Download Analysis of large and small colony L5178Y tk−/− mouse lymphoma

Mutagenesis vol.13 no.5 pp.461^174, 1998
Analysis of large and small colony L5178Y tic1' mouse lymphoma
mutants by loss of heterozygosity (LOH) and by whole
chromosome 11 painting: detection of recombination
Melissa C. Liechty1'*, Jane M. Scalzi1, Kenneth R. Sims1,
Herbert Crosby Jr1, Diane L. Spencer2, Lisa M. Davis1,
William J. Caspary2 and John C. Hozier1
'Applied Genetics Laboratories Inc., 1335 Gateway Drive, Suite 2001,
Melbourne, FL 32901, USA and 2Laboratory of Environmental
Carcinogenesis and Mutagenesis, National Institutes of Health,
Research Triangle Park, NC 2//09, USA
Analysis of 122 spontaneous large and small colony mutants
derived from L5178Y tk+l~ mouse lymphoma cells at 28
heteromorphic microsatellite loci on chromosome 11
showed that extensive loss of heterozygosity (LOH) is
common in both large colony and small colony mutants,
eliminating most chromosome 11 loci as candidates for a
putative growth control locus. These results, in conjunction
with historical cytogenetic data, suggest that a putative
growth control locus lies distal to the thymidine kinase
(Tkl) gene, near the telomere. Thirty seven mutants were
hybridized with a chromosome 11-specific whole chromosome painting probe for analysis of rearrangements. Generally, painting confirmed earlier observations that large
colony mutants are karyotypically normal, whereas small
colony mutants frequently have detectable rearrangements.
A point probe distal to Tkl revealed no evidence of
chromosome breakage in small colony mutants that
appeared normal on whole 11 painting and had no LOH.
Therefore, the molecular difference between large and
small colony mutants remains unknown. Models to explain
large and small colony mutants consistent with our findings
are presented, including loss of a putative growth control
gene, differential mechanisms of chromosome breakage/
repair and second site mutations as explanations for small
colony mutants. Painting revealed translocations and aneuploidy and showed that non-disjunction was not a common
explanation for complete LOH. The most common finding
was that large regions of LOH do not result from deletions,
demonstrating that these cells can detect recombination
events as well as previously observed chromosomal
rearrangements, deletions and point mutations.
Introduction
In 1972, Clive and co-workers reported the development of a
mouse cell line heterozygous at the thymidine kinase {Tkl)
locus on chromosome 11 (Clive et al, 1972, 1979; Hozier
et al, 1981; Liechty et al., 1993). These cells, L5178Y tk+l~
mouse lymphoma cells, clone 3.7.2C, have been used to study
mechanisms of mutagenesis and to assess the mutagenic
activity of chemicals. Both chemical and physical agents can
induce trifluorothymidine (TFT) resistance in this cell line by
inactivating the second Tkl allele (Moore-Brown et al., 1981).
The basis for the mouse lymphoma assay (MLA) is the ability
to select for tk~*~ mutants in a background of tk+l~ non-mutant
cells. This heterozygous cell line is more responsive to the
range of mutagenic activities of chemicals than those cells
that have the selectable gene on the hemizygous X chromosome
(Clive, 1985; Evans et al, 1986; Moore et al, 1989). World
wide, the mouse lymphoma assay (MLA) is the most widely
accepted mammalian cell gene mutation assay and may eventually be included in the core battery of tests that will be
approved for international harmonization of genetic toxicology
testing practices (Garriott et al., 1995). Extensive molecular
and cytogenetic analyses have shown that these cells detect a
variety of mutations, including point mutations and other small
mutations within Tkl, losses of Tklb (the functional allele),
losses of multiple loci in addition to Tklb and cytogenetically
detectable chromosomal aberrations such as translocations
(Hozier et al, 1981, 1985, 1989; Moore et al, 1985; Blazak
et al, 1986a,b, 1989; Applegate et al, 1990; Liechty et al,
1993,1994). This heterozygous system is important for measuring mutations because it models in vivo mechanisms leading
to chemical induction of neoplasia.
Mutant colonies isolated in the MLA fall into a bimodal
size distribution, with the larger colonies growing at a rate
typical of the tk+l~ cells from which they originated (large
colony mutants) and the smaller colonies growing at a slower
rate (small colony mutants; Moore et al, 1985). Both colony
size classes are produced among both spontaneous mutants
and induced mutants, although the proportion of small colony
mutants produced is mutagen dependent. It has been hypothesized that lesions inactivating Tklh in some mutants may also
affect a second locus that affects growth rate, resulting in
small colony mutants, whereas large colony mutants are
produced when this putative growth control gene is not affected
(Hozier et al, 1982, 1992; Moore et al, 1985, 1987). Early
cytogenetic studies showed that a subset of small colony
mutants has cytogenetically detectable aberrations of chromosome 11, including translocations (usually non-reciprocal) in
which non-11 chromosomal material is fused to a break point
in the distal end of chromosome 11, presumably at or near
the Tkl gene, hi these studies large colony mutants were
characterized as cytogenetically normal (Hozier et al, 1981,
1982, 1985, 1991; Moore et al, 1985; Blazak et al, 1989).
Fluorescence in situ hybridization (FISH) has shown that Tkl
resides at the distal end of chromosome 11, band E1 -2, as would
be expected from the earlier observations of rearrangements
involving this region in many mutants (Hozier et al, 1991).
The majority of mutants isolated in the MLA, whether
spontaneous or induced, have lost Tklb and losses of Tklb are
common in both phenotypic size classes (Applegate et al,
1990; Clive et al, 1990; Glover and Clive, 1995; Liechty
et al, 1996). Southern analysis using an Ncol polymorphism
between the two Tkl alleles has revealed that 70-75% of
spontaneous mutants have lost Tklb, as evidenced by loss of
a 6.4 kb band on blots hybridized with a tk cDNA probe
(Applegate et al, 1990; Clive et al, 1990). Greater or lesser
proportions of induced mutants have lost Tklb, depending on
*To whom correspondence should be addressed. Tel: +1 407 768 2048; Fax: +1 407 727 2643; Email: [email protected]
© UK Environmental Mutagen Society/Oxford University Press 1998
461
M.C.LIechty et al
the identity of the mutagen. Only rarely has a perturbation in
banding pattern suggestive of a partial loss of Tklb been
observed in the many mutants analyzed, suggesting that loss
of the entire 77t7b allele is not uncommon in tkr1' mutants.
The molecular distinction between large and small colony
mutants was not clear, since 71fc7b appeared to be completely
lost in many mutants in both size classes. Cancer is a genetic
disease controlled in part by genes that mediate cell growth.
Therefore, identification of genes and/or mechanisms that
affect cell growth in the MLA may be helpful in understanding
this disease. To that end, we developed tools to measure the
extent and nature of losses in mouse lymphoma cells. We
used PCR analysis of microsatellite sequences to survey
chromosomes 11 of the L5178Y tk+l~ cell line for simple
sequence repeat polymorphisms (SSRP). Several thousand of
these PCR-based SSRPs have been described in the mouse
(Love et al, 1990; Hearne et al, 1991; Dietrich et al, 1992,
1996; Copeland et al, 1993a,b). We identified 28 SSRPs
throughout the length of chromosome 11 (96% of the chromosome 11 genetic map), including one in 77:7, which are
polymorphic in this cell line (Liechty et al, 1994, 1996).
In this study, we have used these SSRPs to analyze 122
spontaneous mutants for loss of heterozygosity (LOH). These
mutants were isolated using the in situ mutagenesis protocol,
which captures more of the small colonies than the suspension
protocol (Rudd et al., 1990; Spencer and Caspary, 1994;
Spencer et al., 1994). We further analyzed 37 of these mutants
by whole chromosome 11 painting (Liechty et al., 1995). The
painting probe can reveal certain features of the mutants that
are not discernible by LOH analysis, such as chromosome
rearrangements and chromosome copy number, and can be
used to determine if extensive LOH results from deletion. The
two chromosome 11 homologs on which Tkl resides are
essentially normal, but are distinguishable by a centromeric
heteromorphism (Hozier et al., 1982). The homolog with the
smaller centromere has been designated chromosome lla,
while that with the larger centromere has been designated
chromosome lib (Sawyer et al., 1985). The individual homologs of chromosome 11 can be distinguished in a painted
preparation by this heteromorphism. Cytogenetic studies have
revealed rearrangements involving the distal end of chromosome 1 lb in many tic1' mutants, but no corresponding involvement of chromosome lla, suggesting that the functional Tkl
allele resides on the lib homolog. This has been confirmed
by isolation of tic1' mutants that have lost chromosome lib
and duplicated chromosome lla (Hozier et al., 1992). LOH
analyses of such mutants have allowed us to identify which
of each pair of alleles lie on each homolog and have allowed
us to confirm that only lib alleles are lost in mutants (Liechty
et al., 1994, 1996).
The combined results of LOH analysis and chromosome 11
painting are presented here. We discuss the results in terms of
understanding the underlying mechanisms leading to lesions
in Tkl and possible mechanisms contributing to the growth
rate phenotype.
Materials and methods
Cells and culture medium
L5178Y tk+'- cells, clone 3.7.2C (Chve et al, 1972) and spontaneous
tk^~ mutants were routinely cultured in suspension in RPMI-1640 medium
supplemented with 10% heat-inactivated horse serum, 0.25 mg/ml L-glutamine,
0.05% Pluronic F68, 107 |ig/ml sodium pyruvate, 95 U/ml penicillin and
95 Jig/ml streptomycin. Spontaneous tic4' mutants were recovered using the
462
in situ protocol for the MLA (Rudd et al., 1990; Spencer and Caspary, 1994;
Spencer et al., 1994). Before each mutagenesis experiment, the cells were
incubated for 24 h in RPMI-1640 supplemented as above plus methotrexate
(0.1-0.3 ng/ml), thymidine (9 |ig/ml), hypoxanthine (15 |ig/ml) and glycine
(22.5 Hg/ml) to kill pre-existing TFT-resistant (TFT) cells. The cells were
then incubated for 72 h in the same medium without methotrexate. Culture
medium and all supplements were purchased from Life Technologies
(Gaithersburg, MD). Semi-solid cloning medium contained 0.25% granulated
agar (Becton Dickinson & Co.).
Accumulation of TFT colonies in situ
Several sets of mutant plates were prepared as follows. Samples of 0.5X10*
cells were added to 50 ml semi-solid culture medium and poured into two
plastic 100 mm culture dishes, allowed to solidify at room temperature, then
incubated at 37°C in 5% CO2. Approximately 40 h after plating, a 10 ml
overlay of semi-solid medium containing TFT (280 ng/dish, final concentration
8 |ig/ml) was added to each of the dishes, which were then incubated for an
additional 8 days. Using an inverted microscope with an eyepiece micrometer,
the size of mutant colonies was measured to classify the colonies as large or
small. Individual mutant colonies were then plucked from the agar using
sterile pasteur pipettes. Six colonies or fewer were randomly plucked from
each dish. Cells from each colony were dispersed into 2 ml fresh culture
medium in 12-well culture dishes. When confluent, cell cultures were
transferred to culture tubes with 10 ml fresh medium and incubated.
Preparation of DNA for LOH analysis
Genomic DNA for use as PCR template was prepared by centrifuging the
mutant cell cultures derived from the individual colonies described above.
Cells were washed twice in sterile TEN (10 mM Tris, pH 7.8, 25 mM EDTA,
150 mM NaCl) and suspended in TEN at 15X106 cells/145 ul. An equal
volume of 3% sarcosyl, 50 mM EDTA, pH 8.0, 1 mg/ml proteinase K was
added to lyse the cells and the lysate was incubated overnight at 55°C. The
lysate was incubated with 150 |ig/ml RNase for 1 h at 55°C, then with an
additional 250 Hg/mf proteinase K for 1 h at 55°C. The lysate was extracted
twice with phenol and dialyzed extensively against IX TE (10 mM Tris,
pH 8.0, 1 mM EDTA).
LOH analysis
Genomic DNA was used as template for PCR amplification. Primers for
amplification of microsatellite sequences on chromosome 11 were obtained
from Research Genetics Inc. (Huntsville, AL). DNA from tk+'~ L5178Y
3.7.2C mouse cells and from a mutant which has lost all of chromosome lib
containing the Tklb allele was also PCR amplified as a control. Amplification
reaction mixtures were prepared by combining 10 ul 2X PCR Master
(Boehnnger Mannheim) containing 20 mM Tris-HCl, 100 mM KC1, 3 mM
MgCl2, 0.05 U/ul Taq DNA polymerase, 400 uM each dNTP, 0.01% Brij 35,
pH 8.3, 5 pmol each primer, 40 ng template DNA and H2O to a final volume
of 20 |il. Reactions were performed in a Perkin Elmer Model 9600 thermal
cycler by touchdown PCR. An initial 94°C denaturation for 2 min was
followed by two cycles of 94°C denaturation for 20 s, 61°C annealing for
20 s and 72°C extension for 20 s. The annealing temperature was decreased
by 1°C for nine additional two-cycle sets until two cycles were performed at
an annealing temperature of 52°C. Then 12 additional cycles were performed
using an annealing temperature of 50°C, followed by a 72°C extension for
5 min. After amplification, 15 |il each PCR product was combined with 2 \i\
25% Ficoll tracking solution and the PCR products were resolved by
electrophoresis on 10% polyacrylamide gels for 2—4 h at 200 V. Gels were
post-stained with ethidium bromide for visualization of the PCR products. A
100 bp ladder DNA marker from Pharmacia Inc. was used to estimate the
sizes of the PCR products.
Sequence analysis
Selected mutants were subjected to sequence analysis as described in Liechty
et al. (1993). Briefly, RNA was isolated from each mutant and cDNA
sequencing template was prepared by RT-PCR using a primer specific for
Tkl. Double-stranded sequencing was performed using Sequenase v.2.0
(Amersham Corp., Arlington Heights, IL) and specific primers designed for
Tkl. Both alleles of Tkl were sequenced simultaneously from each mutant.
Preparation of metaphase chromosomes for painting
Metaphase chromosomes suitable for painting were prepared from each mutant
by treating 15 ml cell suspension with 0.02-0.2 ug/ml colcemid for 15—120
min at 37°C in an atmosphere of 5-10% CO2. Then, cells were pelleted by
centnfugation at 100-300 g for 4-7 min. The supernatant was decanted and
the pellet resuspended in the residual 0.5 ml fluid. An aliquot of 8-10 ml
0.075 M KC1, pH 6.0-6 7, was added slowly with gentle agitation and the
suspension was incubated at room temperature for 20 min. The suspension
was again centrifuged at 100-300 g for 4-7 min. The supernatant was decanted
and the pellet resuspended in the residual 0.5 ml fluid. Then 8—10 ml Camoy's
Large and small colony L5178Y tk~'~ mouse lymphoma mutants
fixative (3:1 methanol:glacial acetic acid) was added slowly with gentle
agitation and the suspension was incubated at 37°C for 20 min. The fixed
preparations were pelleted, resuspended in fresh fixative and incubated for an
additional 5 min. Fixation was repeated twice more. Slides with metaphase
spreads were prepared by dropping fixed cells onto clean glass slides. Slides
were aged for 1—4 weeks at room temperature prior to hybridization.
Chromosome 11 painting
Chromosomes on slides were denatured by incubating for 5 min at 75°C in
70% formamide, 2X SSC (IX: 150 mM NaCl, 15 mM sodium citrate,
pH 7.0). Prepared chromosome 11 probe (biotinylated and combined with
competitor DNA in 50% formamide, 2X SSC hybridization solution) was
denatured at 75°C for 5 min, preannealed for 1.5 h at 37°C and applied to
the slides at 10 ng/nl. The slides were hybridized overnight at 37°C and
washed twice in 2X SSC, once in IX SSC and once in 0.5X SSC for 10-15
min per wash at 42-45°C. Hybridized probe was detected with FTTC-labeled
antibodies as described previously (Hahn et al., 1992; Lane et al., 1992). The
band 1 IE-specific probe and D//M/r6°-specific point probe were directly
labeled by random priming with Cy3-conjugated dCTP (Amersham). These
probes were handled as for biotinylated probes except for elimination of the
final antibody detections. Instead, the slides were counterstained with DAPI.
Chromosome measurements
To determine the minimum deletion size that could reliably be observed by
chromosome 11 painting, it was necessary to determine the range of normal
variability in chromosome lengths occurring during preparation of metaphase
spreads on slides. The lengths of painted chromosomes 11 were measured in
60 spreads prepared from L5178Y 3.7.2C cells. As an internal standard for
size in each spread, the length of chromosome 13 in a 12; 13 Robertsonian
translocation chromosome that is characteristic of this cell line was also
measured (Sawyer et al., 1985). Chromosome 13 was chosen because the
translocated chromosome is readily identifiable in spreads, it is similar in size
to chromosome 11 and it is unlikely to be affected in mutants. The ratio
between the length of chromosome 11 and chromosome 13 was calculated
for each spread and the standard deviation for 60 spreads was determined.
For mutant analysis, similar calculations were performed. Mutants for which
the mean ratios, as determined from at least 10 spreads, were greater or less
than one standard deviation from the expected normal ratio were considered
to have chromosomes 11 that were clearly longer or shorter respectively
than normal.
Results
To determine the extent of LOH in large and small colony
mutants, we examined 122 spontaneous TFT mutants derived
from L5178Y mouse cells at 28 heteromorphic microsatellite
loci. Figure 1 shows the positions of these loci on the
chromosome 11 linkage map. A standard chromosome 11
idiogram is shown aligned with the linkage map for orientation,
although the correlation of physical and genetic maps in mouse
is inexact. The number of large and small colony mutants
in each LOH category examined are shown arrayed along
chromosome 11 to provide an illustration of the distribution
of extent of LOH among mutants.
We first screened the mutant DNAs for a complex polymorphic microsatellite sequence that we designated DllAgll,
which lies in Tkl in intron F between exons 6 and 7 (Gudas
et al, 1992; Liechty et al, 1996). Analysis was by PCR using
primers, designated Agl2, specific for DllAgll (Liechty et al,
1996). Two alleles were seen in 36 (30%) of the mutants,
indicating no LOH at the DllAgll locus in this group (see
Figure 2 for a summary of all LOH results). At least eight other
loci distributed throughout chromosome 11 were examined in
these 36 mutants to verify that there was no LOH in these
mutants. We tentatively concluded that these mutants harbored
intragenic mutations in the Tklb allele. Fourteen of these
mutants were subjected to sequence analysis of the entire 77:7
coding region. In one of the 14 mutants, no mutations were
observed, in two mutants, only TkP sequence and no Tklb
sequence was observed in the RT-PCR product, in 10 mutants,
there were single base changes and in one mutant, there was
1
2
3.1
3.2
3.3
4
5
10—
20 —
1.1
30—
1.2
1.3
2.1
2.2
2.3
40 — • Mit15. Mit29, Mit30
•Mit4
LLLLLS
50—
1.1
1.2
1.3
1.1
1.3
2.1
2.3
60 —
1.2 70 —
1.2
2.2
8 0 ^
• Mit8 • Mit35, Mit36 '
Mit41 :
Mit54 Mit67
Mit58
Mit59• Nds7 •
• Mit13 =
JLLLLL
) LLLSSSS
)SSSSSS
-VM12Mit128
Mit42 •Mit103
•Mit48, Mit49:
•Tk1(Agl1)-
D LLLSSSSSS
»SSS
i L
Fig. 1. Linkage map of chromosome 11 showing microsatellite loci that are
heteromorphic in the L5178Y tk+l~ cell line. Locus names have been
abbreviated, i.e., Mit62 refers to DllMit62, etc. Map positions are shown in
centiMorgans (cM) to the left of the locus names and are according to
Watkins-Chow et al. (1996). The ideogram on the left provides an
approximation of where these loci lie on banded chromosomes. On the far
right, mutants with LOH extending from DllAgll to the region between
bracketed loci are indicated for each interval by the letter L (for large
colony mutant) or S (for small colony mutant); each letter indicates one
mutant. Data are shown for 63 spontaneous mutants isolated using the in
situ protocol for the mouse lymphoma assay. An additional 23 mutants (not
shown) lost alleles at all loci and 36 mutants (also not shown) retained all
alleles.
a large insertion (data not shown). Thus, for 11 of the 14
mutants sequenced, the presence of intragenic mutations within
Tklb was confirmed. Twenty seven of the 36 presumptive
point mutants were large colony and nine were small colony.
Four of the 14 mutants sequenced were small colony mutants;
three of these had single base changes and one yielded no
Tklb sequence from the RT-PCR product.
The remaining 86 mutant colonies lost heterozygosity at the
DllAgll locus, suggesting the loss of all or at least part of
the Tklb allele. To assess the extent of damage in these 86
spontaneous mutants, we assayed the remaining polymorphic
microsatellites (Liechty et al, 1994; Figure 1) along mouse
chromosome 11 from D11MU62, 1 cM distal to the centromere,
to DllAgll, which resides within the Tkl gene 78 cM from
the centromere (Liechty et al, 1994, 1996; Watkins-Chow
et al, 1996). We had no informative microsatellites for the
most centromeric region or the region distal to the Tkl gene.
Twenty three (27%) of the 86 mutants that lost heterozygosity
at the 717 locus also lost heterozygosity at every locus
examined and may have originated by loss of the lib chromosome through non-disjunction or by mitotic recombination (see
Figure 2). Of these, nine were small colony mutants and 14
large colony.
One large colony mutant lost heterozygosity only at
DllAgll. The 62 remaining mutants with LOH at DllAgll
lost at least one microsatellite besides DllAgll, with the region
463
M.C.LIechty et al
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Large: 27 1 0
Total: 36 1 3
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9 50
14 72
1 3 13 23 122
1 1 6
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Painted:
Small
Large
8
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0 2
10
5
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4 0 0 1 1 0 0
2 0 0 0 1 2 0
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15.
37
Fig. 2. Summary of extent of LOH among all mutants examined. Each bar represents a class of mutants, based on LOH. Black areas represent loci where
LOH did not occur. White areas indicate the extent of LOH on chromosome lib in centiMorgans (cM), as shown on the scale on the right. In each category,
LOH extended from DIlAgll in Tkl to the locus indicated above each bar inclusive and included all intervening loci tested. The numbers below the bars
represent the number of mutants found in each category, subdivided into small and large colony mutants. There was no clear difference in LOH between large
and small colony mutants that could reveal a difference in these two mutant categones. For this reason, intermediate size mutants that could contribute to
ambiguity between the two mutants classes were eliminated from the tally; the remaining mutants representing the smallest and largest 25% of the mutant
population are shown at the bottom.
of LOH varying from DIlAgll only to regions including
DIlAgll and loci as far away as D11MU2 and D11MU63.
These mutants could have originated as interstitial or terminal
deletions or through mitotic recombination events. Of these,
32 were small colony mutants and 30 were large colony
mutants. In every case in which LOH was observed in these
86 mutants, the alleles lost, including DIlAgll, were from
chromosome lib, the homolog bearing the functional Tklb
aJlele, as would be expected if the loss were related to loss
of Tklb.
We performed both LOH analysis and chromosome 11
painting on a total of 37 mutants, including 15 large colony
mutants and 22 small colony mutants. The number of mutants
painted from each LOH category is shown in Figure 2. Table
I summarizes the results of LOH and painting analyses for
these mutants. The mutants analyzed included presumptive
point mutants (i.e. LOH was not observed at any of the test
loci) and mutants with varying degrees of LOH, from loss of
Tklb alone to loss of alleles at all tested loci. In every mutant
painted, regardless of the extent of LOH, there were at least
two chromosome 11 equivalents in each cell revealed by
chromosome painting; mutants containing only one copy of
chromosome 11 were not observed. In only one case, mutant
42b (Figure 3B), a large colony mutant, was a pair of
chromosomes 11 with one obviously shortened homolog
464
Table I. Mutants subjected to painting analysis categorized by size
phenotype and extent of LOH
Total
mutants
Normal
karyotype
Abnormal
karyotype
Large colony mutants
No LOH
LOH to DllNds7 or less
LOH proximal to DllNds7
Complete LOH
15
0
6
6
3
11
0
5
6
0
4
0
1
0
3
Small colony mutants
No LOH
LOH to DUNds7 or less
LOH proximal to DllNds7
Complete LOH
22
8
8
5
1
12
5
4
2
1
10
3
4
3
0
LOH, loss of heterozygosity; No LOH, LOH observed at no test loci; LOH
to DllNds7 or less, LOH observed from Tkl to loci no more proximal than
DllNds7 (see Figure 1; losses of this extent are too small to be definitively
ascribed to deletion by whole chromosome painting); LOH proximal to
DllNds7, LOH observed from Tkl to loci more proximal than DllNds7
(deletions resulting in losses of this extent are apparent in painted
preparations); Complete LOH, LOH observed at all tested loci
observed. We previously reported only one other mutant that
has obviously less than two chromosome 11 equivalents, C.I 17
(Liechty et al, 1995; Moore et al., 1985). Painting shows that
Large and small colony L5178Y tk~l~ mouse lymphoma mutants
A
142a
fi
No
11a
DHMil69
Tki
lib
11a
Fig. 3. Recombination and duplication of regions of chromosome 11 in large colony mutants. (A) Mutant 142a. (Far left) Idiograms of chromosome l l a
(white) and l i b (black). LOH from Tkl to DllNds7 on l i b is indicated by white. (Near left) 142a hybridized with a MMU (mouse) 11 whole chromosome
painting (WCP) probe. There is no evidence of deletion as the cause of LOH. Instead, chromosome l i b is longer than normal. (Near right) 142a hybridized
with a probe for band B l (see Figure 1) and a point probe for DllMit69 that is distal to Tkl. Although Bl would be expected to lie outside the region of
LOH, hybridization of two bands on chromosome 1 l b (arrows) shows that this region has been duplicated in this mutant Simultaneous deletion of the end of
the chromosome, including Tkl and DllNds7, is unlikely because the marker distal to Tkl is still present. (Far right) Idiograms of l l a and l i b showing the
location of the B l (dotted band) and point probes (dotted circles). The location of the region of LOH (white) is likely to be as shown, but is not certain.
Because there has been a duplication, it is not clear from these analyses whether LOH results from recombination or deletion. (B) Mutant 42b. (Left)
Idiograms showing LOH of l i b sequences from Tkl to D11MH62 (i.e. all tested loci). (Right) 42b hybridized with MMU11 WCP. l l a and l i b centromeres
are heteromorphic (Hozier et at., 1982), but painting shows that both chromosomes 11 have identical sired centromeres. This observation suggests that
chromosome 11 b was lost and l l a was reduplicated, accounting for loss of all l i b markers. One of the l l a chromosomes is shorter than normal, but it is not
known what region of this chromosome has been lost.
this small colony mutant has two chromosomes lla, one of
which has lost approximately half the chromosome and has a
non-11 fragment translocated to it.
Of the 15 large colony mutants painted (Table I), four (27%)
produced abnormal paints. The remaining 11 mutants were
normal, i.e. each metaphase spread had two painted chromosomes 11 which appeared normal in length, one being an lla
and the other an lib, as determined by the centromeric
heteromorphism. The painting probe requires extensive competition with unlabeled mouse Cotl DNA to prevent nonspecific binding of probe to the centromeres of all the
chromosomes. Where competition was insufficient, chromosome 11 centromere morphology could not be determined. In
such cases, centromere morphology was determined from
slides that were Giemsa stained following hybridization. All
the large colony mutants painted had suffered some degree of
LOH, as determined by PCR analysis of heteromorphic loci
on chromosome 11. Table II shows the extent of LOH and the
results of chromosome 11 painting for each large colony
mutant tested.
Of the four large colony mutants yielding abnormal paints,
two mutants, 8a and 18b, were karyotypically unstable (data
not shown): mutant 8a had apparent iso-lls (i.e. two copies
of chromosome 11 joined by a centromeric fusion) in addition
to an apparently normal lla in many spreads. It is not clear
whether the apparent iso-11 represents an l l a - l l a fusion, an
lib—1 lb fusion or an lla—lib fusion, since there is no
apparent demarcation between the fused centromeres. An l i b
centromeric fusion to a non-11 chromosome in addition to an
apparently normal lla was also observed in a few spreads.
This mutant had LOH at all tested loci and therefore was
expected to have lost chromosome lib, yet painting showed
normal spreads of this mutant containing both chromosomes
lla and lib. Mutant 18b had different aberrations in different
spreads, including apparently different translocations of non11 material to the distal end of lib, either telomere to telomere
or telomere to centromere. This mutant had also suffered LOH
at all tested loci, yet retained both chromosomes lla and lib,
based on the centromeric heteromorphism.
Two large colony mutants had stable chromosome 11 aberrations. These mutants are illustrated in Figure 3. Mutant 142a
had an apparently normal chromosome lla, but a chromosome
lib that was clearly abnormally long, despite LOH extending
from Tkl to DllNds7, -25% of the recombinational length of
the chromosome. All of the abnormally long chromosome lib
was of 11 origin, however, as determined by painting of the
entire chromosome with the 11-specific painting probe. Painting
with a band-specific subchromosomal probe showed a duplication outside the region of LOH on chromosome lib. Mutant
42b had two copies of chromosome lla and no lib. One of
the two chromosomes was apparently normal length, while
the other was visibly shorter.
465
M.CLiechty et al
Table IL Extent of LOH and results of chromosome 11 painting in large
colony mutants
Table III. Extent of LOH and results of chromosome 11 painting in small
colony mutants
Mutant
Mutant
LOH
LOH to DllNds7 or distal
17b
Tkl only
33b
Tkl-DllMitW3
131a
Tkl-DUMitlO3
110a
Tkl-DUMitli
37b
Tkl-DllNds7
142a
Tkl-DUNds7
LOH proximal to DllNds7
29a
Tkl-DUMit8
137a
Tkl-D1IMU8
133a
Tkl-DUMM
21a
Tkl-DUMitl9
29b
Tkl-DllMitl9
48b
Tkl-DllMit63
Complete LOH
8a
All1
18b
All
42b
All
Karyotype
Normal
Normal
Normal
v
Normal
Normal
l i b longer than normal (see Figure 3)
Normal (Figure 6)
Normal
Normal
Normal
Normal (Figure 6)
Normal (Figure 6)
lla and apparent iso-11 in some spreads
Translocation of non-11 to l i b in some
spreads; karyotypically unstable
Two lias, no lib. One shorter than normal
(Figure 3)
Mutants are listed in order of increasing extent of LOH. For partial LOH,
the losses are inclusive of the indicated loci. Four (27%) of 15 large colony
mutants had abnormalities revealed by painting.
•All, LOH observed at all test loci.
Of the 22 small colony mutants painted, 10 (45%), including
three putative point mutants and seven mutants with LOH,
produced abnormal paints. These results are summarized in
Table I. Table m shows the extent of LOH and the results of
chromosome 11 painting for each small colony mutant tested.
The three putative point mutants were classified as such
because they had no LOH at the tested loci and appeared
normal when examined by Southern blotting. These mutants
are of particular interest because they have chromosomal
abnormalities revealed by painting that may explain the small
colony phenotype.
Among these small colony mutants with no LOH was
mutant 30c. This mutant appeared normal in many spreads,
but in some spreads, like 142a above, it had an apparently
normal chromosome l l a and a chromosome l i b that was
abnormally long, although all of 11 origin, as determined by
chromosome 11 painting (see Figure 4A). Mutant 32c had a
non-11 translocation on the distal end of chromosome l i b in
some spreads. The size of the non-11 translocation varied from
large to undetectable in different cells. Similar karyotypic
instability has previously been reported in small colony mutants
(Hozier et al., 1983, 1985). Mutant 143a appeared normal in
many spreads, with chromosomes lla and lib present In
some spreads, there was an iso-11 chromosome, with no other
11s present. In banded preparations the two centromeres in
the iso-11 appeared identical. It is not clear what role an
isochromosome would play in mutation at Tkl and it may be
the result of a secondary event. A point mutation in 143a in
the Tklb coding sequence consisting of a T—>C transition in
codon 156 has been confirmed by sequence analysis. These
three mutants are illustrated in Figure 4.
A variety of abnormalities, as illustrated in Figure 5, were
observed in small colony mutants with LOH. Mutant 35b
(LOH from Tkl to D11MU49; Figure 1) had an apparently
normal chromosome l i b and an apparent iso-11 chromosome,
466
LOH
Point mutants"
2a
None*
5b
None
16b
None
17a
None
30c
None
32a
32c
143a
None
None
None
Karyotype
Normal'
Normal
Normal
Normal*
One 11 homolog abnormally long in some
spreads (see Figure 4)
Normal
Non-11 translocation on l i b (Figure 4)
Apparent iso-lls, no other 11s, in some
spreads (Figure 4)
LOH to DlWds7 or distal
35b
la
14b
112a
Tk]-DllMit49
Tkl-DUMit49
Tkl-DllMitlO3
Tkl-D!lMitlO3
Tkl-DUMitlO3
130a
123a
139a
TkJ-DllMitlO3
Tk]-D!lMitl2
Tkl-DUMitl3
24a
LOH proximal to DUNds7
118a
Tkl-DllMit4
3b
Tkl-D11MH21
36b
9b
10b
Tkl-DIlMitl9
Tkl-DlIMil63
Tkl-DUMit63
Complete LOH
41b
All8
Normal
Apparent Iso-11, normal l i b (Figure 5)
lib abnormally long (Figure 5)
Normal
11 translocation on non-11, lla and lib
normal (Figure 5)
Normal
Normal
lib abnormally long
Normal (Figure 6)
2 lias, non-11 translocation on l i b
(Figure 5)
Normal
Two lias, no lib (Figure 5)
Two lias, no lib
Normal
Mutants are listed in order of increasing extent of LOH. For partial LOH,
the losses are inclusive of the indicated loci. Ten (45%) of 22 large colony
mutants had abnormalities revealed by painting. Abnormalities are described
in detail in the text.
"Point mutants, no LOH observed; None, LOH not observed at any test loci;
All, LOH observed at all test loci; a, mutants with large colony sibs.
although, as for mutant 8a, it is not clear from painting whether
this chromosome is an iso- 11 a, iso-11 b or 11 a-11 b centromeric
fusion. Like mutants 142a and 30c, mutants la and 139a (this
mutant not shown) have a chromosome l i b that is clearly
abnormally long, but is all of 11 origin, as determined by
chromosome 11 painting. Mutant 112a has apparently normal
chromosomes 11, but has a small region of 11 origin translocated to a non-11 chromosome. Mutant 3b has two apparently
normal copies of chromosome lla and has a non-11 translocation on the distal end of chromosome lib. The l i b portion of
this translocation chromosome appears normal length (Figure
5B). Mutants 9b and 10b are identical and may be sibs. These
mutants each have two chromosomes lla and no chromosomes
lib. They both have LOH at all but two test loci.
Twelve large colony mutants and seven small colony mutants
had LOH but normal chromosomes 11. Examples of such
mutants are illustrated in Figure 6.
Discussion
The results presented here reveal the utility of heteromorphic
microsatellite repeats in analyzing the extent of lesions found
in tic1' mutants. LOH implies chromosome loss by any of
several mechanisms, including non-disjunction, deletion or
recombination (such as mitotic recombination or gene conver-
Large and small colony L5178Y tk~*~ mouse lymphoma mutants
Fig. 4. Karyotypically unstable small colony mutants with no LOH (A) Mutant 30c. Most spreads are normal (left), but in some (right) longer than normal
chromosomes lib indicate that a duplication has occurred on this chromosome. (B) Mutant 143a. Most spreads are normal (left), but some spreads (right)
have iso-lls, chromosomes 11 joined at the centromere. (C) Mutant 32c. Many spreads are normal (left), but many clearly have a non-11 translocation on the
distal end of lib (middle). The translocation varies in length from cell to cell (compare middle with right, arrows).
sion). Whole chromosome painting in conjunction with LOH
analysis can distinguish among these mechanisms. Mutants
without detectable LOH would be expected to be the consequence of point mutations at the Tkl locus or deletions,
recombinations or gene conversion-like events limited to a
region between the closest flanking heteromorphic loci.
In cases where LOH is a consequence of rearrangements,
LOH analysis provides better resolution of the extent of the
467
M . C L I e c h t y et al
Fig. 5. Small colony mutants with LOH and aberrations detectable by whole chromosome painting. To the left of each photograph are ldiograms of each
mutant showing chromosome l l a (white) and l i b (black). Regions of LOH on l i b arc indicated by white and are inclusive of the loci shown on the right.
Hatched regions are of 11 origin, but of which homolog is uncertain. Dotted regions are non-11 chromosomes. To the right of each idiogram is a
corresponding painted spread. (A) Mutant 35b. This mutant has an apparent iso-lla and a normal l i b with LOH at Tkl, D11MU48 and D11MM9. If the LOH
resulted from deletion, the deletion would be too small to be detectable by whole chromosome painting. (B) Mutant la, with LOH from Tkl to DllMitlO3. A
deletion of this size would not be detectable by whole chromosome painting. Instead, chromosome 1 lb is longer than normal, suggesting that a duplication
has occurred on this chromosome. Mutant 139a (not shown), with LOH from Tkl to DHMitli, appears similar to la, i.e. there has been a duplication on
chromosome l i b . (C) Mutant 112a, with LOH from Tkl to D11MU103. Both chromosomes 11 look normal, but a small region of chromosome 11 origin is
found on another non-11 chromosome (arrow) and probably originated from l i b when TkJb was inactivated. (D) Mutant 3b, with a large region of LOH from
Tkl to DllMit21, has two chromosomes l l a and a normal length l i b with a large non-11 translocation on the distal end (arrow). (E) Detail of mutant 9b,
with LOH at all loci except DllMit62, has two chromosomes l l a and no l i b , although it has both l l a and l i b alleles of DllMit62.
lla
1lb
D
118a
i
uuFig. 6. Large and small colony mutants with large regions of L O H and normal chromosomes I I . These mutants provide evidence that LOH results from
recombination. Idiograms of chromosome l l a (white) and l i b (black) show the extent of LOH in each mutant White regions on l i b represent replacement
of l i b alleles by l l a alleles. (A) Large colony mutant 29a. (B) Large colony mutant 29b. (C) Large colony mutant 48b. (D) Small colony mutant 118a.
468
Large and small colony L5178Y tk'4' mouse lymphoma mutants
lesions than analysis of banded chromosomes: the unit of
resolution for analyzing breakpoints in banded chromosomes
is a chromosome band (or less), which in mouse generally
consists of 10-20 Mb DNA. The resolution possible with LOH
analysis is determined by the spacing between two adjacent
polymorphic loci. The smallest interval in which we observed
LOH breakpoints was 1 cM, ~2 Mb. The largest region in
which breakpoints were mapped but not resolved was 12 cM,
between DllMit63 and D11MU19.
As shown in Figure 1, the identified polymorphic loci are
more densely distributed in the distal portion of chromosome
11 near the Tkl gene. This is because of our more extensive
search for polymorphisms in this region. Some loci, such as
D11MU48 and DUMit49, cannot be ordered with respect to
each other on the basis of linkage analysis (Dietrich et al.,
1996), although the order of most of the other microsatellite
loci is known. Tkl has been mapped by means of somatic cell
genetics, cytogenetic analysis, in situ hybridization and linkage
analysis, but has not been specifically mapped with respect to
the microsatellite loci. The available mapping data suggest
that the Tkl gene lies ~1 cM distal to DUMH48 and D11MM9
(Evans et al, 1996; Watkins-Chow et al., 1996). Of 86 mutants
that lost a Tkl allele, 85 also lost an allele at both D11MU48
and D11MM9, consistent with close linkage of these three
loci. The other mutant retained both alleles of D11MU48 and
D11MU49.
Thirty six of the 122 mutants lost alleles at none of the loci
examined, including DllAgll within Tkl. We confirmed the
presence of both alleles of Tkl by Southern blot analysis of
DNA from the 36 mutants showing no LOH (Applegate et al.,
1990). These results suggest that these mutants harbor either
small rearrangements that include Tkl or point mutations
within the Tkl gene.
Sequence analysis confirmed or suggested intragenic
mutations in 13 of the 14 mutants analyzed; in two mutants,
no Tklb sequence was observed in the RT-PCR product,
suggesting that Tklb was not expressed in those mutants. Lack
of expression could result from mutations in Tkl outside the
coding region or outside Tkl but near enough to disrupt
transcriptional signals. Ten mutants had single base changes
and one had a large insertion. Nine of the 36 presumptive
point mutants were small colonies. The 27 large colony mutants
are consistent with the expectation that intragenic lesions in
the Tkl gene would not affect a putative growth control
gene and therefore the mutant would have normal growth
characteristics. The nine small colony mutants may be a
consequence of an intergenic event spanning the growth control
gene and sequences affecting expression of Tkl without
disrupting the structural gene, but lying within a region lacking
markers, or may result from another mutation at a secondary
site involved in growth control. Three of four small colony
mutants sequenced had single base changes and one had no
Tklb sequence in the RT-PCR product, possibly because 77c7b
was not transcribed, resulting in no cDNA from this allele to
be sequenced, or because some of the Tklb sequence was
missing from the chromosome. We analyzed eight of these
mutants by chromosome 11 painting (see Table HI for a
summary). Five of the eight painted normally. However,
painting analysis of one of these mutants, 32c, (see Figure
4C) revealed a translocation of non-11 material to the distal
end of chromosome lib, showing that certain major rearrangements are not detected by LOH analysis. Mutant 32c could be
an example of a mutant with a break having occurred distal
to the most distal test locus. Other small colony mutants that
paint normally may have suffered intergenic lesions that are
too small to be detected by either LOH analysis or chromosome
11 painting. Two of these small colony 'point' mutants were
subjected to hybridization with a probe for DllMit69, the
most distal microsatellite locus identified on chromosome 11
and distal to Tkl at 80 cM, although not heteromorphic in
these cells. If a break occurred very near the end of the
chromosome in these mutants, it could account for the small
colony phenotype and might be detectable as loss of a
hybridization site for the DllMit69 probe. DllMit69 hybridization was observed in both these mutants, however (data not
shown), suggesting that these mutants are more Likely to have
arisen as a result of intragenic mutation in Tkl and a second
mutation at a separate growth control locus than by mutation
of a growth control gene linked to Tkl. Figure 7 presents
models for generation of large and small colony mutants,
including models consistent with formation of small colony
mutants with no LOH (Figure 7D and E). Mutant 32c can be
explained as having lost the end of lib, including the putative
growth control gene and enough of Tkl to inactivate it, while
retaining both DllAgll and the Ncol RFLP with Tkl oriented
on the chromosome as shown in Figure 7Db.
Eighty six mutants were found to have LOH at DllAgll.
Sixty three of these mutants had LOH at some but not all loci.
The extent of loss was variable: one mutant lost only the
Tklb allele, some mutants lost only the Tklb allele and loci
immediately proximal and others lost alleles ranging from the
Tkl gene to loci nearer the centromere, up to and including
D11MU2 and D11MH63. This group of mutants was divided
almost equally between small and large colonies (32 and 31
respectively). We can explain this group of mutants either by
the inclusion or exclusion of the growth control gene resulting
in the small and large colony phenotype respectively (see
Figure 7). The growth control gene may lie distal to Tkl if
the mutant results from a single mutation or may lie elsewhere
in the genome if it is affected as a secondary mutation.
Twenty three of the 86 mutants (19% of the 122 mutants
examined) lost alleles at all the microsatellites tested. These
results are consistent with whole chromosome loss through
non-disjunction, with deletion of a large part of the chromosome
or with mitotic recombination. In this cell line, non-disjunction
can be distinguished from the other mechanisms by whole
chromosome painting because of the centromeric heteromorphism in chromosome 11. A non-disjunction mutant would have
only chromosome lla, or two copies of lla if the remaining
chromosome were reduplicated, and the centromeres of those
two chromosomes would be identical in size. Four such
mutants, one small colony (41b) and three large colony (8a,
18b and 42b), were subjected to painting analysis. One large
colony mutant (42b) had two copies of lla and no l i b
chromosomes, clearly arising by non-disjunction, but the other
three mutants had both lla and lib centromeres, suggesting
a mechanism other than non-disjunction for their generation.
Nine of the 23 mutants that lost alleles at all loci were small
colony and 14 were large. If non-disjunction did not occur,
we can postulate intergenic lesions in the mutants. However,
for the small colony mutants, the lesions would include the
putative growth control gene and for the large colony mutants
they would not (see Figure 7 for models).
The two peaks in the bimodal distribution of colony sizes
in the MLA overlap to some extent, allowing the possibility
that intermediate size colonies could be misclassified (Moore
469
M.C.LIechty et al
A
Cm
A
B
™
Ncol Agl1
C
'HI
B
A
G c
-Tel
111
Cm
BREAKAGE/REPAIR
SMALL
Tl(1
A
B
C
NcdAflli
;
i
i
3
SINGLE POINT MUTATION
LARGE
Cm
Q
WILD TYPE CHROMOSOME 11b
A
B
_ *
!
Tkl
, 9l1
Nco IA
9
!
Cm
1
• • ! • • • «•
GC
Loss
GENERATION OF NEW TELOMERE
Tkl
B
C Ncol Aflli
GC
<*
' n I 111
Tel
!
OR
D
B
C
ni
C
Tk1
Ncol Agl1
'HI
-Tel
in
E
Cm
SECOND SITE MUTATION
SMALL
Tk1
C Ncol ,
A
B
til
Cm
A
B
c
Ncol Afll1
-i
;
-Tel
111
GC
LOH
SMALL
B
Cm
CO-MAPPING GROWTH CONTROL GENE
Tk1
Ned Agl1
TRANSLOCAT1ON
GC
111
-Tel
GC /
H I ill—i-; -Tel
LOH
Fig. 7. Models illustrating possible mechanisms leading to small and large colonies. (A) A schematic of the wild-type tk + chromosome 11 as found in the
L5178Y mouse tk+l~ heterozygote. Cm, the centromere; Ncol, the site of a RFLP diagnostic for loss of the Tkl gene; Agll, the polymorphic simple sequence
repeat within Tkl located between exons 6 and 7; vertical bars, the seven exons in Tkl; GC, the putative growth control gene; Tel, the telomere. The Tkl
gene must be oriented with the 5'-end toward the telomere to account for mutants such as 32a, if the breakage/repair model is correct. The chromosome is
not drawn to scale. (B) Point mutations limited to Tkl generate large colony mutants. (C) A growth control gene maps near but distal to Tkl. The region of
LOH is defined by the brackets. These mutants would exhibit LOH at DIIAgll. Either large or small colony mutants are generated, depending on whether
LOH occurs at the growth control gene. The LOH could result from a deletion or recombination event. If a deletion, Tklb and the growth control ailele would
be lost, leading to slow growth; if a recombination, Tklb would be replaced by Tkl", which contains a point mutation inactivating the gene. Since we do not
know the condition of the homologous growth control ailele, the mutant's growth could be slow or normal. (D) Breakage and loss of the distal end of
chromosome 11. The growth control gene and Tklb could be lost or a break (indicated by the vertical striped line) at the 5'-end of the Tkl gene could
inactivate the Tklb ailele without disrupting either the Ncol pattern of Southern blots or resulting in loss of a DIIAgll ailele. Loci distal to DIIAgll would
show LOH. If the broken fragment is not translocated to another chromosome and a growth control gene is on that fragment, a slow growth mutant would
result. This type of mutation could account for the small colony mutants described in this paper that appear to have suffered no LOH, if a break occurred
distal to all heteromorphic markers. The break could be resolved in any of several ways, including: (a) generation of a new telomere resulting in a shortened
chromosome; (b) translocation of chromosomal material from another chromosome (indicated by the striped horizontal line) to the broken end. We would
expect either of these possibilities to result in slow growth, because of loss of the growth control gene or because of the breakage itself. Resolution of the
break by recombination could replace the growth control ailele with its homolog. Recombination could lead to normal growth recovery depending on the
status of the growth ailele on the homologous chromosome. (E) Mutation at a second site that has a growth control effect could result in small colony
mutants, regardless of the type of mutation occurring at Tkl.
et al., 1985). This would compromise analysis of the LOH
data with respect to mechanisms leading to the small and large
colony phenotype. Because the data showed no clear difference
between large and small colony mutants with respect to the
extent of LOH, we considered the possibility that the overlap
between small and large colony sizes could have resulted in
incorrect assignment of colonies into one class or the other.
For that reason, we eliminated the intermediate size mutants
and evaluated only the smallest 25% and largest 25% of the
mutants collected. This subset of the data, shown in Figure 2,
still showed no definable difference in LOH between small
and large colony mutants. Misclassification of initially quiescent cells as small colony mutants was unlikely because of
the manner in which the in situ protocol is performed:
mutagenized cells are seeded into soft agar and allowed to
express mutations before the selective soft agar overlay is
added (Spencer et al., 1994), ensuring that slowly growing
mutants and mutant progenitors are equally likely to be
correctly scored, even if expressed at different times. Therefore,
we conclude that the similar pattern of LOH between large
and small colony mutants was likely to result either from
470
different mechanisms of mutation induction or from differences
in growth control gene involvement in the lesions, rather
than from assignment of mutants to the wrong colony size
phenotype. Glover and Clive (1995) have observed a similar
spectrum of mutations among small and large colony spontaneous mutants isolated using the suspension protocol. This
suggests that both the in situ and the suspension protocols
recover mutants with similar patterns of LOH, although the
relative frequencies of small and large colonies differ.
It has been hypothesized that small colony mutants are the
consequence of intergenic lesions affecting the Tkl gene and
a putative growth control gene, while large colony mutants
are the consequence of either intragenic lesions limited to the
Tkl gene or intergenic lesions that do not affect the putative
growth control gene (Hozier et al., 1981). This proposal was
based on the early cytogenetic observation that many small
colony mutants had detectable chromosome aberrations, while
large colony mutants were cytogenetically normal (Hozier
et al, 1981, 1985, 1991; Moore et al., 1985; Blazak et al.,
1986a, 1989). This historical evidence predicts that there would
not be extensive LOH in large colony mutants as a result of
Large and small colony L5178Y Hc4~ mouse lympnoma mutants
deletions. Our painting results agree with the historical findings;
there are many large colony mutants that do harbor extensive
regions of LOH, up to and including all the test loci on the
chromosome, but without chromosome aberrations and without
chromosome loss. Therefore, we propose that much of the
LOH measured in mutants, at least cases of extensive LOH,
is unlikely to be the result of deletion, but instead reflects
recombination events that would not be revealed by cytogenetic analysis.
Of the 37 mutants that were painted, 15 had LOH from Tkl
to loci proximal to DllNds7 (i.e. LOH extending from Tkl to
at least DllMit59; see Figure 1). Because of the inherent
inaccuracy in measuring chromosome lengths in painted preparations, less extensive LOH from Tkl (78 cM) to loci no
more proximal than DHNds7 (62 cM) could not be accurately
attributed to either deletion or recombination by measurement
of chromosomes. Map positions for these loci are shown in
Figure 1 roughly to scale, but because there is poor correlation
between the genetic and physical maps in mouse, it is not
certain how much of the chromosome is involved in a given
loss. For LOH that does not extend to loci proximal to DllNds7
(i.e. <~20% of the length of the chromosome) we cannot
determine with assurance by means of painting whether a
deletion or a recombination has occurred. All mutants in this
category had two chromosome 11 equivalents that appeared
to be normal or of greater length. Nine of the 15 produced
paints that appeared normal. Except for mutant 42b (Figure
3), which is missing a small part of one chromosome 11,
mutants containing less than two chromosome 11 equivalents
were not observed, suggesting that mutants lacking one
chromosome 11 are not viable. No mutants with extensive
LOH (from Tkl to loci proximal to DllNdsT) appeared to
result from deletions. Twenty two mutants had less extensive
LOH and could not be characterized as either deletions
or recombinations. Of the total of 14 mutants that painted
abnormally, four had normal length chromosomes lla but
chromosomes lib that were abnormally long. These mutants
may have arisen as a result of unequal recombination. Banded
chromosomes of these over-long 11s have not been analyzed
to confirm what regions of the chromosomes have been
duplicated. Mutants with extensive LOH but normal length
chromosomes may have arisen from two events, deletion of
one region and duplication of another of equal size, but this
is less likely than a single recombination event.
There is ample evidence of association between cancers
and loss of heterozygosity resulting from recombination: on
chromosome 7 in skin tumors (Bremner and Balmain, 1990;
Bianchi etal., 1991), on chromosome 3 in breast rumors (Chen
et al, 1994), on several different chromosomes in ovarian
cancer (Yang-Feng et al, 1993), on chromosome 3 in renal
tumors (van der Hout et al., 1993) and on chromosomes 13
and 17 in small cell lung carcinoma (Mori et al., 1989), in rat
mammary tumors (Gollahon et al., 1995), in mouse forestomach tumors (Ushijima et al., 1995), in Wilms tumor
(Mannens et al., 1988; Coppes et al, 1992; Baird et al, 1994)
and in retinoblastoma (Zhu et al., 1992). In numerous cases,
LOH is known to be associated with tumors, but it is not
known whether the LOH results from deletion or recombination; deletions have frequently been assumed.
We have examined only spontaneous mutants, but similar
observations have been made in spontaneous and induced
mutants derived from the human lymphoblastoid tk+l~ TK6
heterozygous cell line developed by Thilly and co-workers
(Liber and Thilly, 1982). Recombination has been observed in
spontaneous and X-ray induced TK~ mutants from TK6 cells
(Li et al, 1992) and at several loci on chromosome 15 in
Saccharomyces cerevisiae mutants induced by UV radiation
and by several chemical agents (Acuna et al., 1994). Li et al.
(1992) reported data consistent with either a growth gene or
telomeric disruption for spontaneous and induced mutants from
TK6 cells. They reported that 75 of 80 slow-growth mutants
that they isolated showed regions of LOH that could include
the telomere. They used four probes on human chromosome
17, one on the short arm and the other three on the long
arm, where the TK gene resides. None of the mutants lost
heterozygosity within the short arm. Although five of the
slowly growing mutants showed no LOH at any of the sites
they examined, the remaining 75 slow-growth mutants showed
LOH at D17S24, which had been mapped distal to the TK
gene. Since they did not have a probe distal to D17S24, we
do not know whether the telomeric region also showed LOH.
Thus, as for L5178Y cells, the possibility remains that lesions
in the region distal to D17S24 could be responsible for the
slow-growth phenotype by harboring a growth gene or by a
disruption of the telomere requiring repair before normal
growth could be resumed.
If there is a growth control gene, we predict that it is distal
to Tkl (Figure 7), based on the finding that mutants that have
lost Tkl and more proximal loci, up to and including DllMit62,
are of either the small or large colony phenotype; therefore,
the putative growth control gene must lie outside the heteromorphic loci examined. The early cytogenetic observations of
small colony mutants with translocations involving the distal
region of chromosome 11 suggests that the putative growth
control gene would lie very close to Tkl, making the telomeric
region a more likely candidate for the growth control gene
than the centromeric region. Since the Tkl gene itself is within
~2 cM of the telomere, this growth control gene would have
to lie very close to Tkl.
An alternative model, focusing on chromosome breakage
rather than a growth control gene, might explain the difference
between growth phenotypes. This model invokes a process of
chromosome damage and repair. In this model, a cell with
chromosome damage would suffer arrested growth until the
cell repairs the damage (Baker et al., 1987; Weinert and
Hartwell, 1988; Lock and Ross, 1990), in which case the
colony is small because cell division was interrupted for a
time. This hypothesis is consistent with several observations
of small colony mutants. The first is that chromosome aberrations are cytogenetically detectable more frequently in small
colony than in large colony mutants (Hozier et al., 1981, 1983,
1985; Moore et al, 1985; Blazak et al, 1986a, 1989). The
second is that some mutants with cytogenetically detectable
aberrations are karyotypically unstable, because they revert
over time to a cytogenetically normal karyotype, while
remaining tic1- (Hozier etal, 1981, 1983, 1985; Moore et al,
1985; Blazak et al, 1986a). This karyotypic instability may
lead over time to an underestimation of the true proportion of
small colony mutants that have chromosome 11 abnormalities.
The third observation is that some small colony mutants, after
being removed from semi-solid medium and allowed to grow
in suspension, re-acquire a normal growth phenotype while
remaining tkr1'. This would occur if cell growth were arrested
while repairing the chromosome or regenerating a telomere.
A third model invokes mutation at a locus removed from
Tkl (and probably not on chromosome 11, on the basis of
471
M.C.Llechty et at
LOH analysis) to account for at least some of the slow growth
mutants. Small colony mutants that have point mutations, no
LOH and normal karyotypes are more easily explained by this
second site model than by the constraints of the model requiring
a putative growth control gene to be located in the small
region between DllMit69 and the telomere. Whether either of
these models is correct, they are not incompatible with the
breakage/repair model, which may better explain some mutants,
particularly translocation mutants that eventually recover
normal growth characteristics.
A corollary of the hypothesis of small and large colony
mutant origin is that mutant growth phenotype might predict
the mechanism of action of a test compound: point mutagens
would be expected to induce intragenic lesions limited to the
Tkl gene (and, therefore, result in large colonies), while
clastogens would be expected to induce intergenic lesions that
would affect other genes in addition to the Tkl gene. These
intergenic lesions would include the putative growth control
gene at least some of the time and, therefore, the frequency
of small colony mutants would be higher if the test compound
were a clastogen than if it were a point mutagen (Hozier et al,
1981; Sawyer et al., 1985; Auletta et al, 1993; Kirkland,
1993). However, the data presented here reveal both small
colony and large colony mutants with very extensive losses of
heterozygosity over almost the entire chromosome and also
both small colony and large colony mutants with virtually no
losses other than at the Tkl gene itself. While these observations
are compatible with the concept of a growth control gene,
they are incompatible with the speculation that size of colony
by itself can reliably predict whether a test compound is acting
as a point mutagen or as a clastogen. However, if the damage/
repair model is correct, mutagenic compounds might be divided
by their mechanisms of damage induction, based on small or
large colony phenotype. Thus large colony-inducing compounds might cause lesions that are repaired more quickly
than small colony-inducing compounds. Conversely, small and
large colony mutants may arise by similar mechanisms, but
are distinguished by whether the growth control gene is
affected. If this is the case, similar patterns of LOH may be
(and are) observed in both small and large colonies; the lack
of polymorphic markers for the growth control gene or for
any loci distal to Tkl prevents distinction between large and
small colony mutants.
The TK6 cell line also has a slowly growing mutant
population (Yandell et al., 1986; Liber et al, 1989). In the
human genome, TK is found at the distal end of the long arm
of chromosome 17 near the telomere, just as 77:7 is found near
the telomere on mouse chromosome 11. Thus, a mechanism
involving breakage of chromosome ends which could be
responsible for some small colony mutants in L5178Y mouse
cells may have a human cell counterpart. However, the fact
that slow growing TK6 mutants are generally cytogenetically
normal (Kodama et al., 1989) suggests that if breakage occurs,
repair by translocation, as appears to have occurred in mouse
mutant 32c, is not common in the human cell line. TK6 cells
are wild-type for Trp53, which is essential for maintenance of
genomic stability, whereas Storer et al. (1997) have found that
Trp53 is heterozygous in L5178Y tk+/~ cells. This difference
could account for failure to recover small colony translocation
mutants in TK6.
Recently, Mitchell (1997) described a model in which Trp53
is a secondary site for mutation in L5178Y cells, accounting
for the genetic difference between large and small colony
472
mutants. Munke and Francke (1987) mapped Trp53 by in situ
hybridization to band 11B2-C, whereas Hozier et al. (1991)
mapped Tkl to band HE. These loci are 39 cM apart on the
chromosome 11 linkage map (Watkins-Chow et al., 1996).
The heteromorphic microsateOite loci that we examined span
the region both proximal to and between Trp53 and Tkl. If
LOH at Trp53 occurring concomitantly with Tkl mutation
were a factor in determining mutant colony size, then we
would expect to see a change in colony phenotype in those
mutants that have undergone LOH at the Trp53 locus, as
compared with those that have not. We did not observe such
a change in phenotype in our data. However, Trp53 mutation,
or mutation at other loci affecting mutability, could result in
mutation at secondary sites to account for small colony mutants.
Storer et al. (1997) showed that the existing p53 mutation in
L5178Y cells is likely to result in production of dysfunctional
p53 protein, which would make these cells particularly susceptible to induction of multiple mutations. Li et al. (1994)
observed a greater frequency of mutations at randomly selected
microsatellite loci in TK6 clones that had been selected for
mutations at TK than in unselected clones, suggesting that in
many cases, mutations at one locus may be accompanied by
other mutations elsewhere in the genome. Xia et al. (1994)
also concluded that a mechanism independent of tk mutagenesis
was required to explain the distribution of small and large
colony mutants in TK6 and related WIL2-NS cell lines.
Although TK6 cells are wild-type for p53, some other locus
may exert a similar effect on mutation in TK6 and/or L5178Y
cells. The karyotypic variability that we observed in several
mutants (i.e. 30c, 143a and 32c, as shown in Figure 4) and
the apparent multiple events occurring in individual mutants
(i.e. a large tract of LOH and a translocation in mutant 3b, as
shown in Figure 5D) are evidence of multiple mutations in
these mutants.
The MLA is a widely used genetic toxicology assay. Since
L5178 Y cells are mutated at Trp53 and appear to be particularly
susceptible to mutation and to multiple mutation, the question
arises as to whether this cell line is an appropriate indicator
of the mutagenic potential of chemicals or whether its greater
sensitivity to mutation may lead to false positive results, i.e.
positive results unsupported by other assays. Extremes of pH
and osmolality in testing or too low a limit on relative total
growth (RTG, a measure of toxicity) can result in false
positives (Oberly and Garriott, 1996). Amacher et al. (1980)
recommended that RTG be limited to a minimum of 20%,
rather than the 10% generally used. The National Toxicology
Program, extending RTG to 3% to 'salvage' some cultures,
may have contributed to the reputation of the MLA for
generating false positives. Re-evaluation of years of data from
Lily Research Laboratories at 20% RTG reduced the number
of false positives without significantly reducing sensitivity, i.e.
the ability to detect mutagens (Oberly and Garriott, 1996).
Thus, adjustment of the criteria used to evaluate results of the
MLA can reduce the incidence of false positive findings.
Comparisons of assay systems have shown that in vitro
short-term tests for genetic toxicity, including the MLA, can
be used to predict rodent carcinogenicity (Tennant et al., 1987;
Zeiger et al, 1990). When care is taken to recover and detect
small colony mutants, the results of the MLA are comparable
with those for rodent carcinogenesis bioassays, which are used
to identify agents expected to be carcinogenic in humans
(Mitchell et al, 1997). Mutation or loss of p53 is found in
many human and rodent tumors. Thus, similar mechanisms
Large and small colony L5178Y 1k~4~ mouse lymphoma mutants
involving mutation of p53 may account for the ability of the
MLA to predict results of rodent bioassays and the MLA may
be particularly suited to identification of compounds that result
in p53 mutation. This, in addition to the ability of the MLA
to detect a wide variety of mutations, adds to its utility in
detecting many kinds of mutagens.
The observation of recombination in the mutants studied is
important, because it indicates that the MLA may be used as
an assay for recombination. The MLA is already important as
a mammalian cell gene mutation assay in the USA and Europe.
Discussion is currently under way regarding harmonization of
genetic toxicology testing practices world wide. If a mammalian
cell gene mutation assay is included in the core battery of
tests approved world wide, the MLA is likely to be that test.
It has already been established that the MLA detects a variety
of mutations that have been shown to be relevant in the
etiology of human cancers. These include point mutations,
deletions, chromosomal abnormalities such as translocations,
numerical changes in chromosomes such as losses and duplications and LOH. Now we have evidence that the MLA can
detect recombination as well. Together, our LOH and painting
data suggest that recombination is a significant mechanism in
the generation of mutants in the mouse lymphoma assay. This
is a significant finding, for it will allow the MLA to be used
to assess recombination in addition to its use to assess
clastogens, point mutagens and agents causing deletion events.
Acknowledgements
This work was supported by contracts N44-ES-92003 and N44-ES-32002
awarded by the National Institute of Environmental Health Sciences.
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Received on October 6, 1997; accepted on March 18, 1998