Cisplatin increases meiotic crossing-over in mice.
ABSTRACT Genetic mapping of traits and mutations in mammals is dependent upon linkage analysis. The resolution achieved by this method is related to the number of offspring that can be scored and position of crossovers near a gene. Higher precision mapping is obtained by expanding the collection of progeny from an appropriate cross, which in turn increases the number of potentially informative recombinants. A more efficient approach would be to increase the frequency of recombination, rather than the number of progeny. The anticancer drug cisplatin, which causes DNA strand breakage and is highly recombinogenic in some model organisms, was tested for its ability to induce germ-line recombination in mice. Males were exposed to cisplatin and mated at various times thereafter to monitor the number of crossovers inherited by offspring. We observed a striking increase on all three chromosomes examined and established a regimen that nearly doubled crossover frequency. The timing of the response indicated that the crossovers were induced at the early pachytene stage of meiosis I. The ability to increase recombination should facilitate genetic mapping and positional cloning in mice.
- [show abstract] [hide abstract]
ABSTRACT: A total of 57 different microsatellite variants have been typed in one or more of five different sets of recombinant inbred (RI) mouse strains. The present report concentrates on markers for Chromosomes (Chrs) 10, 16, 18, 19 and X. These markers extend the regions swept in these RI strains, provide reference markers for integrating RI and conventional maps, and provide additional estimates of genetic distances. Multilocus maps, based on maximum likelihood analysis of present and previously published RI SDPs on five chromosomes, are presented. Unexpectedly, three microsatellite markers, previously assigned to Chr 10, detected polymorphic fragments mapping to other chromosomes.Mammalian Genome 09/1995; 6(8):493-8. · 2.42 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: To identify environmental carcinogens there is a need for inexpensive and reliable short-term tests that can be used to predict the carcinogenic potential of any given substance with high accuracy. The Ames assay, which is based on the induction of mutations in Salmonella typhimurium, is the most extensively used short-term test but certain human or animal carcinogens exist that are persistently undetectable as mutagens with the Ames assay or with other short-term tests. There is a need for a short-term test to detect those carcinogens that are missed by the Ames assay. Carcinogenesis is in many cases associated with genome rearrangement. Because of this association a system screening for intrachromosomal recombination that results in genome rearrangement has been constructed for potential use as a short-term test in the yeast Saccharomyces cerevisiae. Evaluation of this recombination system shows that it is readily inducible by a variety of mutagenic as well as non-readily inducible by a variety of mutagenic as well as non-mutagenic carcinogens, including carcinogens that are not detectable by the Ames assay or by various other short-term tests, such as safrole, urethane, ethionine, auramine, methylene chloride, carbon tetrachloride, cadmium sulfate, aniline, dimethylhydrazine, aminotriazole, acetamide, thiourea and DDE. The present report shows the data for these as well as for additional agents, their response profiles with different concentrations of the agents and the protocol for the DEL system.Carcinogenesis 09/1989; 10(8):1445-55. · 5.64 Impact Factor
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ABSTRACT: Smaller chromosomes have higher rates of meiotic reciprocal recombination (centimorgans per kilobase pair) than larger chromosomes. This report demonstrates that decreasing the size of Saccharomyces cerevisiae chromosomal DNA molecules increases rates of meiotic recombination and increasing chromosome size decreases recombination rates. These results indicate that chromosome size directly affects meiotic reciprocal recombination.Science 05/1992; 256(5054):228-32. · 31.03 Impact Factor
Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 8681–8685, August 1997
Cisplatin increases meiotic crossing-over in mice
WILLIAM H. HANNEMAN, MARIE E. LEGARE, SHANNON SWEENEY, AND JOHN C. SCHIMENTI*
The Jackson Laboratory, Bar Harbor, ME 04609
Communicated by Mario R. Capecchi, University of Utah School of Medicine, Salt Lake City, UT, June 5, 1997 (received for review
March 10, 1997)
mammals is dependent upon linkage analysis. The resolution
achieved by this method is related to the number of offspring
that can be scored and position of crossovers near a gene.
Higher precision mapping is obtained by expanding the
collection of progeny from an appropriate cross, which in turn
A more efficient approach would be to increase the frequency
of recombination, rather than the number of progeny. The
anticancer drug cisplatin, which causes DNA strand breakage
and is highly recombinogenic in some model organisms, was
tested for its ability to induce germ-line recombination in
mice. Males were exposed to cisplatin and mated at various
by offspring. We observed a striking increase on all three
chromosomes examined and established a regimen that nearly
doubled crossover frequency. The timing of the response
indicated that the crossovers were induced at the early
pachytene stage of meiosis I. The ability to increase recom-
bination should facilitate genetic mapping and positional
cloning in mice.
Genetic mapping of traits and mutations in
A prime achievement of the genome project has been the
establishment of high-density genetic maps of humans and
mice (1, 2). The maps, and simple sequence repeat markers
that were used to construct them, have led to a revolution in
genetic analysis. These reagents form a scaffold for the cre-
ation of physical maps, consisting of ordered clones spanning
the entire genome. These advances have simplified and accel-
erated the pace at which genes or traits can be localized and
With the advent of such resources, the limiting factor in
positional cloning of genes has become the collection of
informative families (for humans) or conducting of crosses (for
mice) in which the gene of interest is segregating. Now that
extensive collections of molecular markers along chromo-
somes are available, the bottleneck in high-resolution mapping
is the ability to obtain recombination breakpoints that delimit
a gene to a workably small interval. Ideally, this is well under
0.5 centimorgans. It is not uncommon for mapping crosses in
mice to involve several thousand progeny to achieve such
resolution. This is expensive and time consuming, especially if
the phenotype is incompletely penetrant or difficult to assay or
if crossovers are especially rare in the critical region.
An alternative to increasing cross size as a means to improve
mapping resolution is to boost recombination frequency, ei-
Recombination occurs in both mitotic and meiotic cells.
Although it is required for accurate chromosome segregation
in meiosis, this is not the case for mitotic crossing-over, which
is postulated to be a consequence of DNA repair (3). Because
certain types of DNA damage induce recombinational repair,
this property has been exploited in yeast, Drosophila melano-
gaster, and mammalian cell cultures to develop assays for
screening potential mutagenic or carcinogenic agents (4–7).
The chemotherapeutic agent cis-platinum(II)diammine di-
chloride (cisplatin, CP) is highly recombinogenic in assays with
Candida albicans, Saccharomyces cerevesiae, and somatic cells
of D. melanogaster (8, 9). RecA mutants of Escherichia coli and
Rad52-deficient yeast are hypersensitive to this DNA-adduct-
forming drug, implying a need for efficient recombinational
repair to survive its cytotoxic effects (10). Yeast deficient for
the excision repair gene RAD3 are also hypersensitive, imply-
ing that both excision and recombinational repair are required
to remove CP lesions. Similarly, human cells require ERCC-1
for repair of CP-induced damage (11). Presumably, the exci-
sion of interstrand CP crosslinks creates double-strand breaks,
which in turn stimulate recombinational repair (11–15). In-
deed, CP is known to induce double-strand breaks in Droso-
phila meiotic chromosomes and to disrupt synaptonemal com-
plexes in mice (16, 17).
These combined observations raised the possibility that CP
could induce meiotic recombination in mammals. We have
found that CP stimulated intrachromosomal meiotic gene
conversion in a transgenic mouse assay that enables the
detection of such events in spermatids (refs. 18 and 19; W.H.H.
and J.C.S., unpublished observations). Given the association
between gene conversion and crossing-over, we hypothesized
that this drug might also induce crossing-over in mice. We
report that male mice treated with CP undergo meiotic
crossing-over at levels up to twice that of controls. This drug
regimen should be useful in genetic mapping experiments in
mice whereby the number of meioses that must be screened to
localize a gene can be halved.
MATERIALS AND METHODS
Mouse Breeding and CP Treatment. DBA?2J females, more
than 2 months of age, were induced into estrous by adminis-
tration of pregnant mare’s serum and human chorionic go-
nadotropin as described (20). Because the females were more
than 2 months old, they did not superovulate with the treat-
ment. CP (10 mg?kg; Sigma) was injected intraperitoneally
immediately after dissolving at 1 mg?ml in 0.9% NaCl. Control
males were injected with the same volume of 0.9% NaCl.
Treated and control mice were taken from the same batches
of age-matched animals obtained from the production facility
at The Jackson Laboratory. Each male was placed into a cage
with two hormonally treated females (also age-matched be-
tween control and treated matings) at specified times after CP
injection and removed the following day. Drug treatment and
housing of the mice were performed with approval of the
Institutional Animal Care and Use Committee according to
American Association for the Accreditation of Laboratory
Animal Care guidelines.
DNA Isolation and Microsatellite Marker Typing. DNA was
isolated from brains of newborn pups as described (21).
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.
© 1997 by The National Academy of Sciences 0027-8424?97?948681-5$2.00?0
PNAS is available online at http:??www.pnas.org.
Abbreviation: CP, cisplatin.
*To whom reprint requests should be addressed. e-mail: firstname.lastname@example.org.
Microsatellite loci were typed in a modification of a reported
procedure (22). The PCR mixture contained 5 ?l of brain
DNA diluted 1:50 in water, 0.22 ?M primers, all four dNTPs
(each at 0.8 mM), 0.5 unit of Taq polymerase (Perkin–Elmer),
2.25 mM MgCl2, and 1? PCR buffer (Perkin–Elmer). Ampli-
fication was performed in a total reaction volume of 30 ?l, plus
a 10 ?l oil overlay, in 96-well plates on a thermal cycler (MJ
Research, Cambridge, MA). After denaturing at 97°C for 1
for 30 sec, 55°C for 30 sec, and 72°C for 30 sec, ending with a
5-min incubation at 72°C. PCR products were separated in
agarose gels (3% of a 3:1 NuSieve mixture from FMC) and
stained with Sybr green (FMC). The double and triple cross-
overs were typed twice to ensure accuracy.
The complete allele typing data are available via the World
Wide Web at: http:??www.jax.org??jcs?cisplatin.html.
Statistical Analysis. ?2and P values were derived from the
2?2contingencytest(assistedbythesoftware FISHER 2byKaz
Matsuki). Two values were compared in each test class:
recombinant and nonrecombinant. However, because there
were double and triple crossovers in CP-treated animals, the
nonrecombinant class was defined as (no. offspring ? no.
crossovers). G tests yielded nearly identical results.
RESULTS AND DISCUSSION
CP Increases Crossing-Over on Multiple Chromosomes. To
test for possible effects of CP on crossing-over, a set of F1
hybrid males (C57BL?6J ? DBA?2J) was exposed to a single
CP dose of 10 mg?kg and mated 28 days later to DBA?2J
females that were hormonally induced into estrous, and their
offspring were scored for recombination at multiple intervals
on three chromosomes. Sperm produced at this point would
have been at the mid-meiosis I prophase stage of spermato-
genesis at the time of treatment (ref. 23; discussed below).
Compared with age-matched controls, CP-treated males ex-
hibited no reduction in fertility; nearly all males from both
groups yielded productive matings with similar litter sizes.
DNA was extracted from pups of both groups and typed at
microsatellite loci along three randomly chosen chromo-
somes—chromosomes 10, 16, and 18. The results are summa-
rized in Fig. 1. The crossover frequency (no. crossovers?no.
progeny) in offspring of CP-treated males was elevated by
and mated 4 weeks thereafter. The control group was sham-injected with vehicle (0.9% NaCl). Results shown are for chromosomes 10, 16, and
18, as indicated. The thick vertical line represents each chromosome, and F indicates the position of the centromere. Microsatellite loci that were
typed in the crosses are indicated at the right of each chromosome, whereby the prefixes D10Mit, D16Mit, and D18Mit have been abbreviated with
M. The numbers listed in the intervals between microsatellites are recombination percentages in those particular intervals. Results from two
independent crosses with chromosome 10 are shown (the second cross in CP-2). The results from the first cross are presented immediately above
those from the second cross. The overall recombination frequency between the most distal and proximal markers are shown under the horizontal
lines near the bottom. The results of ?2analysis in a two-way contingency test are shown at the bottom. These reflect the probability that the overall
recombination frequency differs between control and treated groups. Independently summarized percentages for both experiments involving
chromosome 10 are given, along with the overall combined percentage as indicated. Contingency test results are given for both the first experiment
and the combined data on chromosome 10. The lengths of each chromosome as depicted do not reflect true physical or genetic size.
Crossover frequencies in CP-treated and control males. The treated group refers to male mice that were injected with CP at 10 mg?kg
8682Genetics: Hanneman et al.Proc. Natl. Acad. Sci. USA 94 (1997)
more than 75% on each chromosome, and all the increases
were statistically significant by the ?2test (Fig. 1). When the
data for all chromosomes was combined, the recombination
frequency rose from 35% per chromosome in the untreated to
62% in the CP-treated group—a highly significant increase
(?2? 12, P ? 8 ? 10?6). To assess whether progeny from any
individual male in the treated group contributed dispropor-
tionately to the data, a recombination index (total no. cross-
overs?no. pups sired) was calculated for each animal. Only one
of the control males had a higher recombination index than
treated animals (Fig. 2).
Timing and Repeatability of CP-Induced Recombination in
repeatability of the effect, (ii) ascertain the developmental
timing of the recombination induction, and (iii) determine
observed over time within individual animals. One set of males
(CP-2) was injected with CP as above and mated 3, 4, and 8
weeks later. A second sham-treated control group (CON-2)
was concurrently tested, and additional CP-treated mice were
mated 5 weeks after exposure. Crossing-over on chromosome
10 was measured in progeny from these experiments.
The results confirmed the sharp increase in recombination
induced by CP. The CON-2 group displayed a recombination
the CP-2 group mated after 4 weeks showed a frequency of
79% (Fig. 1), representing a more than 2-fold increase over the
controls. Interestingly, this was significantly higher (P ? 0.045)
than the original cohort (60%). The only difference between
the two experiments was that CP-2 males were also mated 1
week before the 4-week sampling point. One possible expla-
from the testis (24), the ejaculates from males of the first group
would have contained a mixture of sperm descended from
precursors at different developmental stages. The possibility
was considered that the mating of CP-2 males 3 weeks after
exposure may have enabled them to clear sperm whose pre-
cursors were postmeiotic at the time of CP exposure, thereby
effectively enriching the ejaculate for sperm that were derived
from CP-exposed pachytene spermatocytes. However, this
effect was not observed for chromosomes 16 and 18. Recom-
bination between the proximal and distal markers in CP-2 was
not statisically different from the CP-1 values on these chro-
mosomes (chromosome 16, 65% vs. 57%, P ? 0.47; chromo-
some 18, 60% vs. 50%, P ? 0.3. Data are available on the
World Wide Web at http:??www.jax.org??jcs?cisplatin.html).
When the data from all three chromosomes are combined, the
recombination fraction in CP-1 was 0.618 (89 of 144) and that
of CP-2 was 0.62 (89 of 143). Thus, the overall level of induced
crossovers appeared to be highly repeatable. It must be
considered, however, that cryptic double crossovers might not
have been detected on chromosomes 16 and 18 in CP-2.
The combined data reveal a peak of CP-induced recombi-
nation on chromosome 10 in gametes from males exposed 4
weeks before mating (Fig. 3). Importantly, this effect was
within the group (CP-2) that was sequentially mated. The
crossover rate in CP-2 rose from 57% at 3 weeks (see below)
to 79% at 4 weeks before declining at 8 weeks to a level (44%)
that was not significantly different than the combined fre-
quency (35%) of the two control groups (P ? 0.37; Table 1).
Spermatozoa present 8 weeks after treatment would have been
at the spermatogonium stage when exposed, so it appears that
CP did not have a marked effect upon mitotic recombination
in stem cells. The inductive effect of CP disappeared within 7
days after the peak at 4 weeks after treatment; males mated 5
weeks after exposure did not differ significantly (P ? 0.45)
from the combined controls (Table 1). These results suggest
that the spermatogenic cell types subject to induction of
recombination are those that require no more than 4–5 weeks
to develop into mature spermatozoa in the ejaculate.
Inspection of crossover distributions revealed some unusual
patterns. It is possible that the stimulation of recombination by
CP was nonuniform along the length of the chromosomes. For
example, although there was little difference between control
and treated in proximal chromosome 10 between D10Mit28
and D10Mit186, distal recombination frequencies were mark-
edly higher (Fig. 1). Typing of larger data sets with additional
markers should reveal whether CP has preferred sites of action
observation was made with regards to the offspring of CP-2
males mated 3 weeks after exposure. This group had a higher
crossover frequency on chromosome 10 relative to controls
(Table 1; the small sample size renders the results not highly
significant, P ? 0.073). However, the majority of crossovers (8
of 13) occurred in the D10Mit3–D10Mit186 interval, and 5 of
these arose in a single male’s litter of 7. This could not be
explained by a ‘‘jackpot’’ event in stem cells, because offspring
from this animal assayed at later time points (4 and 8 weeks)
did not show elevated recombination in the same interval. It
is unclear whether this curiosity was related to CP treatment.
The timing of recombination events in mammalian meiosis
is poorly characterized, in contrast to yeast. Crossing-over is
phase, but it is not clear just when these events are initiated.
Because the CP-induced crossing-over occurred during a
defined window of time, this information may be useful in
elucidating this issue. On the basis of the time course of
spermatogenesis in the mouse established by classical anatom-
ical and mutagenesis studies, CP-induced recombination
events, presumably a consequence of causing double-strand
breaks, can be traced to primary spermatocytes that have
progressed about 2 days into pachynema (23, 25, 26).
In yeast, double-strand breaks are detectable at the same
time as lateral axes of the synaptonemal complex form (3).
They disappear by the end of the zygotene and early pachytene
nation index [(total number crossovers for all chromosomes)?(number
pups sired ? 3)] was calculated and plotted for each of the CP-treated
and control males that yielded the data in Fig. 1 (excluding CP group
2 for chromsome 10). F, CP-treated males; E, controls.
Recombination indices for individual males. A recombi-
Genetics: Hanneman et al.Proc. Natl. Acad. Sci. USA 94 (1997)8683
stages, when the synaptonemal complex axes align to form the
tripartite structure. Thus in yeast, initial events of recombi-
nation occur well before exchange in pachynema. Our results
suggest that CP initiates recombination later than spontaneous
double-strand break formation and disappearance in yeast
meiosis. However, because the rate of spermatogenesis can
vary between strains of mice and the time required for passage
from the testis to the ejaculate (estimated to be 7.5–8 days) is
not concretely established, our conclusion that CP induces
recombination in early pachynema must be considered an
It has been reported that preleptotene?leptotene spermato-
cytes are much more sensitive to CP-induced chromosome
aberrations than are spermatocytes at the zygotene or
pachytene stages (ref. 27; preleptotene spermatocytes require
about 4 days to progress to pachynema), but it is not clear that
these clastogenic events are the same that would stimulate
crossing-over. Recent evidence from mice suggests that by the
early to midpachytene stage, events have progressed to a point
where they can be resolved rapidly into chiasmata (28). This
suggests that spontaneous initiation of recombination in sper-
matocytes occurs in the early pachytene stage or sooner. This
observation is generally consistent with our results. It is
possible that CP can be exploited as a tool to further refine the
window of time during which recombination is initiated during
meiosis in mice.
Recombination and Interference. Crossing-over is essential
to ensure accurate disjunction of homologous chromosomes
during meiosis. In most organisms, the number of crossovers
per meiosis is relatively constant. Recombinational interfer-
ence ensures that each chromosome pair has at least one
crossover. Otherwise, larger chromosomes would have multi-
ple crossovers, and smaller ones would often have none (29,
30). The results presented herein indicate that induction of
recombination by CP did not occur by elimination of inter-
ference. A total of 67 crossovers on chromosome 10 were
observed in the two 4-week treatment groups. This included
one triple and four double crossovers (there were none in the
of the 67 crossovers among the 96 offspring predicts signifi-
cantly more nonrecombinants and double recombinants than
were observed (Table 2). The overrepresentation of single
recombinants implies that interference (or another mecha-
nism) operates to distribute crossovers evenly between chro-
mosomes, avoiding nonrecombinants that would lead to pos-
It remains to be seen whether interference represents an
absolute barrier to an upper level of recombination or whether
controls are combined from two experiments. All other data points, except for week 5, were collected from the same males mated at different times
Time course effects of CP on recombination. Data are the crossover values (mean ? SEM) for chromosome 10. Data reported for
Time course of recombination induction in
Time course of recombination induction in CP-treated mice. Six
males were treated with CP and mated 3, 4, and 8 weeks later. An
independent set of males that were mated 5 weeks after CP exposure
is also shown. The number of recombinants in chromosome 10
intervals are listed. M is an abbreviation for D10Mit. Recombination
percentages (shown in parentheses) equal (total no. crossovers) ?
*Most of these recombinants were contributed by a single male (see
No. of crossovers (%)
(n ? 23)
(n ? 48)
(n ? 48)
(n ? 41)
8684 Genetics: Hanneman et al.Proc. Natl. Acad. Sci. USA 94 (1997)
more effective drug treatments can increase crossing-over to
an extent limited only by chromosomal stability and meiotic
progression. A physical mechanism of interference is implied
by the existence of ZIP1, a yeast synaptonemal complex
protein whose absence abolishes interference (29). This sug-
gests that interference may put an upper level to the number
of recombination events on a single chromosome, unless there
is experimental modification of synaptonemal complex com-
Utility in Genetic Analyses. The ability to increase crossover
frequency can be of considerable practical benefit to mouse
genetics. It can markedly decrease time, cost, and effort in
conducting large crosses or in creating mapping reagents such
as advanced intercross lines (31). It should be kept in mind that
because CP is known to cause chromosome aberrations during
at certain stages of spermatogenesis (27), there is a possible
risk of creating mutations in the progeny of treated males.
However, we are unaware of any specific locus test data for CP.
CP more than doubled crossing-over (on chromsome 10) in
these experiments, but it is possible that other protocols could
be developed that further increase the frequency. In the
present work, a single drug was tested at a single dosage, in one
genotype of mice, at 7-day sampling intervals. Furthermore,
the effect of CP on females was not investigated, so the current
benefit is restricted to recombinational mapping in males.
Adjusting the treatment parameters or using different strains
and both sexes of mice (perhaps containing mutations in
even higher levels.
We thank Eva Eicher, Rosemary Elliot, Wayne Frankel, and Mary
Ann Handel for critical reviews and advice on the manuscript. This
work was supported by grants from the National Institutes of Health
(GM45415), American Cancer Society (CN-118), and National Sci-
ence Foundation (Presidential Young Investigator Award) to
J.C.S. W.H.H. and M.E.L. are recipients of postdoctoral fellowship
(ES05743) and Physician Scientist (ES00251) awards, respectively,
from National Institute on Environmental Health Sciences.
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Distribution of crossovers in offspring of
No. of crossovers
Distribution of crossovers in offspring of CP-treated males. Data are
a composite of the two groups of CP-treated males for chromosome
10 (Fig. 1). XO?Chr is the number of crossovers on chromosome 10
in any single offspring. The next two columns are the observed (Obs.)
and expected (Exp.) numbers of animals with the corresponding
number of crossovers. Expected values are calculated by using the
Genetics: Hanneman et al. Proc. Natl. Acad. Sci. USA 94 (1997)8685