D16S138, D16S63), (D16S80,
excluded, leaving a maximum of 30 observations on each D16S144, D16S45, D16S82, D16S81, D16S143), and
probe in the first experiment and 66 in the second (Table 1). (D16S10, D16S177) of the short arm, and for the two clusters
Retention and cotransference frequencies were converted to (D16S107, D16S179, D16S10, D16S177, UVO) and (TAT,
estimates of association (6) and corresponding logarithm of D16S14) of the long arm. For other markers, local support >2
odds (lod scores) (Z) and submitted to multiple pairwise (corresponding to odds of 100:1) is strong support for order.
analysis by the MAP program (32, 33) as described by Many loci, especially near the centromere, give isolated
Lawrence et al. (12). In a given analysis only paracentric loci reactions discordant with flanking markers. D16Z3 and
(in the same chromosome arm) were mapped, to satisfy the D16S123 show a remarkably high frequency of positive (0.23)
assumption that maximal retention is terminal. and negative (0.16) isolated reactions, respectively.
The order and support shown in Table 1 were based on
RESULTS separate estimates of the asymptote L for each experiment
Experiments 1 and 2 were analyzed separately and found to and chromosome arm (Table 2). L denotes the conditional
give consistent locus orders. Therefore, the data were pooled probability that a locus separated from its centromere be
106 Medical Sciences: Ceccherini et al.
Table 2. x2tests (E = 0, p = 1, K = 1)
X2 value
p arm q arm
Hypothesis 1 2 1 2 Total
L= P 331.00 133.48 221.88 61.22 747.58
L = Pmin 216.42 125.82 116.28 37.07 495.59
L 211.18 87.69 115.58 21.91 436.36
Value of L 0.178 0.170 0.160 0.160
1, First experiment; 2, second experiment.
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 assump-
tions 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 inconse-
quential. 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 re-
actions is complex and not attributable solely to error (12).
Estimates of map length in centirays (cR) are dosage-
dependent 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 sup-
ported by our radiation hybrid data have been identified in
different regions of the chromosome 16 (Table 1). In partic-
ular 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 Ratio x2
p 39 152 39 0.26 257.19
q 59* 235 48 0.20 154.83
df, Degrees of freedom.
*Including region distal to D16S5.
Proc. Natl. Acad. Sci. USA 89 (1992)
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 prefer-
ential 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 cen-
tromere, 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 heterochro-
matin, 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 dis-
tances 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 consid-
ered, 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 long-
range 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 pulsed-
df field gel electrophoresis data available for the same intervals
of the short arm. In particular, as shown in Table 4, the
256 physical distance between D16S84 and D16S94 has been
238 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
 Medical Sciences: Ceccherini et al. Proc. Natl. Acad. Sci. USA 89 (1992) 107

Table 4. Location database for chromosome 16

Radiation
Cytogenetic Genetic map, cM Physical hybrid map,
Locus Region assignment Male Both sexes Female map, Mb Mb
pter pter 0.0 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 1.10 1.7
D16S84 D p 7.0 1.0 2.00 1.7
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
D16S101 K 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 ql2-q13 54.4
D16S124 T ql3-q21 61.7
D16S107 Q ql3-q21 65.8
D16S179 p q 66.6
D16S10 Q ql3-q22.1 57.2 67.1
D16S177 Q q 67.3
Uvo T q22.1 68.1
D16S4 S q22.1 60.0 68.8
TAT x q22.1 60.0 72.1
D16S14 w q22.1 72.9
D16S162 w ql2-qter 73.8
D16S15 VWXY q22-q24 77.3
D16S176 x q 81.2
D16S5 x 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)-
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, radi-
ation 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 data-
bases 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).
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