Copyright 0 1991 by the Genetics Society of America
A Tester System for Detecting Each of the Six Base-Pair
Substitutions inSaccharomyces cerervisiae by Selecting for an Essential
Cysteine inIso-1-Cytochrome c
Michael Hampsey
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, Louisiana 71130
Manuscript received November 8 , 1990
Accepted for publication January IO, 199 1
ABSTRACT
A collection of isogenic yeast strains that is specifically diagnostic for the six possible base-pair
substitutions is described. Each strain contains a single, unique base-pair substitution at the Cys-22
codon of the CYCl gene, which codes for iso-l-cytochrome c. These mutations encode replacements
of the functionally critical Cys-22 and render each strain unable to grow on mediacontaining
nonfermentable carbon sources (Cyc-). Specific base-pair substitutions, which restore the Cys-22
codon, can be monitored simply by scoring for reversion to the Cyc+ phenotype. These strains revert
spontaneously at very low frequencies and exhibit
specific patterns of reversion in response to different
mutagens.Only true ( C Y C P ) revertants were recovered after 7 days on selection medium. T h e
following mutagen specificities were observed:ethyl methanesulfonateand N-methyl-N’-nitro&nitrosoguanidine, G.C + A .T; 4-nitroquinoline-l-oxide,
G.C -+ T - A and G - C -+ A - T ; diepoxybutane, A.T + T A, A.T + G C and G .C + T A; 5-azacytidine, G. C -+ C .G . Methyl methanesulfonate induced all six mutations, albeit at relatively low frequencies, with preference for A - T T.
A and A . T + G.C. Ultraviolet light was the most inefficient mutagen used in this study, consistent
with its preference for transition mutations atdipyrimidine sequences reported in other systems. This
tester system is valuable as a simple and reliable assay for specific mutations without DNA sequence
analysis.
-
A
NALYSIS of mutagen specificity is essential for
understanding the molecular basis of mutagenesis. The most convenient testersystems for determining mutagenic potential and specificity are based on
genetic reversion analyses. YANOFSKY,ITO and HORN
(1966) utilized a set of trpA mutants to investigate
mutagenic mechanisms in Escherichia coli. Mutants
from the extensive collection of Salmonella typhimurium histidine auxotrophs were developed by AMES,
( 1 973) for detecting both frameLEEand DURSTON
shift and base substitution mutations. Their system
was especially significant for having established a correlation between mutagens and carcinogens (MCCANN
et al. 1975). More recently, some of their strains have
been used to detect specific base substitutions either
by phenotypic screeningof hisG revertants (LEVINand
AMES 1986)or by colony hybridization to sequencespecific probes (MILLERand BARNES1986). In yeast,
PRAKASH
and SHERMAN
( 1 973) devised a testersystem
to detect specific mutations based on reversion uf
defined c y 1 mutants.
A more comprehensive pictureof mutagen specificity is generally obtained from tester systems that deAbbreviations: 5-AZ, 5-azacytidine; DEB, (+)-1,2:3,4-diepoxybutane;
EMS, ethyl methanesulfonate; MMS, methyl methanesulfonate; MNNG, N methyl-N’-nitro-N-nitrosoguanidine;NQO, 4-nitroquinoline-l-oxide;UV,
ultraviolet light; bp, base pairs; PCR, polymerase chain reaction.
Genetics 128: 59-67 (May, 1991)
tectforwardmutations.
COULONDRE
and MILLER
( 1 977) utilized the E. coli l a d gene for this purpose.
Their system is based on generation of nonsense codons at 78 different sites, which are subsequently
identified by genetic mapping and suppression analysis. More recently, lacl has been exploited to determine mutational specificity by direct DNA sequencing, both in E. coli (FARABAUGH
et al. 1978; CALOS
and MILLER1981; MILLER1985; BURNS,ALLENand
GLICKMAN
1986; BURNS,GORDONand GLICKMAN
and GLICKMAN
1987; SCHAA1987; SCHAAPER, DUNN
PER and DUNN1987), and in human cells (HSIAet al.
1989). A number of other forward mutation systems,
based on convenientmethodsforretrieval
and sequencing of DNA, have been developed. These include the lambdac l gene (WOOD,SKOPEK
and HUTCHINSON 1984); hybrid M131ac (LECLERC
and ISTOCK
1982); the E. coli gpt and supF genes in mammalian
systems (HAUSER
et al. 1986; ASHMAN
and DAVIDSON
XU and DAVIDSON
1988); and the
1987; GREENSPAN,
endogenous aprt gene of Chinese hamster ovary cells
(GROSOVSKY
et al. 1988). Two forward mutation systems, both designed with methods to readily retrieve
altered DNA, have been developed in yeast. One is
based on inactivation of the SUP4-o nonsense suppressor gene (PIERCE,GIROUXand KUNZ 1987), and the
60
M. Hampsey
other on inactivation of the URA3 gene (LEE et al.
1988).
A drawback to the tester systems described above
is that in no case can mutational alterations be ascertained without additional genetic or biochemical manipulations. A method to unambiguously define mutations based on an easily scorable mutant phenotype
would be extremely valuable. Recently, two such systems were described in E. coli. FOSTERet al. (1987)
established an assay for G . C + T. A transversions
based on selection for an essential serine in plasmidencoded P-lactamase. MILLER and coworkersdescribed a set of lac2 mutants to detect allsix base
substitutions (CUPPLES
and MILLER 1989), as well as
specific frameshift mutations (CUPPLESet al. 1990),
based on selection for essential active site residues in
P-galactosidase.
In this paper I describeaconvenient
system to
detect each of the six base substitutions in yeast. The
system is based on the critical requirement forcysteine
at position 22 of iso-l-cytochrome c, encoded by the
CYCl gene. Six isogenic strains, each containinga
unique, single base substitution in codon-22, were
constructed. These strains do not grow on media
containing glycerol as the sole carbon source (Cyc-)
and revert to Cyc+ only by defined substitutions that
restore the Cys-22 codon. Therefore, reversion to the
Cyc+ phenotype is diagnostic for specific base substitutions.
encoded iso-2-cytochrome c in their strains. Glucose at 0.1 %
seemed to be optimal in that higher concentrations did not
further stimulatereversionfrequencies,butresulted
in
higher background growth of the mutants. YPD, YPG and
minimal media containing either 2%glucose or 3% glycerol
are standardyeast media (SHERMAN,
FINKand HICKS 1983).
Minimal medium was supplemented with uracil (20 ,.tg/ml)
and leucine (30 ,.tg/ml) as required. Medium containing 5fluoroorotic acid was described by BOEKEet al. (1987).
Chemical mutagens were purchased from Sigma Chemical Co. and were used without additional purification.
In vitro mutagenesis: Oligonucleotide-directed in vitro
mutagenesis was performed according to the technique of
KUNKEL,
ROBERTS
and ZAKOUR (1987) using bacteriophage
Ml3-CYC1-B/H. MlJ-CYCl-B/H is M13mp19 containing
the 2.5-kb BamHI-Hind111 CYCP DNA fragment. Singlestranded, uracil-containing DNA was obtained from bacteriophage MI3-CYCl-B/H propagated in E. coli strain CJ236
[dutl ungl thil relAl/pCJ105 (Cmr)]. Sequence changes in
the Cys-22 codon were directed with the following four
oligonucleotides:
ORB-6, TCTACAATCCCACACCG;
ORB-7, TCTACAACGCCACACCGT;
ORB-8, CTACAAAGCCACACCGT; and
ORB-9, CTACAAGGCCACACCGTG.
T h e underlined bases indicate the directed mutations. Oligonucleotides were synthesized using the Biosearch model
8650 DNA Synthesizer and were purifiedby polyacrylamide
gel electrophoresis as described by LO et al. (1 984). After
completion of the mutagenesis procedure, mutations were
confirmed by DNA sequencing.
Gene transplacements: T h e CYCI+ gene and the four
site-directed mutations in M 13-CYCl-B/H were subcloned
from RF DNA as 2.5-kb BamHI-Hind111 fragments into the
et al. 1979).The two
yeast integrating vector YIp5 (STRUHL
MATERIALSANDMETHODS
Cys-22 alterations, previously isolated as 246-bp EcoRI-KpnI
fragments in M13mp19 (HAMPSEY,DAS andSHERMAN
Yeast strains, media and chemicals: Strains YMH11986), were first transferred as EcoRI-KpnI fragments to
YMH7 are isogenic derivatives of strain S260-11B (MATa
pM19 and then subcloned as 2.5-kb BamHI-Hind111 DNA
cycl-706::CYH2 cyc7-67 ura3-52 leu2-3, 112 cyh2) and differ
fragmentsinto
YIp5. pM19 contains the c y 1 BamHIfrom each other only by single base substitutions within
Hind111 fragment with a unique Sac1 site between the EcoRI
codon-22 of the cycl gene. Strain S260-11B and the cycland KpnI sites (M. HAMPSEY,
unpublished) that was used as
706::CYHZ allele were described previously by BAIMand
a marker to confirm EcoRI-KpnI fragment replacements.
SHERMAN(1988).Diploid strainsYMH51-YMH57were
Transplacement of the cycl alleles into the normal chroconstructed by crossing strains YMH1-YMH7, respectively,
mosomal locus was performed by integrative transformation
with strain B-7462 (MATa cycl-1 cyc7-67 ura3-52 hisl-1 canland subsequent excision as describedby BAIMand SHERMAN
100) and selecting for diploids on minimal medium supplemented with uracil. T h e cycl-1 (SINGHand SHERMAN 1978; (1988), except thateviction of the resident cyc1-706::CYH2
allele and flanking plasmid DNA was obtained by first
and F. SHERSTILESet al. 1981) and cyc7-67 (T. CARDILLO
selecting for 5-fluoroorotic acid resistance (BOEKEet al.
MAN, unpublished data) alleles are completedeletionsof
1987) and subsequently screening for cycloheximide resisttheir respective structural genes. T h e symbol Cyc is used to
ance.
Single copy transplacement at the c y 1 locus was dedenote phenotypes, correspondingtoeitherthe
ability
termined for each strain by Southern blot analysis and the
(Cyc') or inability (Cyc-) to grow on mediacontaining
codon-22 mutations were confirmed by reisolating and seglycerol as the sole carbon source.
quencing cycl DNA fragments (data notshown).
Revertants of strains YMH52-YMH57 were selected on
Retrieval of DNA fragments encompassing cycl codonYPGD medium, consisting of 1% yeast extract, 2% peptone,
22: T h e 246-bp EcoRI-KpnI fragments, which include the
3% glycerol, 0.1% glucose and 2% agar. Glucose is imporcoding information for amino acids 2-83, were cloned ditant in this medium to obtain efficient and reproducible
rectly from total yeast DNA into M13mp19 as described
reversionfrequencies.
Initial growth under nonselective
previously (HAMPSEY,
DAS and SHERMAN1986).Alternaconditions apparently stimulates reversion frequencies, an
observation reported initially by FINK and LOWENSTEIN tively, DNA was amplified from total yeast DNA by the
polymerasechain reaction, using theGeneAmp kit and
(1 969); this effect presumably reflects a
requirement for
DNA Thermal Cycler manufactured by Perkin Elmer Cetus
DNA replication in order to convert mutagen-induced leand synthetic oligonucleotides oJN-24 and ORB-27. (oJN-24
sions into mutations. Although SHERMANet al. (1974) reis the CYCl noncoding strand sequence from -74 to -57
ported that initial growth on nonselective medium did not
and ORB-27 is the coding strandsequence from 391 to 372;
increase reversion frequencies of cycl mutants, this is prob+1 is the A of ATG initiation codon.) Amplified DNA was
ably a consequence of
the residual growth affordedby CYC7-
61
Mutagenicity Tester Strains
then subcloned as 246-bp EcoRI-KpnI fragments into
M13mp19.
DNA sequence analysis: Single-stranded DNA was isolated from M13mp19 derivatives containing either 2.5-kb
BamHI-Hind111or 246-bp EcoRI-KpnI cycl DNA fragments.
Sequencing was performed according to the dideoxy-terminator method, following the protocol supplied with the
United States Biochemical sequencing kit. At least two independent clones from PCR-amplified DNA were sequenced to avoid potential PCR artifacts. Codon-22 sequences were determined from BamHI-Hind111 clones using
the primer oJN-24 (described above); EcoRI-KpnI fragments
were sequenced using the M 13 universal primer.
Estimation of iso-1-cytochrome c levels: Iso-1-cytochrome c amounts were determined bylow temperature
(-1 96") spectroscopic examination of intact yeast cells as
described by SHERMAN
and SLONIMSKI
(1964). The spectroscope was obtained from Central ScientificCo. (Franklin
Park, IL) and was mounted on a standard monocular laboratory microscope. A 1000 W projector lamp was used as
the light source.
Reversion analysis: Strains YMH52-YMH57 were
grown for 2 days to approximately 7 X 10' cells/ml in YPD
medium at 30°, harvested, washed and resuspended at 1 X
lo9 cells/ml in 50 mM potassium phosphate buffer, pH 7.0
(KP buffer). Chemical mutagenesis (except 5-AZ) was performed at concentrations of 1 X 10' cells/ml in KP buffer
at 23" as described below for each mutagen. Cell survival
following each mutagenic treatment was monitored by plating appropriately diluted cells on YPD plates and scoring
colonies after 2-3 days at 30". Spontaneous and induced
revertants were selected on YPGD medium at 2-5 X lo7
cells per plate and scored after 7 days at 30".
EMS,MMS and DEB were added to cell suspensions at
final concentrations of 2, 0.5 and 0.2%, respectively. After
incubation for the indicated times, samples werewithdrawn
and added to an equal volume of sterile 10% sodium thiosulfate to inactivate the mutagens. Cells were collected by
centrifugation, washed twice, and spread on YPGD plates.
MNNG and NQO were dissolved in acetone at 10 mg/
ml immediately before use, diluted in KP buffer to 4 mg/
ml and 1 mg/ml, respectively, and added tocell suspensions
at final concentrations of 40 pg/ml and 10 pg/ml. Following
incubation for theindicated times, sampleswere withdrawn,
treated and washed as described above for the other mutagens, and spread on YPGD plates.
UV mutagenesis was done by irradiating cells spread
directly on YPGD medium. Irradiation was performed in
the dark using a 254-nm lamp (UVP, Inc., San Gabriel,
California) mounted 15 cm above the surface. Plates were
incubated in the dark to avoid photoreactivation.
5-AZ was prepared immediately before use at 10 mg/ml
in minimal medium (2% glucose) supplemented with uracil,
and filter sterilized. The indicated concentrations of 5-AZ
were prepared by dilution using the same medium. Cultures
were inoculated with approximately lo4 cells and incubated
with vigorous aeration for 24 hr to densities of 2-4 X lo7
cells/ml. Cultures were collected by centrifugation, washed
twice, and spread on YPGD plates.
RESULTS
Cys-22 mutants: Seven isogenicyeast strains, differing only by single point mutations in the
Cys-22 codon
of CYCl, were constructed by molecular genetic techniques, as described in theMATERIALS AND METHODS.
These strains are presentedin Table 1. Strain YMH1
TABLE 1
Yeast strains and their corresponding mutational specificities
Haploid
Diploid
strain
strain
Codons 21-23"
YMHI YMH51 CAA TGCCAC
YMH2 YMH52 CAACGC CAC
YMH3 YMH53 CAAAGC CAC
YMH4
YMH54
CAAGGC
CAC
YMH5 YMH55 CAA T S C CAC
YMH6 YMH56 CAA T T C CAC
YMH7 YMH57 CAA TAC CAC
Amino
acid 22
Cys
Arg
Ser
Gly
Ser
Phe
Tyr
Mutational
specificity
G.C+A . T
A . T T+ . A
G . C T+ . A
G . CC+. G
A . TC
+.G
A . TG
+ .C
DNA sequence changes in codon-22 are underlined. Codons
21 and 2 3 are included to depict the immediate sequence context
flanking the mutational targets. For the complete sequence of the
CYCl structural gene see HAMPSEY,
DASand SHERMAN
(1986).
contains the normal amount
of iso-1-cytochrome c
observed in a C Y C P strain,asdetermined
by low
temperature (- 196 ") spectroscopic analysis, and exhibits a Cyc+ phenotype. Strains YMH2-YMH7
are
completelydeficient iniso-1-cytochrome c a n d a r e
phenotypically Cyc-. Hemizygous ( c y c l / c y c l - 1 ) diploid
derivatives,designatedYMH51-YMH57,wereobtained by crossing each haploid strain with strain B7462 (Table 1). Consistent with the haploid phenotypes,strainYMH51
is Cyc+ andstrainsYMH52YMH57 are Cyc-.
Generation of revertants: Strains YMH52YMH57 were tested by monitoring reversion to Cyc+
in response to seven different mutagens. Spmtaneous
reversion was measured as a control in each mutagenesis experiment.Intotal,approximately1.2
X lo9
untreated cells for each strain were plated on
selective
medium during the course
of these experiments, from
which spontaneous reversion frequencies were determined. All six strains revert spontaneously only at
very low frequencies (Table 2). T h e G. C + A .T
transition and G . C + T. A transversion exhibited the
highest spontaneous reversion rates, occurring atfrequencies of 1.3 X lop8 and 1.8 X lo-'. The A - T +
G - C transition andG - C + C - G transversion arose at
barely detectable frequencies, occurring at about 2 X
1O-' and 1 X 1OP9, respectively. No A . T + T . A o r
A . T + C G transversions were detected during the
course of these experiments, corresponding to sponof <1 X
GIROUX
taneous
reversion
frequencies
et al. (1988) reported the same pattern of spontaneous
base substitutions at theSUP4-o locus, suggesting that
this particular region of cycl does not undergo aberrant spontaneous mutation.
EMS and MNNG are alkylating agents whose primary mutagenic lesions are believed to be @-ethylguanineand@-methylguanine,respectively.These
mutagens induce primarilyG - C -+ A . T transitions in
several different organisms (PRAKASHand SHERMAN
1973; COULONDRE
a n d MILLER 1977; BURNS, ALLEN
a n d GLICKMAN1986; KOHALMI a n d KUNZ 1988;
62
M. Hampsey
TABLE 2
Reversion frequencies after mutagenic treatments
Revertants per lo7 survivors'
Survivalb
YMH52
Mutagen"
(G.C
+A . T )
@)
Exposure
YMH53 ( A . T
T.A)
-+
YMH54 (G.C
YMH55 (G.C
YMH56 ( A . T
YMH57 ( A . T
+T.A)
4C.G)
+ C.G)
+G.C)
-
None
(100)
0.13
<0.01
0.18
0.01
co.01
0.02
E M S (2.0%)
15 min
30 min
60 min
120 min
93
97
88
83
16 f 2.9
31 f 10
41 f 8.5
90 f 19
0.0
0.0
1.1 f 1.1
4.2 f 3.6
1.2 f 1.0
0.5 f 0.5
3.8 f 1.3
4.4 k 0.7
1.7 f 1.5
1.0 f 1.0
0.7 f 0.6
0.0
0.0
0.0
0.4 f 0.3
2.2 f 2.0
0.2 k 0.3
1.4 f 1.2
3.3 k 1.0
3.4 f 2.0
M N N G (40 rg/ml)
30 min
60 min
120 min
88
83
68
18 f 3.0
53 f 11
135 f 26
0.0
0.2 f 0.4
1.0 f 1.1
1.1 f 1.0
1.7 f 2.1
1.0 f 1.1
0.0
0.2 f 0.4
0.0
1.1 f 1.0
0.0
0.0
1.2 f 1.1
1.0 f 1.0
2.2 f 0.3
MMS (0.5%)
15 rnin
30 min
60 min
84
64
30
0.6 f 0.7
1.6 f 0.8
6.9 f 7.0
2.0 f 0.6
11 f 5.6
29 f 9.0
0.8 f 0.7
3.9 f 2.6
9.4 f 3.6
0.0
0.2 zk 0.4
2.9 f 2.6
0.4 f 0.4
1.3 f 1.5
4.0 f 4.5
2.2 f 0.8
9.0 f 5.2
27 f 4.9
uv
15 sec
30 sec
45 sec
91
59
35
1.8 f 1.5
3.0 f 2.2
5.9 f 3.5
0.0
0.0
0.0
0.0
0.3 f 0.5
4.3 r 3.7
1.1 f 1.2
2.1 f 1.9
3.7 f 3.2
0.4 f 0.7
0.0
0.0
0.0
0.0
0.0
30 min
60 min
120 min
54
35
21
9 3 f 4.2
19 f 3.1
27 f 6.1
1.3 f 1.7
3.3 f 2.9
0.0
24 f 5.5
53 f 17
89 k 23
0.0
1.2 f 1.1
1.6 f 2.8
0.0
0.0
0.0
1.0 rt 1.0
1.2 f 1.4
0.0
30 min
60 min
120 min
98
91
79
0.4 f 0.7
1.2 rt 1.2
0.5 f 0.5
12 f 3.2
23 f 6.1
51 f 6.7
9.0 k 1.7
19 f 7.5
46 f 12
0.0
0.5 f 0.9
0.0
0.0
0.0
0.0
18 f 2.9
49 10
103 f 14
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.3 k 0.6
1.1 f 1.4
1.2 f 0.4
3.5 f 1.7
8.9 f 5.8
14 f 4.9
23 f 13
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
5-AZd
~
2.5 mg/ml
5.0 mg/ml
7.5 mg/ml
10.0 mg/ml
~
~~
~
~~
~
~
~
~
~~
~~
~~~
*
~~~
~
Mutagenic treatments were performed as described in the MATERIALS AND METHODS.
* Survival frequencies are presented for strain YMH52 and are theaverage of three independent experiments. N o significant differences
in survival frequencies were found among thesix strains.
Reversion frequencies were determined by countingrevertant colonies and normalizing to IO' survivors. Spontaneous reversion
frequencies were determined as controls for each mutagenic condition and were derived from a total of about 1.2 X 10' cells plated for each
strain during thecourse of these experiments. Mutagen-induced reversion frequencies were determined fromthree independent experiments,
representing 6-15 X lo7 cells plated for each condition, and are presented as the mean f the standard deviation.
Strains were grown in the presence of the indicated concentrations of 5-AZ for approximately 12 generations before plating on selection
medium.
GREENSPAN,
Xu and DAVIDSON
1988). This specificity
predictsthat YMH52 shouldexhibit the strongest
response to EMS and MNNG. Indeed, a 2-hr exposure
to 2% EMS induced reversion of YMH52 at 9 x
corresponding to approximately 700-fold induction
of the G. C + A. T transition (Table 2). An even
stronger response to MNNG was observed, resulting
in>lOOO-fold induction following 2-hr exposure to
40 pg/ml MNNG. The other four strains exhibited
only very weak responses to both mutagens.
MMS,likeEMS,
is amonofunctional alkylating
agent. Unlike EMS, however, MMS was reported to
be rathernonspecific with respect to induction of base
substitutions in yeast (PRAKASH
and SHERMAN 1973).
Consistent with this result, MMS induced reversion of
all six tester strains. However, a clear preference for
the A.T + T - A transversion and A T + G C transition was apparent. A 2-hr exposure to 0.5% MMS
induced both mutations at >1000-fold above back-
-
ground. These data suggest that the primary mutagenic lesion induced byMMS occurs at A .T basepairs.
In the most comprehensive study of UV specificity
in yeast to date,KUNZet al. (1987) found that 93%of
UV-induced mutations at the SUP4-o locus are single
base substitutions and that 85%
of these are transitions
with a clear preference for G C+ A. T over A. T +
G.C.Furthermore, UV-induced mutations are sequence context dependent.Ninety-nine percent of all
substitutions occurred at sites of adjacent pyrimidines
with the mutation arising primarily at the 3' position
of the dipyrimidine (KUNZet al. 1987). Similar specificity for UV has beenfoundatother
yeastloci
et al. 1986; LEE et al. 1988)and in other
(IVANOV
organisms (WOOD,SKOPEKand HUTCHINSON
1984;
MILLER 1985; SCHAAPER, DUNN and GLICKMAN 1987;
HSIAet al. 1989). Inspection of the sequence context
flanking the target base-pairs within codon-22 (Table
Mutagenicity Tester Strains
1) reveals that neither of the transition-specific sites is
part of a dipyrimidine sequence. Each of the four
transversion-specific sites is part of adipyrimidine
sequence, although only the sites specific for G.C +
T .A and A T + C G occupy the 3' position. These
are the two most infrequently detected base substitutions identified by KUNZ et al. (1987), representing
only 3.2% of all UV-induced base substitutions at
SUP#-0. This information suggests that the cycl tester
strains should exhibit only a weak response to UV.
Indeed, noneof the six strains was efficiently reverted,
even at a UV dose thatresulted in only 35% cell
survival (Table 2).
NQO-induced mutagenesis has been studiedmainly
in the l a c l (COULONDRE
and MILLER 1977) and cycl
(PRAKASH and SHERMAN 1974;
PRAKASH,
STEWART
and SHERMAN 1974)systems. In both studies NQO
was reported to induce primarily G C += A T transitions and G.C 3 T A transversions. Similar specificity is reported here. T w o hours of exposure to 10p g /
ml NQO resulted in a 500-fold induction of G C +=
T. A and a 2 0-fold
1
induction of G . C += A. T. Little
o r no reversion of the other strains was observed.
Interestingly, the relative G. C + T A and G .C +=
A. T response is opposite to that reported in lacl and
at the other sites at c y c l , suggesting thatsequence
context can affect the relative efficiency of NQOinduced mutations.
DEB is a bifunctional alkylating agent (reviewed by
EHRENBERG
and HASSAIN1981) and an effective mutagen inyeast (OLSZEWSKA
and KILBEY 1975). Although its mutagenic potential has been investigated
in yeast rad mutants (ZUK et al. 1980; ZABOROWSKA,
1982; LECKA-CZERNIK
et a l .
ZUK and SWIETLINSKA
1984), the specificity of DEB-induced mutations has
not been reported. The bifunctional nature of DEB
suggests that itmay induce a broader spectrum of
mutations than the monofunctional alkylating agents.
Following a 2-hr exposure to 0.2%DEB, three of the
tester strains were efficiently reverted. The A. T +
G - C transition was induced at a frequency of
and the A. T + T A and G C += T A transversions
at about5 X
(Table 2). These frequenciesrepresent >5000-fold induction of A. T + G - C and
A. T + T A and about 250-fold induction of G. C +
T .A. Essentially no stimulation of the other three
substitutions occurred.
The base analog 5-AZ was reported by ZIMMERMANN and SCHEEL
(1984) to induce both mitotic recombination and point mutation inyeast. In the s.
typhimurium tester systems, 5-AZ induced exclusively
G. C + C G transversions (LEVINand AMES 1986;
MILLER and BARNES1986). The same specificity,
along with much weaker induction of G. C + T .A
transversion, was found in the l a c 2 tester system (CUPPLES and MILLER 1989). These results suggest that
-
63
strainYMH55shouldrespond
to 5-AZ,which was
indeed observed (Table 2). Very weak stimulation of
G. C += T. A also occurred, at a rate barely above
background.
Analysis of revertants: If reversion analysis of the
strains described here is to be diagnostic for specific
mutations it is essential either that all revertants are
true (CYCl+) revertants or that true revertants can
readily be distinguished from pseudorevertants. This
consideration was addressed by examining iso- 1-cytochrome c levels for many of the revertants isolated in
this study. The rationale here is that true revertants
will display the normal amount of iso-1-cytochrome c,
whereas suppression of Cys-22 defects by second-site
mutations is likely to be partial and would yield pseudorevertants that contain diminished amounts of iso1-cytochrome c. Low temperature (- 196") spectroscopic examination of over 400 revertants that arose
after 7 days of incubation on glycerol medium, including representative revertants of each strain induced
by each of the mutagens in this study, revealed only
strains containing the normal amount of iso-l-cytochrome c with normal spectral properties (data not
shown). Furthermore, sequence analysisof
CYCl
DNA isolated from 21 independentrevertants, including revertants induced by each of the mutagens
used in this study, revealed only the C Y C P sequence
(data not shown).
I wish to note that a second class of revertants of
strain YMH52 arose after prolonged incubation
on
selective medium, discernable as tiny colonies after
12-1 5 days. They did not arise spontaneously but
were induced by each of the mutagens in this study
except 5-AZ. The strongest response was elicited by
2-hr exposure to 2%EMS, resulting in revertants at a
frequency of about lop6. These revertantswere phenotypically distinct fromtheotherrevertantsand
from the C Y C P strain, YMH51, in that they grew
markedly slower on YPG medium and contained less
than 5% of the normal amount of iso-1-cytochrome c.
Their slow growthphenotype allowed them to be
readily distinguished from truerevertants. Therefore,
they did not interfere with the specificityof strain
YMH52 for detecting theG.C += A . T transition and
they are not represented in Table 2. Further characterization of one of these revertants revealed that it
retains the sequence CGC at codon-22 with no other
changes in the structural gene (R. BERROTERAN
and
M. HAMPSEY, unpublished data).
No pseudorevertants
of strains YMH53-YMH57 were found, even after
prolonged incubation on selective medium,
DISCUSSION
The critical requirement for cysteine at position 22
of iso-1-cytochrome c is a fundamental premise of this
system. This requirement is supported by the follow-
64
M. Hampsey
ing evidence. First, Cys-22 is evolutionarily invariant
throughout the phylogenetic series of nearly 100 eukaryotic cytochromes c (HAMPSEY,
DAS and SHERMAN
1986, 1988). Second, the crystal structure of iso-lcytochrome c (LOUIE,HUTCHEON
and BRAYER1988;
LOUIE and BRAYER 1990)
shows that Cys-22 forms a
covalent thioether linkage tothehemeprosthetic
group, providing astructural basis forthe Cys-22
requirement and suggesting that this requirement is
critical. Third, all known replacements at position 22,
which include serine, glycine, arginine, phenylalanine
and tyrosine (HAMPSEY,DAS andSHERMAN1986,
1988; Table l), abolish iso-l-cytochrome c function.
This information suggests that only cysteine at position 22 will allow efficient growth on media containing
nonfermentable carbon sources. T h e observation that
all revertants of strains YMH52-YMH57 that arise
after 7 days on selective medium contain either the
CYCI+ DNA sequence and/or contain iso-l-cytochrome c that is indistinguishable in amount and spectral characteristics from thatof CYCI+ strains validates
this premise.
The cycl tester strains were designed to minimize
the possibility of obtaining Cyc+ revertants by mutational pathways other than the single base substitutions that restore the normal Cys-22 codon. Each of
the base substitutions in codon-22 is a missense mutation (Table 1) and is therefore not suppressible by
classical nonsense suppressors. These
strains
are
homozygous for the cyc7-67 allele, which is a deletion
of theentirestructuralgeneencoding
iso-2-cytochrome c (T. CARDILLO
and F. SHERMAN,
unpublished
data);therefore,revertants can not arise either by
mutations that result in overexpression of iso-2-cytochrome c or by recombination between the cycl and
C Y C 7 genes, both of which have been described previously as classes of revertants of cycl mutants (MCKNIGHT, CARDILLOandSHERMAN198 1; ERNST,
STEWART and SHERMAN 1981). Furthermore, revertants also can not arise by reversion of the cycl-1 allele
o r by recombination between cycl and c y c l - I , since
cycl-I is a deletion of the entire C Y C l region (SINGH
and SHERMAN 1978;
STILESet al. 1981).
This system is preferable to the one described by
'RAKASH and SHERMAN (1973) for detecting
specific
se substitutions in yeast. Their system, also based
"eversion of cycl mutants, was comprised of nine
translation initiation codon mutants andtwo nonsense
mutants.Althoughtheir
system provided the first
detailed characterization of mutational specificity in a
eukaryotic organism,each of their strainscould revert
by more than one pathway. Consequently, reversion
of specific mutants did not necessarily correlate with
specific base substitutions. Rather, they had to rely on
patterns of reversion and protein sequence analysis to
elucidate mutationalspecificities. By contrast, theCys-
22 missense mutants described here revert by only a
single pathway. Furthermore, these strains are isogenic, allowing for the relative rates of allsixbase
substitutions to be compared in a single genetic background.
The response of these strains to several different
mutagens was determined (Table 2). Predictable patterns of reversion were obtained with mutagens whose
specificities have been described previously. Furthermore, the low spontaneous reversion frequencies for
all six strains provided sufficient sensitivity to detect
relatively weak stimulation of other base substitutions.
For example, althoughEMS induced primarily the G .
C + A . T transition, the other five base substitutions
were also detected(Table 2). These strains should
therefore be valuable for detecting the potential and
specificityof relatively weak mutagens,althougha
more extensive analysis than is presented here would
be required to accurately determine induction over
background.
The specificities of 5-AZ and DEB in yeast had not
beendescribed previously, althoughrecent studies
using S. ty~himurium(LEVIN andAMES1986; MILLER
and BARNES1986) and E. coli (CUPPLES
and MILLER
1989) demonstrated that5-AZ induced either primarily or exclusively G.C + C . G transversions. 5-AZ
displayed the same specificityin this tester system,
inducing almost exclusively reversion of strain
YMH55 (Table 2). Interestingly, DEB efficiently induced three different base substitutions, A. T + T .
A, A. T + G .C and G . C + T .A; little or no stimulation of the other substitutions was detected (Table
2). These data suggest that DEB induces at least two
different DNAlesions andthat alkylation of A - T
base-pairs may be the primary mutagenic lesion.
A limitation to any reversion-based tester system is
that mutational alterationsare detectedin the context
of a single DNA sequence. Sequence contextis known
to affect mutationalspectra in response tocertain
mutagens, first recognized in studies of the bacteriophage T 4 rZZ locus by BENZER(1961).A less than
ideal context presumably accounts for theweak effect
of UV in this study (see RESULTS).
In general, a detailed analysis of mutagen specificity should rely on
detection of mutations at multiple sites and is most
reliably determined in forwardmutation
systems.
Nonetheless, thepotentialproblem
associated with
context effects can be diminished in reversion systems
by scoring for mutationsat multiple sites. The known
structuralrequirementsfor
functional iso-1-cytochrome c provides a basis for expanding thecycl tester
system for this purpose. Both Cys-19 and Cys-22 COvalently bind heme in thioether linkages, and His-23
and Met-85 function as axial ligands to the heme iron.
T h e structural roles of these residues and their evolutionary conservation strongly suggest that all four
Strains Mutagenicity Tester
amino acids are critical for iso-lcytochrome c function. [The only phylogenetic variance at these four
positions is in the cytochromes c from Crithidia and
Euglena (HAMPSEY,
DASand SHERMAN
1986) where
alanine occupies the equivalent of position 19. However, alanine does not functionally replace Cys-19 in
iso-l-cytochrome c (M. Hampsey, unpublished data).]
Since histidine and cysteine are encoded by only two
codons and methionine is encoded by a single codon,
all of which require at the DNA level both A - T and
G - C base pairs, additional mutants specific for detection of all six base substitutions can be constructed.
Accordingly, simplereversion analysis wouldscore for
mutational specificity in four different sequence contexts. Construction and testing of these strains are in
progress.
Hemizygous ( c y c l / c y c l - l ) diploids were usedin this
study in order to mask the effects of recessive mutations that could interfere with the reversion analyses.
Diploid strains YMH52-YMH57 were found to be
more resistant than haploid strains YMH2-YMH7
(Table 1) to the lethal effects ofthe various mutagens
(haploid data not shown). These strains also yielded
revertants of more uniform colonysize. Moreover,
reversion frequencies were more reproducible using
the diploid strains (haploid reversion frequencies are
not presented), an effect also reported by PRAKASH
and SHERMAN
(1973). Haploid strains, on the other
hand, have the potential to be used to uncover recessive mutations identifying genetic loci that affect mutagenic potential and specificity. For example, haploid
strains could be used to identify mutator genes that
affect specific basesubstitutions. MILLERand co-workers have exploited their lac2 tester system for this
purpose. By screening for colonieswithincreased
reversion rates to Lac+, they haveuncovered two new
NGHIEMand
mutator loci in E. coli, mutM (CABRERA,
MILLER 1988) and mutY (NCHIEM et al. 1988), that
T A transversions. Only recently
generate G-C
has a mutator gene specific for a single base substitution been described in a eukaryotic system. The yeast
rad18 mutator was shown by KUNZ, KANC and KoHALMI (199 1) to specifically increase G. C + T .A
transversions.
In summary, the cycl-based tester system described
here offers a simple, inexpensive,and sensitive means
to survey mutagen specificity with respect to single
base substitutions in a eukaryotic organism. This system can be used to rapidly screen a large number of
potential mutagens or to screen for mutagens that
induce specific base substitutions. The effects of different physiological variables on mutagenic potential
and specificity can readily be evaluated. This system
may also prove to be valuable for isolating new mutator strains that could provide additional insight into
-
-
65
the components affecting the fidelity of DNA replication and repair.
I am gratefulto FREDSHERMAN
(University of Rochester) for his
generous gift of yeast strains. I thank DAVIDGROSSand TINA
HENKIN for comments on the manuscript and RHONDA BERROTERAN for preparing synthetic oligonucleotides. This work was
supported, in part, by U.S. Public Health Service grant GM39484
and by a Junior Faculty Research Award from the American Cancer
Society.
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