Mutagenesis vol.13 no.5 pp.487^97, 1998
Spontaneous and ENU-induced mutation spectra at the ell locus
in Big Blue® Rat2 embryonic flbroblasts
David E.Watson1>2, Michael L.Cunningham2 and
Kenneth R.TindaU1-3
'Molecular Mutagenesis Group, Laboratory of Environmental
Carcinogenesis and Mutagenesis and 2Laboratory of Pharmacology and
Chemistry, National Institute of Environmental Health Sciences, Research
Triangle Pa*, NC 27709, USA
Big Blue® Rat2 embryonic fibroblasts carry the X-Liz
shuttle vector which is also present in the Big Blue® mouse
and rat Mutations in the Big Blue® systems have most
often been measured at the lacl locus. However, a method
for positive selection of mutations at the A, ell locus
was recently described. This assay appears to have many
advantages over the use of lacl as a mutational target,
but it has yet to be well characterized in mammalian
mutagenesis studies. The objective of these studies was to
determine the spontaneous and ethylnitrosourea (ENU)induced mutant frequencies (MFs) and mutational spectra
at ell using Big Blue® Rat2 embryonic fibroblasts. The
average spontaneous MF was 13 ± 1.4 X 10~5. The average
induced MF was 60 ± 10 x 10- J 10 days following a 30 min
treatment with 0.1 mg/ml ENU. Eighty four independent
spontaneous mutants were sequenced: 23 (27.4%) were
frameshift mutations and 61 (72.6%) were base substitutions. Two spontaneous frameshift hotspots were detected,
both in mononucleotide runs. G:C—>A:T transitions were
the most common type of base substitution in ell; of these
71% occurred at CpG sites. The ENU-induced mutational
spectrum at ell (44 mutants) consisted of 42 base substitutions (95.5%) and two -1 frameshift mutations (4.5%).
Compared with the spontaneous spectrum, the ENUinduced spectrum had significantly fewer frameshift
mutations (4.5 versus 27%) and base substitutions occurred
predominantly at A:T base pairs (71 versus 34%). Overall,
the spontaneous ell mutational spectrum reported here
differs slightly from spontaneous spectra reported at the
Big Blue® lacl locus, but the mutational spectra and
base substitution MFs following treatment with ENU were
comparable at both loci. These data support the continued
use of ell as a selectable marker in mutagenesis studies
involving cells or tissues that carry a A. transgene.
Introduction
The development of transgenic rodents which contain stably
integrated prokaryotic genes that are readily recovered for
mutational analysis has facilitated the study of mutagenic
processes in mammals (Mirsalis et al, 1995). The most widely
used transgenic in vivo mutation assay is the Big Blue® mouse
(B6C3F1 or C57B16), which carries 30-40 copies of the X.Liz shuttle vector integrated into a single genomic site. A
similar Big Blue® transgenic rat (Fisher 344) with 30-40
integrated copies of A.-Liz has also been described (Kohler
et al, 1991). The use of these animals and cell lines derived
from them has improved our understanding of both spontaneous
and chemically induced mutagenesis through the generation
of mutant frequency and mutational spectra at lacl. Databases
containing thousands of lacl mutations have been established
(de Boer, 1995; Cariello etal., 1997; Hutchinson and Donnellan, 1997), including those resulting from the in vivo action
of alkylating agents (Mirsalis et al., 1993; Walker et al,
1996), polycyclic aromatic hydrocarbons (Kohler et al, 1991;
Gorelick et al., 1995; Shane et al., 1997) and aromatic amines
(Ushijima et al, 1994; Hayward et al, 1995). Thus a great
deal is known about lacl as an in vivo target for mutation in
Big Blue® animals. A limitation to the use of lacl as a
mutational target gene is that the procedure by which lacl
mutants are identified, purified and sequenced is time consuming and expensive and can be complicated by the existence of
ex vivo mutants. While these latter mutants can usually be
eliminated as sectored plaques, careful scoring is critical to
obtaining accurate, quantitative results (Hayward et al, 1995;
Piegorsch et al, 1995; Shane et al, 1997). Recently the ell
locus was used as an alternative mutational target in Big
Blue® mice (Jakubczak et al, 1996). Likewise, several other
transgenic in vivo mutation assays based on X also carry ell.
Thus the ell gene may be widely applicable to several
in vivo mutation assays, allowing for direct system to system
comparisons as well as providing an additional genetic marker
for comparisons within a system (e.g. lacl versus ell).
The ell assay selects for ell mutants by exploiting die
critical role of the ell gene product in the commitment of X
to lysogeny following infection of a permissive Escherichia
coli host. The ell gene product is unstable in E.coli because
of rapid proteolysis by FtsH, a protease encoded by the E.coli
gene hflB. A different E.coli gene, hflA, encodes the HflKC
membrane complex, which modulates the protease function of
FtsH (Kihara et al, 1997). The E.coli strain used in the ell
assay has mutations in both hflA and hflB, which results in
greater longevity of the ell gene product. This condition favors
formation of lysogens by X phage having wild-type ell. Such
lysogens are indistinguishable in the E.coli lawn. In contrast,
X phage which cany an inactivating mutation specific to ell
will form plaques in the E.coli lawn when incubated at 24°C
(Jakubczak et al, 1996).
Mutant plaques are readily identified, cored, plaque purified
and sequenced in a process similar to that employed for
analysis of lacl mutations in X phage. The ability to directly
select for mutants confers important technical advantages, as
well as savings of expense, time and effort. In addition, ex
vivo mutations are not a problem with the ell assay because
of the immediate commitment to lysogeny or lysis following
infection. Mutations at ell following lysogeny will not result
in plaque formation except in the unlikely event of mutations
occurring in both cl and ell in the same phage (Jakubczak
et al, 1996). It is also noteworthy that ell (294 bp) is less
than one third the length of lacl (1080 bp), thereby allowing
for efficient DNA sequence analysis.
^To whom correspondence should be addressed. Tel: +1 919 541 3275; Fax: +1 919 541 1460; Email: [email protected]
© UK Environmental Mutagen Society/Oxford University Press 1998
487
D.E. Watson, M.I,.Cunningham and K.R.Tlndall
Jakubczak et al. (1996) showed that loci and ell have
comparable mutant frequencies in DNA from the bladder of
Big Blue® B6C3F1 mice. Moreover, they observed comparable
increases in mutant frequency at lacl and ell in bladder DNA
following in vivo exposure of mice to the bladder carcinogen
p-cresidine. These are encouraging data which indicate that
the ell locus may be a useful alternative mutational target in
the Big Blue® system, but the assay has yet to be validated by
different laboratories using different experimental conditions.
To contribute to the characterization of ell as a useful target
for in vivo mutation studies we determined the spectrum of
84 independent mutations that arose spontaneously at the ell
locus in Big Blue® Rat2 fibroblasts. In addition, we examined
both the mutant frequency and the mutation spectrum following
treatment of Big Blue® Rat2 cells with the alkylating agent
TV-ethyl-Af-nitrosourea (ENU). The data from these experiments
support the continued use of ell as a selectable marker in
mutagenesis studies involving cells or tissues that carry a A.
transgene.
Materials and methods
Escherichia cob bacteria
G1250 (derived from XLl-Blue MRA strain; Stratagem:) [A(mcrA)183,
A(mcrCB-hsdSMR-mrr)173, endAl, supE44, thi-1, gyrA96, relAl, lac° supF,
hflAy.TnS, hflB29 TniO, Tl*] was a generous gift from Dr John Jakubczak
(NCI, Bethesda, MD). Overnight cultures of G1250 were grown in 5 ml
LBMM (LB broth containing 0.2% w/v maltose and 10 mM MgSO4) at 37°C
using a vertical rotary incubator at high r.p.m.. The next morning 10 ml
LBMM were added, the cells incubated for another 4-5 h and the optical
density of solutions was determined (600 cm). At the end of this second
incubation bacterial growth had saturated and had the same optical density as
overnight cultures (typically 1.2). Bacterial solutions were diluted to an OD
of 1.0 prior to use. Bacteria (0.1 ml) were infected with X phage (see below),
mixed with 3 ml TB1 top agarose and plated onto 40 ml TB1 plates. Plates
for mutant selection were incubated for 48 h at 24°C; titer plates were
incubated overnight at 37°C. For 1 1 TB1 bottom agan 10 g bacto-tryptone;
5 g NaCl; 15 g bacto-agar; 1 mg thiamine. For 1 1 TB1 top agarose: 10 g
bacto-tryptone; 5 g NaCl; 7 g agarose; 1 mg thiamine.
Big Blue9 Rat2 embryonic fibroblasts
Big Blue* Rat2 embryonic fibroblasts (Stratagene, La Jolla, CA) were cultured
in 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) containing 10%
dialyzed fetal bovine serum (Hyclone Laboratories Inc., Logan, UT), 2 mM
L-glutamine (Gibco BRL Life Technologies, Grand Island, NY) and
IX Antibiotic-Antimycotic (100 U/ml penicillin G, 100 U/ral streptomycin
sulfate and 0.25 |ig/ml amphotericin B; Gibco BRL Life Technologies). Under
these conditions the population doubling time was ~24 h. Cells were
subcultured as follows: rinsed with Hank's balanced salt solution (HBSS),
disaggregated with 2 ml 0.25% trypsin (Gibco BRL, Gaithersburg, MD),
suspended in medium and diluted (1:5) for replating. Cells not replated were
centrifuged for 5 mm at 500 g, washed with phosphate-buffered saline, pH
7.4, centrifuged again and stored frozen in microfuge tubes at -135°C prior
to DNA extraction.
DNA extraction, packaging and mutant selection
Genomic DNA was extracted from pellets containing 2-5 X106 cells using
Recoverease DNA Isolation Kits (Stratagene, La Jolla, CA). The resulting
DNA solutions were stored at 4°C. X-Liz shuttle vectors were rescued from
genomic DNA by adding 10 |il Transpack packaging extract (Stratagene) to
10 p.1 DNA solution and incubating at 30°C. After 90 mm an additional 10 |Xl
Transpack were added, followed by another 90 min incubation at 30°C.
Packaging reactions were stopped by addition of 560 |il SM buffer (50 mM
Tris, 10 mM MgSO4, 0.001% gelatin, 0.1 M NaCl, pH 7.5). Tubes were
vortexed aggressively and pipetted repeatedly to reduce the viscosity of the
packaged DNA solution.
To select for c/I mutants 100 (il packaged DNA solution were mixed with
0.1 ml G1250 bacteria (OD 1.0; described above), incubated for 15 min at
37°C, mixed with 3 ml TB1 top agarose, plated onto a TB1 plate and grown
at 24°C for 48 h. Five TB1 plates were used for each sample, for a total
plating of 500 nl packaged DNA. To determine the number of plaque forming
units on the mutant selection plates 20 \l\ of each of three 100-fold dilutions
were plated onto three TB1 plates (equivalent of 0.2 |il X phage solution/
488
plate). Plates were incubated overnight at 37°C. The average number of
plaques on the three titer plates was multiplied by 2500 to estimate the number
of plaque forming units on the ell mutant selection plates. Mutant frequencies
were calculated as the number of mutant plaques (plaques on TB1 plates
grown at 24°C for 48 h) divided by the estimated total number of plaque
forming units on these plates.
Reconstruction experiment
The reconstruction experiments tested the ability of mutant X phage to form
plaques in the presence of a vast excess of wild-type X phage. DNA of
unexposed Big Blue* Rat2 fibroblasts were used as the source of ell wildtype and mutant X phage by coring plaques from TB1 plates which had been
incubated at 37 and 24°C respectively. These cored plaques were suspended
in 1 ml SM buffer, vortexed, incubated for 1 h at room temperature and
centrifuged for 1 mm at 14 000 g to pellet the agar. The supernatant was
diluted lO^-lO8 and replated on TB1 plates to allow for plaque purification
of both the ell wild-type and ell mutant phage Plaques from the 108 dilution
were cored, suspended in 1 ml SM buffer, vortexed, incubated for 1 h at room
temperature and centrifuged to pellet the agar plug. Lysis plates were prepared
for each plaque-purified X phage; 20 jxl of each supernatant were adsorbed
onto E.coli G1250 and incubated on TB1 plates (described above). Mutant X
phage were incubated for 48 h at 24°C; wild-type X phage were incubated
overnight at 37°C. Stock solutions of the X phage were prepared by overlaying
the surface of each lysis plate with 10 ml SM buffer for 2 h at room
temperature. Titers of ell wild-type and mutant X phage and sequencing of
me ell gene were conducted for each stock solution. Two reconstruction
experiments were conducted that used 50 mutant X phage and up to 105
(Figure 1A) or 106 (Figure IB) wild-type X phage mixed together, adsorbed
onto E.coli G1250, plated and grown under mutant selection conditions (24°C,
48 h) in order to assess the efficiency of recovery of X phage bearing ell
mutations. Note that at very high titers of wild-type X phage ell mutants that
arose during phage stock preparation were accounted for m the final calculation
of the percentage of mutants recovered.
Spontaneous and ENU-induced mutational spectra
To determine the spontaneous mutational spectrum at the ell locus in Big
Blue* Rat2 embryonic fibroblasts a modified fluctuation test was conducted.
Forty eight individual cell populations were established by plating 100 cells
into each well of two 24-well plates. When the cells were nearly confluent
they were transferred to 175 cm2 flasks and again grown to near confluence.
Cell pellets were made from these cell lines, from which DNA was extracted,
mutants were purified and mutant ell genes were sequenced. No more than
five mutants were sequenced from any of the original 48 cell populations and
only independent mutants were included in the mutational spectrum (Table I
and Figure 2), i.e. if the same mutation in ell was observed more than once
in one of the 48 cell populations then that mutation was counted only once
in the spectrum.
One of the Rat2 cell populations obtained from the spontaneous mutation
work was then selected as the cell line for use in the ENU treatment studies.
This Rat2 cell population was chosen for further study because it had a low
spontaneous ell mutant frequency One day prior to exposure 10* Rat2 cells
were added to each of 21 175 cm2 flasks. The following day the cells were
rinsed with HBSS and each flask received 50 ml medium containing 15 mM
HEPES, pH 7.0. In addition, ENU-treated cells received 5 mg ENU (0.1 mg/
ml final concentration). Cells were treated for 30 min (Zimmer et aL, 1996).
At the end of the treatment cells were washed three times with HBSS and
fresh culture medium was added to these flasks. Cells were grown for 10
days and subcultured every 2-3 days. On day 10 following treatment cells
were harvested and the mutant frequency and mutational spectra were
determined.
Plaque purification, PCR and ell sequencing
Mutant plaques were cored and delivered into microfuge tubes containing
0.1 ml SM buffer. Tubes were vortexed and allowed to sit for 1 h at room
temperature. The samples were then microfuged for 1 min and 2 ul supernatant
from each sample were delivered onto the surface of a 1 cm2 section of a
TB1 plate containing 0.1 ml E.coli G1250 and 3 ml TB1 top agarose. Plates
were incubated at 24 °C for 48 h. Typically 24 mutants were plaque purified
on a single TB1 plate.
For PCR amplification of the ell gene plaque-purified mutants were cored
and processed as described above except that X phage were released in 0.1 ml
water instead of SM buffer. PCR amplification was carried out on 10 jal
supernatant. The other constituents in the PCR were as follows: 5 U AmpliTaq
Gold (Perkin Elmer), 250 nM dNTP, 10 \i\ 25 mM MgCl2, 10 jxl 10X PCR
buffer (Perkin Elmer), 4 pmol each primer, final volume 100 uJ. Primers
(Research Genetics, Huntsville, AL) were: c//pl, 5'-AAA-AAG-GGC-ATCAAA-TTA-AAC-C-3'; c//p2, 5'-CCG-AAG-TTG-AGT-ATT-TTT-GCT-G-3'.
Using a Perkin-Elmer 9600 or 2400 me PCR program was: step 1, 95°C
ell mutation spectra
terminator reaction kit FS; Perkin Elmer). The sequencing program consisted
of 25 cycles of 96°C 30 s, 50°C 15 s, 60°C 4 min, then a hold at 4°C.
Products were precipitated with 2 nl 3 M sodium acetate, pH 5.2, and 50 |ll
95% ethanol. Tubes were vortexed, placed on ice for 10 min, centnfuged for
30 min at 14 000 g and the supematants discarded. The pellets were washed
once with 250 JJ.1 70% ethanol, centrifuged for 15 min at 14 000 g and again
the supematants discarded. The samples were resuspended in 5 (il deionized
formamide, 1 u.1 tracking buffer and heated for 2 min at 90°C. Aliquots of
1.5 nl each sample were loaded onto a 5% acrylamide gel for sequencing in
an ABI Prism 377 (Perkin Elmer), ell sequences from the mutant plaques were
compared with the wild-type ell sequence (GenBank accession no. J02459)
(Schwartz et al, 1978).
Results
0
5«JO
10000
20000
4O000
60000
80000
10OOO0
pfu per plate
Figure 1B.
—i
5OO0O
r
i
1
f
*
'
100000
150000
MOOOO
HOMO
SOOOOO
'MOM)
'
I0O00OO
pfu per plate
Fig. 1. Reconstruction experiments. Percent recovery of mutant X phage
plaques versus the number of accompanying wild-type X phage. In two
independent experiments 50 mutant X phage and up to 100 000 wild-type X
phage (A) or up to 1 000 000 wild-type X phage (B) were mixed, adsorbed
onto E.coli G1250, plated and grown under mutant selection conditions
(TB1 plates, 48 h, 24°C).
10 mm; step 2, 31 cycles of 95, 55 and 72°C for 1 mm each; step 3, 72°C
10 min; step 4, 4°C hold.
PCR products were processed for DNA sequence analysis using QIAquick
PCR purification kits (Qiagen Inc., Chatsworth, CA). The ell gene was
sequenced with a dye-terminator cycle sequencing kit containing AmpliTaq
DNA polymerase FS (ABI Prism, Warrington, UK). The reaction mixture
(20 nl final volume) consisted of 2 nl purified PCR product (in 10 mM TrisHC1, pH 8.0), 3.2 pmol either primer and 8 \i\ sequencing reaction mix (dye-
Reconstruction experiment
To assure quantitative recovery of ell mutants reconstruction
experiments were conducted using plaque-purified mutant and
wild-type X phage. The mutant X phage contained a +1
frameshift (+1 G, nt 179-184) in the sense strand of the ell
gene, as determined by DNA sequence analysis (data not
shown). The ell structural gene of the wild-type X phage had
no mutation. In these experiments 50 mutant X phage and up
to 106 wild-type X phage were mixed, adsorbed onto E.coli
G1250, plated and grown under mutant selection conditions
(24°C, 48 h). These reconstruction experiments tested the
ability of mutant X phage to form plaques in the presence of
a vast excess of wild-type X phage.
In two independent experiments formation of plaques by
the mutant X phage was unaffected by the presence of up to
105 or 106 wild-type phage (Figure 1A and B respectively). It
should be noted that the recovery data were corrected for the
presence of mutant plaques in the wild-type phage stock,
which occurred at a frequency of 8.0X 10~5. These ell mutants
arose spontaneously during phage stock preparation.
Spontaneous mutational spectrum
ell mutants which arose spontaneously in the genomic DNA
of 32 Big Blue® Rat2 cell lines were sequenced and a maximum
of five independent mutations from each cell line were included
in the spontaneous mutational spectrum (Figure 2 and Tables
I and II). Of 104 mutant plaques that were purified and
sequenced five had no detectable mutations at ell; of these
four were independent. Among the remaining 99 ell mutants
83 (95.4%) contained independent mutations. The other 16
mutants were excluded from the spectrum because they were
observed more than once in an expanded cell population and
therefore may have been of clonal origin. One double mutant
was observed: C ^ G (nt 232; Leu->Pro) and A->G (nt 243,
silent).
Frameshift mutations accounted for 23 of 84 (27.4%) of the
mutations in the spontaneous mutational spectrum. All but one
of these frameshift mutations occurred at two mutational
hotspots, both in homonucleotide runs. The first hotspot was
in a run of guanines (sense strand, nt 179-184) in which single
base addition or single base deletion frameshift mutations were
observed with equal frequency (Table I). The second hotspot
was in a run of adenines (sense strand, nt 241-246), in which
eight single base deletions were observed. Curiously, no single
base additions were observed in this run of adenines (Figure
2). Mutations at these two hotspots accounted for 26.2%
of all independent spontaneous mutations. Only one other
frameshift mutation was observed, a -1 frameshift (-C, nt 167),
which did not occur in a homonucleotide run.
Base substitutions accounted for 61 of 84 (72.6%) of
489
D.E.Watson, M.L.Cunningham and K.R.Tindall
Table I. Spectrum of spontaneous independent mutants at cll in untreated Big Blue® Rat2 embryonic fibroblasts*
Mutation
Number of observations
Percent of total (84)
Mutation frequency1'
Frameshifts
+1
-1
Base substitutions
Transitions
G:C->A:T
at CpG sites1
A:T->G:C
Transversions
G:C->T:A
G:C->C:G
A:T->T:A
A:T->C:G
At G:C base pairs
At A:T base pairs
23
7
16
61
32
21
15
11
29
8
11
3
7
40
21
27.4
8.3
19.1
72.6
38.1
25.0
17.9
13.1
34.5
9.5
13.1
3.6
8.3
65.6
34.4
3.59X10-5
1.09X10"5
2.50XKT5
9.51X1CT5
4.99XKT3
3.28X1O"5
2.34X1CT5
1.72X10-'
4.52 X10"5
1.25X10"5
1.72X10"5
0.47 X10"5
1.09X1CT5
6.24 X10"5
3.28X10"3
•Individual cell populations were established by plating 100 cells/well. These individual populations were grown until enough cells were available to select
for mutants. No more than five mutants were sequenced per population. The same mutation observed more than once in a single population was counted only
once in the spectrum. Of 104 mutants that were sequenced five had no mutation in the cll structural gene and 84 were independent, including one double
mutant.
•"Mutation frequency was calculated for each category of mutation as the product of mutant frequency (13.1 X 10~5) and the mutant fraction of interest. For
example, the mutant fraction of +1 frameshifts was 0.083 (8.3%) so the mutation frequency for +1 frameshifts is [O.O83X(13.1X1O"5)] = 1.09X10"5.
Tifteen of the 21 G:C->A:T mutations observed (71%) were at CpG sites
no
110
AGO
QtC
130
TOG
140
ATT
OCA
MO
TTC
TC*
150
ATQ
1(0
100
TOC
CM
C
GUT
2*0
AAT
240
TCT
270
GM
CM
ATC
CMS
2*0
as
230
taa
aH3
TTC
2M
Fig. 2. Nucleotide map of the cll structural gene with 84 spontaneous
independent mutations (shown above the gene) and 44 ENU-induced
independent mutations (shown below the gene) in Big Blue* Rat2
fibroblasts. Independence of mutants was assured using a modified
fluctuation test design (see Materials and methods). 5'-CpG-3' sites are
underlined.
490
the mutations in the spontaneous mutational spectrum with
approximately equal frequencies of transitions (38.1%) and
transversions (34.5%). The ratio of base substitutions was ~2:1
at G:C (65.6%) versus A:T (34.4%) base pairs. G:C->A:T
transitions accounted for 26% of all spontaneous mutations;
of these 71% occurred at CpG sites. CpG sites are sites of
cytosine methylation. Deamination of 5-methylcytosine yields
thymine, which upon replication produces a G:C—>A:T transition (a more thorough discussion is given below). This
proposed mechanism for the high frequency of G:C—»A:T
transitions observed at CpG sites in Big Blue® studies is
widely cited (Provost et ai, 1993; Hayward et ai, 1995;
Piegorsch et ai, 1995; Shane et ai, 1997).
The significant proportion of G:C-»A:T transitions at CpG
sites among spontaneous mutants arising at both cll and lad
led us to perform a detailed analysis of CpG sites in both
genes (Table III), lad and dl contain 95 and 22 CpG sites
respectively. Two G:C—>A:T transitions can occur at each CpG
site, resulting in missense, nonsense or silent mutations. All
of these possibilities were examined. Missense and nonsense
mutations comprise 59.7 and 61.4% of the possible G:C—*A:T
transitions at CpG sites in lad and dl respectively (Table III).
The other mutations (~40%) are silent. Of the possible missense
and nonsense mutations 41.6 and 22.2% have been reported
at lad (de Boer et al, 1997) and dl (this publication)
respectively. It is also noteworthy that CpG sites occur at
similar frequencies in the two structural genes, constituting
8.8 and 7.5% of the total number of nucleotides in lad and
dl respectively.
ENU-induced mutant frequency and mutational spectrum
Mutant frequencies were determined for control and ENUtreated cells 10 days after treatment. The mean control cll
mutant frequency for Rat2 embryonic fibroblast cells was
13 ± 1.4X10"5 {n = 3; Table IV); the mutant frequency
following ENU treatment was 60 ± 10X10"5 (n = 10),
4.6-fold higher than non-treated cells (Table IV).
Fifty mutant plaques from ENU-treated cells were purified
and the cll genes were sequenced. Forty eight had detectable
ell mutation spectra
Table II. Spontaneous mutation spectrum at the ell locus of Rat2 fibroblasts
Nucleotide
3
16
25
29
34
35
42
49
50
64
73
89
89
100
100
113
115
125
126
145
145
149
155
155
163
164
167
173
179
179
185
190
194
200
209
211
212
214
217
218
219
221
226
232
233
241
243
275
287
292
292
CpG site
Mutation
No. observed
Correct aroino acid
Mutated amino acid
G->T
1
1
1
1
1
1
1
1
2
3
3
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
7
7
1
Met
Lys
Glu
Ala
Arg
Arg
Glu
Leu
Leu
Ala
Gly
Ala
Ala
Gly
Gly
Ser
Gin
Arg
Arg
Pro
Pro
Lys
Ser
Ser
Leu
Leu
Ala
Leu
Trp
Trp
Val
Asp
Asp
Met
Leu
Ala
Ala
Arg
Gin
Gin
Gin
Val
Ala
Leu
Leu
Lys
Lys
Gin
Glu
Stop
He
Stop
Stop
Asp
Stop
Pro
Asp
Val
Ser
Thr
Arg
Val
Glu
Ser
Cys
Leu
Lys
Lys
Ser
Ser
Ala
Arg
Stop
Stop
Phe
Pro
Frameshift
Pro
Frameshift
Frameshift
Gly
His
Gly
Thr
Trp
Pro
Val
Stop
Stop
Pro
His
Ala
Pro
Val
Pro
Frameshift
Lys (silent)
Leu
Ala
Gly
Arg
A-H>T
G->T
C-»A
C->T
G->C
G->T
T->G
T->C
G->A
G->C
C->T
C->A
G->A
G->T
C->T
C^A
G^A
G-»C
C->T
C->G
A->G
C^A
C^G
C->T
T->C
-C
T^C
+ G
-G
T^G
G-^C
1
1
1
T->C
T->G
G->C
C^T
C—>T
C^T
A^C
A->C
T->C
G^C
C^G
T^C
-A
A->G
A--.T
A^C
T-)G
T->A
mutations in the ell structural gene (96%); of these 44 were
independent mutants. The mutational spectrum following ENU
treatment (Tables V and VI and Figure 2) differed substantially
from the spectrum observed in non-treated cells. G:C-»A:T
transitions at CpG sites accounted for only 6.8% of all
mutations and only two frameshift mutations were observed
(4.5%). Both frameshift mutations were single base deletions
of A in the run of adenine at bp 241-246. The bulk of the
spectrum consisted of 42 base substitutions (95.5%).
The spectra were used to calculate frequencies for frameshift
and for each type of base substitution mutation (Table V).
While mutant frequency data provide a quantitative index of
all detectable mutations, mutation frequency data provide a
quantitative measure of each type of mutation.
For example, whereas ENU caused a 4.6-fold increase in
1
1
1
4
3
2
1
1
1
1
1
1
8
1
1
1
1
1
Stop
mutant frequency compared with non-treated cells, there was
a 6.0-fold increase in the base substitution mutation frequency
(Table V). Further analysis revealed that base substitutions
occurred predominantly at A:T base pairs (71%). The base
substitution mutation frequency at G:C base pairs was increased
only 2.6-fold, but the frequency at A:T base pairs was increased
12.5-fold. In fact, the mutation frequencies of the three types
of base substitutions at A:T base pairs were all substantially
increased above the spontaneous frequencies (10.4- to 23.4fold; Table V).
A comparison was made between the effect of ENU on
base substitution mutation frequencies at lad of Big Blue®
mice exposed to ENU in vivo (Walker et a/., 1996) and those
observed at ell in Rat2 fibroblasts (Table VII). Treatment of
Big Blue® mice in vivo with ENU caused increases in base
491
D.E.Watson, M.L.Cunningham and K.R.TindaJI
Table IIL Theoretical analysis of mutations that may arise as a result of G:C-»A:T transitions at 5'-CpG-3' sites in lad and dl
Locus1
CpG sites/locus
Mutable nucleotides
at CpG sites
Possible mutations from GC—>AT transitions at CpG sitesb
Silent
lad
ell
c
190
44
95
22
77 (40.5%)
17 (38.6%)c
Missense
Nonsense
109
24
4d
3f
No. observed
47 (41.6 %) c
6 (22.2%)c
'lad data are for spontaneous and chemically induced mutations (http://darwin.ceh.uvic.ca/bigblue/bigblue.htm); ell data are from this paper and include only
spontaneous mutants.
•The data for silent, missense and nonsense mutations at lad and dl were determined theoretically.
Tiepresents the percentage of G:C—>A:T transitions that result in silent mutations at all CpG sites (i.e. no. of silent divided by the total no. of mutable
nucleotides).
''Three of four possible nonsense mutations have been observed (nt 301, 502 and 1051, but not 1063).
^Represents the percentage of mutations observed among those transitions that could give rise to a detectable mutation (i.e. no. of mutants observed divided
by the sum of the missense + nonsense sites).
f
Two of three possible nonsense mutations have been observed (nt 34 and 214, but not 205).
Table IV. Mutant frequencies (MF) at dl in Big Blue® Rat2 embryonic
fibroblasts 10 day after 30 min treatment with DMEM (control) or DMEM
containing 0.1 mg/ml ENU
Cell population
no.
Treatment
MF(X 10"5)
Group average MF
(X 10-5 ± SD)
1
2
3
4
5
6
7
8
9
10
11
12
13
Control
Control
Control
ENU
ENU
ENU
ENU
ENU
ENU
ENU
ENU
ENU
ENU
14.6
11.9
12.7
55 6
71.2
62.5
52.8
79.0
42.1
64.0
63.7
58.3
52.3
13.1 i 1.4
60 2 ± 10.4
This included examination of issues known to complicate
alternative mutant selection assays, such as the plating density
of X phage and the efficiency of mutant recovery. The second
objective was to characterize the mutations which arise spontaneously in ell in Big Blue® Rat2 embryonic fibroblasts.
The independence of spontaneous mutations was assured by
employing a modified fluctuation test. The third objective was
to determine whether a well-characterized mutagen (such as
ENU) would cause the same types of mutations at ell as have
been observed at other selectable loci.
substitution mutation frequencies at A:T and G:C base pairs
by 10.3- and 2.0-fold respectively. The ratio of the increases
in mutation frequencies at these base pairs (i.e. fold increase
at A:T divided by the fold increase at G:C) in lacl was
5.1. The analogous increases in base substitution mutation
frequencies at A:T and G:C base pairs at ell in Big Blue®
Rat2 fibroblasts were 12.5 and 2.6 respectively. The ratio of
the increases in mutation frequencies at these base pairs in ell
was 4.8 (Table VTT).
Plaque size analysis
The possibility exists that the absence of ell mutations in
mutant plaques might correlate in some way with plaque size.
Therefore, prior to plaque purification 35 mutant plaques from
ENU-treated cells were grouped into three categories based
on plaque size: small (n = 11), medium (n = 13) and large
(n = 11). Only two mutants lacked a mutation in the ell
structural gene; both of these were from small plaques. The
other nine small plaques and all the medium and large plaques
had detectable gene mutations at ell.
Reconstruction experiment
The hflA and hflB mutations in E.coli strain G1250 promote
formation of unidentifiable lysogens by ell wild-type X phage,
whereas ell mutants form readily identifiable plaques in the
bacterial lawn. The reconstruction experiment (Figure 1A and
B) was designed to assess the efficiency of ell mutant plaque
formation when plated with increasing numbers of wild-type
X phage. Up to 106 wild-type X phage can accompany mutant
X phage during infection and growth of the bacteria on a single
100 mm dish without affecting the quantitative recovery of
ell mutant plaques (Figure IB). In contrast, the procedure for
detection of lacl mutants depends upon detection of blue
plaques following (3-galactosidase cleavage of 5-bromo-4chloro-3-indolyl-P-D-galactopyranoside
(X-gal) in the
medium. Detection of blue plaques is dependent both on the
intensity of the blue color and the number of plaques per plate.
The typical Big Blue® lacl mutation assay uses 25X25 cm
plates with a limit of 10 000-15 000 p.f.u./plate to ensure
quantitative recovery of mutants. This large difference in
plating density of X phage is a distinct advantage of the ell
selection assay. The upper limit of wild-type X phage in these
reconstruction experiments is at least 10^ per 100 mm plate,
which greatly exceeds the maximum number of X phage
recovered from genomic DNA samples in our laboratory (up
to ~5Xl0 5 packaged phage/reaction). We conclude that high
titers of wild-type X phage impose no apparent limitation on
the quantitative recovery of ell mutants as described in
these studies.
Discussion
The experiments described here were designed to fulfil several
objectives regarding the use of ell as a selectable marker in
A.-based transgenic mammalian mutagenesis studies. The first
objective was to confirm the utility of the ell assay for positive
selection of mutants in the Big Blue® mutagenesis system.
Quality control
The frequency of false positives in the ell assay was <5%.
Of 104 spontaneous mutants sequenced five lacked detectable
mutations at ell (4.9%). Two of the 50 mutants sequenced
following ENU treatment also lacked detectable mutations in
the ell structural gene (4%). Jakubczak et al. (1996) also
observed this phenomenon of mutant plaque formation in the
492
dl mutation spectra
absence of a detectable mutation at ell, although at a slightly
lower frequency (1 of 61, 1.6%). The identity of these noncll mutants is not known. However, several facts make it
likely that they result from mutations that inactivate the cl
gene. Whereas mutations that inactivate cl, ell or clll can
result in clear plaque formation (Pons, 1984), only cl or ell
mutants can do so on an hfl~ host (Herskowitz and Hagen,
1980). Sequencing confirms that these clear plaque mutants
do not arise as a result of mutations in the ell structural gene.
Therefore, mutations in cl are the most likely source of
the few non-c// clear plaque mutants that we observed.
Furthermore, one need not consider mutations at the ell
promoter (pR) in this system. Inactivating mutations at pR are
lethal to X, since these would also prevent transcription of the
O and P genes, both of which are essential for X DNA
replication (Furth and Wickner, 1983).
ell mutant plaques can vary considerably in size, from ~0.2
to > 1 mm. One concern was that smaller plaques might be
more likely to contain non-mutated ell genes. Therefore, a
total of 35 plaques isolated from the ENU-treatment studies
were categorized according to their relative size: nine of 11
small plaques and all of the medium (13 of 13) and large (11
of 11) plaques had identifiable ell mutations (Table I). The
other 15 mutant plaques used to generate the ENU-induced
mutational spectrum also had identifiable mutations in the ell
gene. Therefore, while it is possible that small plaques may
be more likely to lack mutations at ell, these plaques should
not be excluded in the process of scoring mutants. Further
examination of this issue is ongoing in this laboratory and
may be considered worthy of examination by other laboratories
using the ell assay.
It is noteworthy that we modified the procedure for mutant
frequency and phage titer determination from the protocol
described by Jakubczak et al (1996). The original ell assay
used two strains of E.coli, both infected and incubated at 24°C
for 48 h, for determination of titers (strain G1217) and for
detection of ell mutants (strain G1250). However, we observed
-20% higher titers using G1217 incubated for 48 h at 24°C
than when using G1250 incubated overnight at 37°C (data not
shown). This may be a function of differential efficiencies of
X phage to infect different strains of bacteria. Therefore, we
believe it is more appropriate to determine titers and select
for ell mutants using a single strain of E.coli (G1250) that is
incubated at different temperatures rather than to use different
strains of bacteria incubated at the same temperature. Strain
G1217 was not used in any of these experiments. Instead, the
X phage titer was determined using G1250 plates incubated
overnight at 37CC. It is worth noting that all X-\J\z phage
form plaques at 37°C because of the presence of the c/857
temperature-sensitive mutation.
Spontaneous mutational spectrum at ell in Rat2 fibroblasts
Frameshift mutations. Frameshift mutations were common in
ell. They accounted for 27.4% (23 of 84) of all independent
mutations in the spontaneous mutation spectrum. All but one
of these frameshifts occurred at two hotspots: a run of guanines
(sense strand, nt 179-184) and a run of adenines (sense strand,
nt 241-246). At the run of guanines 14 independent mutants
were observed: seven single base (+1) addition frameshifts
and seven single base (-1) deletion frameshifts. In contrast,
no + 1 frameshifts were seen in the run of adenines; all eight
frameshift mutations were -1 deletions.
Frameshift mutations in homonucleotide runs are thought
to involve strand slippage during DNA replication (Streisinger
et al, 1966). Modifications to the Streisinger model (Kunkel,
1990) and alternative models for frameshift mutagenesis have
been proposed (see Masurekar et al., 1991; Kaiser and Ripley,
1995). Neither the strand slippage model nor any of the
proposed alternatives account for the fact that both +1 and
-1 frameshifts occur at the run of guanines whereas only -1
frameshifts occur at the run of adenines. Aside from base
composition (i.e. A:T versus G:C), no substantial differences
exist between the two homonucleotide hotspots. Both are runs
of six nucleotides, both have purines on the sense strand of
the ell gene and neither has neighboring nucleotides that would
readily explain the differences in the polarity of frameshift
mutations observed at these sites. Furthermore, these runs are
separated by only 56 nt. It will be interesting to see whether
similar results are obtained among ell mutants arising in vivo
in rats and mice.
Base pair substitutions. Base substitutions accounted for 72.6%
(61 of 84) of mutations in the ell spontaneous spectrum, with
approximately equal frequencies of transitions (38.1%) and
transversions (34.5%). The ratio of base substitutions was ~2:1
at G:C (65.6%) versus A:T (34.4%) base pairs.
G:C—>A:T transitions comprised 25% of all mutations in
ell. Of these transitions 71% occurred at 5'-CpG-3' (CpG)
sites. A mechanism has been proposed (Duncan and Miller,
1980) for the frequent occurrence of G:C—>A:T transitions at
CpG sites that involves several steps: (i) methylation of
cytosine to form 5-methylcytosine (5-meC); (ii) spontaneous
deamination of 5-meC to form thymine, concurrently forming
a G:T mispair; (iii) replication of the mispair to produce a
G:C->A:T transition. The fact that 71% of the G:C^A:T
transitions in ell occurred at CpG sites strongly suggests that
CpG sites within ell are methylated.
In addition, the ell gene has a total of 35 possible base
substitutions that will produce nonsense mutations, of which
seven were observed in the spontaneous spectrum. Amber,
ochre and opal nonsense mutations were all observed in this
collection of ell mutants (Table II).
Comparison of spontaneous spectra at ell and lad
Frameshift mutations constitute a substantially greater percentage of spontaneous mutations in ell than in lacl. For example,
whereas frameshifts comprised 27.4% of the spontaneous
mutational spectrum in ell, frameshifts in lacl in the Big Blue®
Rat2 fibroblast cell line were found to comprise two of 18
mutants (7.7%; Zimmer et al., 1996) and three of 22 mutants
(13.6%; Manjanatha et al, 1996). Similar frequencies for
frameshift mutations in lacl are also observed in the Big Blue®
mouse, where the data are more abundant. In Big Blue® mouse
liver (de Boer et al, 1997), which is the largest available
database for lacl in mammals, frameshifts account for 8.9%
(25 of 282) of the spectrum. It appears that the frequency of
frameshift mutations at lacl may be tissue specific, however,
with frequencies ranging from a low of 4.4% in mouse spleen
(four of 90 mutants; de Boer et al, 1998) to a high of 14.9%
in mouse lung (15 of 101 mutants; de Boer et al, 1998).
Frameshift mutations are more common in ell than in
lacl because of the two frameshift hotspots in ell, at two
homonucleotide runs of six bases comprised of either guanine
(nt 179-184) or adenine (nt 241-246). Frameshift mutation
frequency increases with increasing length of a homonucleotide
run (Kunkel, 1990). In lacl the longest homonucleotide runs
are of five bases (two runs of adenine at nt 107-111 and 978493
D.E.Watson, M.L.Cunnlngham and K.R.TIndall
Table V. Spectrum of independent mutants at the cll locus in ENU-treated Big Blue® Rat2 embryonic fibroblasts3
Mutation
No. of observations
Percent
Frameshifts
+1
-1
Base substitutions
Transitions
G:C->A:T
at CpG sites0
A:T->G:C
Transversions
G:C-*T:A
G:C->C:G
A:T->T:A
A:T->C:G
At G:C base pairs
At A:T base pairs
2
0
2
42
19
6
3
13
23
4
2
8
9
12
30
4.5
0.0
4.5
95.5
43.2
13.6
6.8
29.5
52.3
9.1
4.5
18.2
20.5
28 6
Mutation Frequency
2.7 X10"5
<1.4X10" 5
2.7X10"5
57.5X1CT5
26.0 X10"5
8.2 X10"5
4.1X10-5
17.8XKT5
31.5X10"5
5.5X10"5
2.7X1CT3
10.9 X10"5
12.3X10"5
16.4X10"5
41.0X10"5
71.4
Fold increase over control
0.76
6.0
5.2
2.5
1.8
10.4
7.0
44
1.6
23.4
11.3
2.6
12.5
"One of the cell populations from the studies on spontaneous mutation (Table I) was chosen for ENU treatment. Flasks (n = 12) containing 10* cells/flask
were exposed for 30 min to medium containing 0.1 mg/m] ENU. Control flasks were treated identically without ENU. These cells were grown for 10 days
prior to extraction of DNA for mutation analysis. Independence of mutants was assured in the same way as in Table I. Of 50 mutants that were sequenced 44
were independent.
b
Mutation frequency was calculated in the same way as shown in Table I.
c
Three of the six G:C->A:T mutations observed (50%) were at CpG sites.
Table VI. Spectrum of mutations at the cll locus of Rat2 fibroblasts following treatment with 0.1 mg/ml ENU
Nucleotide
1
1
12
34
37
41
47
56
62
85
86
104
107
107
108
118
119
124
139
143
143
146
152
155
161
169
170
170
173
179
191
193
219
221
241
292
CpG site
No. observed
Mutation
A->T
A->G
A-+T
A->C
C->A
A-+C
T-K3
A->T
C-K3
A-^G
T->G
T->G
T->G
T-^A
C->T
T-*A
C^A
T->C
G-»A
T->C
T-^A
T^C
A-KJ
G-»A
T-^C
-A
982). The difference of a single base in these runs (five bases
in lad versus six bases in cll) appears to substantially affect
the frequency of frameshift mutations in these two genes. As
494
Correct amino acid
Mutated ai
Met
Met
Ala
Arg
He
Glu
Ala
Asn
He
Thr
Thr
Val
Asp
Asp
Asp
He
lie
Arg
Tip
lie
lie
Pro
Phe
Ser
Leu
Val
Val
Val
Leu
Trp
Asp
Asp
Gin
Val
Lys
Stop
Val
Leu
Ala: silent
Stop
Phe
Ala
Glu
Thr
Ser
Ser
Arg
Gly
Gly
Ala
Glu
Phe
Ser
Trp
Gly
Thr
Asn
Leu
Tyr
Stop
Pro
He
Ala
Asp
Pro
Ser
Gly
Asn
His
Ala
Frameshift
Arg
a result, frameshift mutations in the spontaneous mutational
spectrum in lad are much less common than in cll.
Base substitutions constitute the majority of the spontaneous
cll mutation spectra
Table VII. Companson of ENU-induced base substitution mutation frequencies at A:T and G:C base pairs in cll of Big Blue* Rat2 embryonic fibroblasts and
in lacl of T cells of Big Blue® mice exposed in vivo
Gene target"
cll
lacF
Base pair
A:T
G:C
A:T
G:C
Base substitution mutation frequency
(X 1CT5)
Fold increase in base substitution mutation frequency
caused by ENU
Control
ENU
At A:T
3.28
6.24
0.60
2.3
41 0
16.4
6.2
4.7
12.5
Ratio (AT/GC)
AtG:C
4.8
2.63
10.3
5.1
204
V// data from this study using Rat2 fibroblasts (Table II); lacl data from Big Blue B6C3F1 mouse (Walker et al., 1996).
b
Base substitution frequencies at A:T base pairs divided by that at G:C base pairs for that gene.
c
lacl data from Walker et al. (19%).
mutational spectrum of cll and lacl in Big Blue® Rat2
fibroblasts (Table 1; Manjanatha et al, 1996; Zimmer et al.,
1996) and in a wide range of tissues from Big Blue® mice (de
Boer et al, 1996) and rats (de Boer et al, 1998). Among the
six types of base substitutions, G:C—»A:T transitions are the
most common in the spontaneous spectrum of both genes in
Rat2 fibroblasts (Table 1; Manjanatha et al, 1996; Zimmer
et al, 1996) and in Big Blue® animals (de Boer et al, 1996,
1998). This high frequency of G:C->A:T transitions at CpG
sites led us to conduct a detailed analysis of the consequences
of all possible G:C—»A:T transitions at CpG sites in these two
genes (Table III).
CpG sites comprise a similar percentage of the total number
of nucleotides in the lacl and cll structural genes: 8.8 and
7.5% respectively. Furthermore, approximately the same percentage of transitions are silent mutations in lacl and cll. 40.3
and 38.6% respectively. Thus the two structural genes are very
similar with regard to both the frequency of occurrence of
CpG sites and the theoretical mutability of these sites.
The similarity between lacl and cll with regard to CpG
dinucleotide content and theoretical mutability would lead one
to hypothesize that G:C—»A:T transitions at CpG sites should
comprise similar percentages of the spontaneous independent
spectra for both genes. Indeed, when the discussion is limited
to Rat2 cells the data for percentage of G:C—>A:T transitions
at CpG sites is approximately the same at both lacl and cll.
G:C—>A:T transitions at CpG sites in lacl comprised 13.6
(Manjanatha et al, 1996) and 19.2% (Zimmer et al, 1996) of
all spontaneous independent mutations and in cll they comprised 18% (Table I). Based on the similarity of these values,
we infer that the methylation status of CpG sites in cll and
lacl of Rat2 fibroblasts is very similar.
There are significant differences, however, when comparing
the in vitro versus in vivo frequency of G:C—>A:T transitions
at CpG sites of either lacl or cll. G:C—>A:T transitions at
CpG sites occur more frequently in the spontaneous spectrum
of both genes in Big Blue1* animals as compared with Rat2
fibroblasts. For example, G:C—>A:T transitions at CpG sites
comprise 63% of the spontaneous mutation spectrum for cll
in Big Blue® mouse mammary epithelium (Jakubczak et al,
1996) and comprise 39-59% of the spontaneous spectrum for
lacl of Big Blue® mouse liver, lung, spleen, bone marrow,
stomach, skin, kidney and bladder (De Boer et al, 1998).
Similarly, G:C—>A:T transitions at CpG sites comprise 45%
of the spontaneous spectrum for lacl in liver of Big Blue®
rats (de Boer et al, 1996). These values from tissues collected
in vivo are very similar to one another, indicating that the
level of methylation of both genes is similar in Big Blue
animals. However, the 2- to 4-fold greater frequency of
G:C—>A:T transitions at CpG sites of both genes in vivo versus
in vitro is substantial. The reason for this difference is not
known, but is likely due to greater methylation of cll and lacl
in Big Blue® animals relative to the level of methylation of
these two genes in Rat2 fibroblasts.
The increased frequency of G:C—»A:T transitions at CpG
sites in vivo versus in vitro accounts for the greater percentage
of transitions in the in vivo versus in vitro spontaneous spectra
for cll and lacl. It is important to note that the high frequency
of G:C->A:T transitions at CpG sites (Jakubczak et al, 1996)
may limit detection of other types of mutations. Almost
certainly the lower frequency of G:C-»A:T transitions at CpG
sites observed in Rat2 fibroblasts allows for more efficient
recovery and quantitation of transversions and of frameshift
mutations (Table I and Figure 2).
In summary, the available data indicate that there is one
major difference between the spontaneous spectra for cll and
lacl: the high frequency of frameshifts in cll that result from
two hotspots at the homonucleotide runs. In contrast, the
spontaneous spectrum of base substitutions appears to be very
simliar for cll and lacl, provided comparisons are not made
between data collected in vitro with those collected in vivo.
Calculation of mutation frequencies
Mutation frequencies (Table V) for the comparisons made in
this and other sections were calculated as the product of the
mutant frequency and the mutant fraction of interest. For
example, the mutant frequency for the ENU-exposed cells was
60.1 X10"5. A-»T transversions comprised eight of 44 (18.2%)
independent mutations of the ENU-induced mutational spectrum. Therefore, the mutation frequency for A—>T transversions
in ENU-exposed cells was [0.18X(60.1X10-5)], or 10.9X
10~5. For A—>T transversions in control cells this was 0.47 X
10"5. Therefore, treatment of Big Blue® Rat2 embryonic
fibroblasts with ENU resulted in a 23.4-fold increase in A—>T
transversions at cll. This was the greatest relative increase in
mutation frequency caused by ENU.
Comparison of ENU-induced mutational spectra at cll and
lacl
The effect of ENU on mutant frequency, mutation frequency
and mutational spectrum at cll in Big Blue® Rat2 fibroblasts
is very similar to that seen at lacl in T cells of Big Blue®
mice exposed in vivo to ENU (Walker et al., 1996). ENU caused
a 6.0-fold increase in base substitution mutation frequency at
cll of Rat2 fibroblasts, resulting in an ENU-induced mutation
495
D.E.Watson, M.L.Cunnlngham and K.R.Tindall
spectrum that was dominated by base substitutions (95.5%).
These substitutions occurred primarily at A:T base pairs: base
substitution mutation frequency was increased at A:T base
pairs by 12.5-fold, whereas that at G:C base pairs was increased
only 2.6-fold. The ratio of the fold increase in base substitution
mutation frequencies at A:T and G:C base pairs in cll caused
by ENU was 4.81 (i.e. 12.5/2.6; Table VU). A nearly identical
pattern was observed at lacl in T cell DNA of mice exposed
to ENU, in which 10.3-fold and 2.0-fold increases in base
substitution mutation frequencies at A:T and G:C base pairs
respectively were observed (Walker et ai, 1996). The ratio of
the fold increase in base substitution mutation frequencies at
A:T and G:C base pairs in lacl caused by ENU was 5.1 (i.e.
10.3/2.0; Table VU), which is remarkably similar to the value
seen at cll (4.81). Thus although a greater ENU-induced
mutagenic response was observed at cll in Big Blue® Rat2
fibroblasts than at lacl in T cells of Big Blue® mice exposed
in vivo, the ratio of base substitutions at A:T versus G:C base
pairs induced by ENU at these two loci are nearly identical
(Table VII).
Similar observations were made in vitro in a study using
lacl in the same cell line as was used here with cll. Base
substitutions at A:T base pairs were not observed in untreated
Big Blue® Rat2 fibroblasts (none of 18) but accounted for
48.6% (18 of 37) of all ENU-induced mutants (Zimmer et ai,
1996). Data from mammalian mutagenesis experiments using
the lacl (Kohler et ai, 1991) or HPRT (Skopek et ai,
1992) loci are consistent with the view that the predominant
mutagenic effect of ENU is at A:T base pairs.
Consistent with the greater mutagenic effect of ENU at A:T
base pairs, the mutation frequency for base substitutions at
CpG sites was increased only 1.75-fold as compared with
the CpG base substitution frequency in the spontaneous cll
spectrum. This value is less than the 2.6-fold increase in
mutation frequency observed for all G:C base pairs in cll.
Finally, the mutation frequency for frameshift mutations at cll
decreased slightly from 3.59X1O"5 in unexposed cells to
2.73X10~~5 in ENU-exposed cells. This decrease in the frequency of frameshift mutations is consistent with previous
data (Kohler et ai 1991; Skopek et ai, 1992), indicating that
ENU does not induce a high yield of frameshift mutations.
Collectively, the qualitative and quantitative data at cll in
Big Blue® Rat2 fibroblasts are consistent with those obtained
for ENU in mammalian mutagenesis experiments conducted
both in vitro and in vivo in which lacl and hprt were used as
mutagenesis targets.
Mechanisms of ENU mutagenesis
ENU forms at least 12 different types of DNA adducts
(Beranek, 1990). As a result, it is difficult to attribute particular
types of mutations to the properties of an individual lesion.
However, ENU is known to form O2- and AG-ethylthymidine,
which promote selective mispairing with thymine (Grevatt
et ai, 1991, 1992; Bhanot et ai, 1992). Data presented here
suggest a causative role for O2- and AG-ethylthymidine in the
ENU-induced 23.4-fold increase in mutation frequency for
A—»T transversions at cll.
Summary
Collectively, the data presented here for spontaneous and ENUinduced mutagenesis in Big Blue® Rat2 embryonic fibroblasts
support the continued use of cll as a selectable marker in
mutagenesis studies involving cells or tissues that carry a k
496
transgene. The shorter gene, the absence of ex vivo mutations
and advantages of assay simplicity, including decreased time
and expense, make the cll assay an efficient method for
detecting gene mutations in Big Blue® cells. It was previously
shown that cll and lacl give comparable mutagenic responses
in bladder DNA of Big Blue® mice following in vivo exposure
to the bladder carcinogen ^-cresidine (Jakubczak et ai, 1996).
The similarity between the mutagenic responses caused by
ENU at cll of Big Blue® Rat2 fibroblasts and lacl in the
T cells of Big Blue® mice exposed in vivo (Walker et ai,
1996) is further evidence that cll is comparable with lacl as
a target gene useful for mutagenesis studies.
Acknowledgements
The authors thank the following people who helped with various aspects of
this research: Dr John Jakubczak for E.coli G1250 and for advice on use of
the cll assay; Dr Robert Sobol for comments during the experimental and
manuscript preparation stages of this work; Dr Jef French for a critical review
of the manuscript; Dr Warren Glaab and Heather Lindsay for technical
assistance; Drs Sid Aaron, Bill Mattes and Dave Zimmer for sharing their
unpublished data.
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Received on December 19, 1997; accepted on February 6, 1998
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