TOXICOLOGICAL SCIENCES 113(2), 401–411 (2010)

doi:10.1093/toxsci/kfp267
Advance Access publication November 1, 2009

Creation of a Hyperpermeable Yeast Strain to Genotoxic Agents through

Combined Inactivation of PDR and CWP Genes
Min Zhang,* Michelle Hanna,† Jia Li,† Susan Butcher,† Heping Dai,* and Wei Xiao†,1
*Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei 430072, China; and †Department of Microbiology and Immunology, University of

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Saskatchewan, Saskatoon, SK Canada S7N 5E5

1
To whom correspondence should be addressed at Department of Microbiology and Immunology, University of Saskatchewan, 107 Wiggins Road, Saskatoon,
SK Canada S7N 5E5. Fax: þ1 (306) 966-4298. E-mail: wei.xiao@usask.ca.

Received August 3, 2009; accepted October 22, 2009

yeast (Jia et al., 2002). Although the sensitivity of this method

We previously established a genotoxicity detection system based
on the transcriptional response of the yeast RNR3 gene to DNA
damage. In order to further improve its sensitivity to genotoxicants,
we have attempted to increase cell permeability by removing cell
wall mannoproteins (CWPs). Here, we report that selected deletion
of pleiotropic drug resistance (PDR) genes encoding membrane
efflux transporters also enhanced cellular sensitivity to treatment
by various genotoxic agents. Furthermore, we have validated our
hypothesis that PDR and CWP protect cells through different
mechanisms by demonstrating that simultaneous inactivation of
the above two pathways resulted in a synergistic enhancement of
assay sensitivity as measured by RNR3-lacZ expression and that
this effect is at the cell permeability level. The quadruple mutation
results in RNR3-lacZ assay sensitivity to tested chemicals that
apparently surpasses the industry standard Ames test. We argue
that this hyperpermeable yeast mutant strain would be suitable for
other chemical-based genotoxic assays.
Key Words: Saccharomyces cerevisiae; RNR3-lacZ; genotoxicity
test; permeability; ABC transporters; cell wall.
Development of microorganism-based, environmentally
safe, and highly sensitive genotoxic testing systems remains
an intensive research area in the field of toxicology. The
budding yeast Saccharomyces cerevisiae is an ideal choice
because this species has been used in a variety of fermentation
industries, including making bread, beer, and wine, and is
hence safe for the environment and public health. Furthermore,
budding yeast is the best-studied model lower eukaryotic
microorganism, which is more comparable to human cells than
bacteria. Surprisingly, despite the presence of a very strong
budding yeast research community and the fact that this species
was the first eukaryotic organism whose genomic sequence was
determined, little attention has been paid to genetic factors that
influence the sensitivity of budding yeast to toxic chemicals.
We previously developed a genotoxicity testing system
based on the transcriptional response of the RNR3-lacZ reporter
gene to a broad range of DNA-damaging agents in budding

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to detect genotoxicants is comparable to the bacterial
chromotest, which is also based on transcriptional response
to DNA damage, in many cases, the RNR3-lacZ testing system
is not as sensitive to the best-studied model and industrial
standard Ames test. The Ames test sensitivity to detect certain
types of carcinogenic chemicals was significantly improved
through several genetic modifications, including the removal of
the lipopolysaccharide coat from Salmonella typhimurium to
make cells more permeable to higher–molecular weight
compounds. We reasoned that yeast genes involved in cell
wall and membrane biosynthesis could be modified genetically
to alter cellular permeability and improve the toxicity test in
yeast. Indeed, deletion of both CWP1 and CWP2 genes
encoding cell wall mannoproteins (CWPs) markedly increased
cell wall permeability and enhanced the RNR3-lacZ assay
sensitivity, particularly to higher–molecular weight compounds
(Zhang et al., 2008). In contrast, deletion of some nonessential
membrane components did not significantly affect the RNR3-
lacZ sensitivity to the testing chemicals (Zhang et al., 2008 and
data not shown), which allowed us to conclude that the yeast
CWPs constitute a major barrier to genotoxic agents.
In addition to the physical barrier provided by cell wall
and membrane, budding yeast also possesses a dynamic
biochemical defense system against xenobiotics called pleio-
tropic drug response or pleiotropic drug resistance (PDR)
(Balzi and Goffeau, 1995). PDR is mediated by a network of
ATP-binding cassette (ABC) transporter proteins that
constitute active efflux pumps for a broad spectrum of
unrelated chemicals. The PDR network currently consists of
10 transcription factors regulating about 70 different target
genes, of which 22 genes have been identified with putative
ABC transporter activity (Ernst et al., 2005; Moye-Rowley,
2003). The three major ABC transporter genes in yeast are
PDR5 (Golin et al., 2007), YOR1 (yeast oligomycin resistance)
(Cui et al., 1996), and SNQ2 (sensitivity to 4-nitroquinoline-N-
oxide) (Servos et al., 1993). PDR5, YOR1, and SNQ2 have
402 ZHANG ET AL.
substrate specificities as well as overlapping functions for some
chemicals (Kolaczkowski et al., 1996, 1998). In addition,
PDR1 and PDR3 encode two zinc finger transcription factors
that are required for the activation of most ABC transporter
genes (Katzmann et al., 1994; Mamnun et al., 2002). Pdr1 and
Pdr3 can form either a homodimers or a heterodimer, which
may play a role in target gene specificity. Indeed, deletion of
PDR1 or PDR3 results in differential drug tolerance; neverthe-
less, loss of both genes aggravates drug hypersensitivity due to
impaired drug transporters (Rogers et al., 2001).
In this report, we systematically examined the effects of the
inactivation of major yeast ABC transporter genes on the
RNR3-lacZ genotoxicity test with selected chemicals. We then
hypothesized that the enhanced sensitivity by deleting PDR
genes is mechanistically distinct from that of deleting CWP
genes and demonstrated that indeed the combined inactivation
of both cellular defense systems resulted in an unprecedented
level of sensitivity to detecting certain toxic chemicals.
MATERIALS AND METHODS
Yeast strains, cell culture, and transformation. Haploid S. cerevisiae yeast
strains used throughout this study are summarized in Supplementary table 1.
FY1679-28C and its isogenic derivatives were received from either Dr. A.
Delahodde (Centre National de la Recherche Scientifique, Paris, France) or
Dr. A. Kolaczkowska (University of Wroclaw, Wroclaw, Poland). The pdr5 snq2
yor1 triple mutant grew slowly in the synthetic selective medium lacking uracil
(data not shown) and hence was not further analyzed.
Yeast cells were grown at 30C in YPD (Sherman et al., 1983). Plasmid
DNA was transformed into yeast cells by a modified lithium acetate protocol
(Hill et al., 1991) and selected on minimal SD medium (Sherman et al., 1983).
Transformants were streaked on a fresh selective plate before being utilized for
further analysis.
Plasmids and mutant strain construction. Plasmid pZZ2 (Zhou and
Elledge, 1992) was obtained from Dr. S. Elledge (Harvard University, Boston,
MA) and utilized for the RNR3-lacZ test as previously described (Jia and Xiao,
2003; Jia et al., 2002).
To create the cwp1D::hisG mutant, plasmid pGEM-CWP1 containing
a 0.72-kb CWP1 open reading frame plus 0.7-kb flanking sequences
(Zhang et al., 2008) was used to delete a 0.62-kb Sty1 fragment within the
CWP1 ORF, which was replaced by a BamHI linker to form pcwp1DB. A
3.8-kb BamHI-BglII fragment from pNKY51 containing the hisG-URA3-hisG
cassette (Alani et al., 1987) was then cloned into the BamHI site of pcwp1DB
to form pcwp1D::HUH. The cwp1D::hisG-URA3-hisG cassette was released by
XhoI-SphI digestion prior to yeast transformation. The cwp1 deletion mutant
was allowed to grow nonselectively overnight and then selected on a plate
containing 5-fluoro-orotic acid as described (Boeke et al., 1984) to obtain the
cwp1D::hisG derivative.
To construct the cwp2D::HIS3 disruption plasmid, a 0.5-kb EcoRI-BamHI
fragment containing the 5# flanking region and a 0.3-kb BamHI-SphI fragment
containing the 3# flanking region of CWP2 ORF was sequentially cloned into
pGEM-T (Promega, Madison, WI) to form pcwp2DB. The 1.16-kb BamHI
fragment from YDp-H (Berben et al., 1991) containing the HIS3 gene was then
cloned into the BamHI site of pcwp2DB to form pcwp2D::HIS3. The cwp2D::HIS3
cassette was released by EcoRI-SphI digestion prior to transformation.
Test chemicals. Genotoxic chemicals used in this study include methyl
methanesulfonate (MMS), 4-nitroquinoline-N-oxide (4-NQO), phleomycin,
which have been previously described in Zhang et al. (2008), and the
two anticancer agents, chlorambucil (304 Da) and cisplatin (300 Da). MMS
(110 Da) represents a low–molecular weight mutagen, 4-NQO (190 Da)
represents a medium–molecular weight mutagen, while phleomycin (1526 Da)
is considered to be a bulky molecule. In addition, L-canavanine (274 Da) and
tetracycline (444 Da) were used to represent nongenotoxic agents. All the
above chemicals were purchased from Sigma-Aldrich (St Louis, MO).
DNA-damage treatment and b-galactosidase assay. The b-galactosidase
(b-gal) assay was performed as described previously (Jia and Xiao, 2004; Xiao
et al., 1993). Briefly, 0.5 ml of overnight yeast culture was used to inoculate
2.5 ml of fresh SD selective medium (Supplemental materials and methods) and
incubation was continued for another 2 h. At this point, chemicals were added
at the concentration indicated and cells were incubated for another 4 h.
Conditions for ultraviolet (UV) irradiation have been previously described (Jia
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et al., 2002). After the incubation, 1 ml of the above unsynchronized log-phase
cell suspension was used to determine cell titer by measuring optical density600 nm,
and the remaining 2 ml yeast cells were precipitated by centrifugation, washed
twice with sterile distilled water, and resuspended in a Z buffer (60mM
Na2HPO4
7H2O, 40mM NaH2PO4
H2O, 10mM KCl, 1mM MgSO4
7H2O, and
40mM b-mercaptoethanol, pH 7.0) for the b-gal assay using orthonitrophenyl-
b-galactoside as the colorimetric substrate. The b-gal activity is expressed in
Miller units (Guarente, 1983).
Toxicity test. Cell survival rates were determined as previously described
(Jia et al., 2002). At the end of incubation and prior to the b-gal assay,
untreated and treated cells were collected by centrifugation, diluted, and plated
on YPD in duplicate. The plates were incubated at 30C for 3 days, and the
number of colonies was counted. The toxic effect is expressed as a percentage
of colonies from treated samples versus untreated samples.
Statistical analysis. All the results presented in this report were an average
of at least three independent experiments and expressed as mean ± SD.
Statistical significance between different strains was determined using a two-
tailed paired Student’s t-test. A p value of less than 0.01 was considered
statistically significant and less than 0.0001 highly significant.
RESULTS
Deletion of PDR Genes Enhances the RNR3-lacZ Sensitivity
To ask whether the inactivation of ABC transporters affects
cell permeability to DNA-damaging agents, three representative
chemicals, MMS, 4-NQO, and phleomycin, were selected in
this study. They are all genotoxic and carcinogenic agents with
different molecular size, chemical structure, and mechanism of
action. The RNR3-lacZ reporter gene assay (Jia et al., 2002) was
primarily employed to assess effects of gene mutations on the
cellular responses to test chemicals, although the cell survival
after chemical treatment can also reflect the toxicity.
As shown in Figure 1, compared with wild-type cells,
deletion of either PDR5 or YOR1 resulted in indistinguishable
responses to all three drugs tested with respect to both RNR3-
lacZ induction and cytotoxicity. In contrast, deletion of SNQ2
resulted in a significant enhancement of RNR3-lacZ induction
by 4-NQO (Fig. 1A) and moderate increases in phleomycin
(Fig. 1B) and MMS (Fig. 1C) induction. Specifically, after
treatment with 0.5 lg/ml 4-NQO for 4 h, snq2 mutant cells
displayed almost 4-fold induction compared with less than
1.5-fold induction in the wild type or other two single mutants
(Fig. 1A). In addition, the maximum induction was increased
from 6-fold in the wild type to 11-fold in the snq2 mutant.
Similarly, the snq2 mutant appears to be slightly more sensitive
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FIG. 1. Sensitivity of pdr single mutants to DNA-damaging agents. (A–C) RNR3-lacZ assays; (D–F) cell killing experiments. (A and D) 4-NQO treatment,
(B and E) phleomycin treatment, and (C and F) MMS treatment. Results are the average of at least three independent experiments with SDs; *p < 0.01. Yeast
strains used: (d) FY1679-28C (wild type), (=) FYMK-1/1 (pdr5-D1), (n) FYAK4 (yor1-1), and (e) FYMK 23/2 (snq2-D1).

to killing by the three DNA-damaging agents examined (Figs.
1D–F). The specificity of Snq2 for 4-NQO is not surprising as
the snq2 mutant was initially identified for its enhanced
sensitivity to 4-NQO (Servos et al., 1993).
The ABC transporters often confer complex overlapping
substrate specificities. It has been reported that if one PDR gene
is deleted, the remaining transporters may compensate, whereas
if all three genes are inactivated, the drug resistance will be
impaired (Rogers et al., 2001). These overlapping functions
prompted us to ask whether simultaneous deletion of two or
more PDR genes will result in an additive or synergistic effect
on drug permeability. As shown in Figure 2, the pdr5 snq2
double mutant displayed an increased sensitivity to 4-NQO–
induced or phleomycin-induced RNR3-lacZ expression (Figs.
2A and 2B). For example, at a 4-NQO concentration of 0.25
lg/ml, the double mutant displayed a fourfold induction,
whereas the induction in the wild type and other single mutant
was barely detectable (Fig. 2A). Similarly, at a phleomycin
concentration of 5 lg/ml, the pdr5 snq2 double mutant
displayed more than twofold induction, whereas the induction
in the wild type and other single mutant was barely detectable
(Fig. 2B). As for MMS, the double mutation also enhanced the
RNR3-lacZ induction, albeit to a lesser extent (Fig. 2C) than
induction by 4-NQO or phleomycin. Consistent with the
RNR3-lacZ induction, the pdr5 snq2 double mutant also
became more sensitive to killing by 4-NQO (Fig. 2D) or
phleomycin (Fig. 2E) compared with wild type and the snq2
single mutant. These observations indicate that although
deletion of PDR5 alone has no effect on membrane
permeability to chemicals like 4-NQO and phleomycin, in
the absence of the major transporter Snq2, Pdr5 plays a backup
role in preventing entry of these toxic chemicals.
The other two double mutants (yor1 snq2 and pdr5 yor1)
also showed increased sensitivity to 4-NQO compared with
wild-type cells; however, the effect was not as obvious as the
snq2 pdr5 double mutation (Figs. 2A and 2D). It is of interest
to note that although deletion of YOR1 in the snq2 background
did not further enhance its sensitivity to 4-NQO, the pdr5
yor1 double mutant displayed an enhanced sensitivity to
4-NQO (Figs. 2A and 2D) despite the fact that neither cor-
responding single mutation had a noticeable effect (Figs. 1A
and 1D). In contrast, these two double mutants showed little
enhancement of sensitivity to phleomycin (Figs. 2B and 2E)
or MMS (Figs. 2C and 2F). Based on the above analyses and
the observed slow growth phenotype of the triple mutant (data
not shown), we chose the pdr5 snq2 double mutant to rep-
resent ABC transporter–deficient strain for the subsequent
investigation.
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FIG. 2. Sensitivity of pdr double mutants to DNA-damaging agents. (A–C) RNR3-lacZ assays; (D–F) cell killing experiments. (A and D) 4-NQO treatment,
(B and E) phleomycin treatment, and (C and F) MMS treatment. Results are the average of at least three independent experiments with SDs; *p < 0.01 and **p <
0.0001. Yeast strains used: (d) FY1679-28C (wild type), (=) FYAK 1/1 (pdr5-D1 yor1-1), (n) FYAK 23/2 (snq2-D1 yor1-1), and (e) FYMK 26/8-10B (snq2-D1
pdr5-D1).

Deletion of PDR3, but Not PDR1, Results in a Compromised
RNR3-lacZ Induction
Budding yeast contains at least five ABC transporter
subfamilies and many of them confer drug resistance (Bauer
et al., 1999). It would be technically challenging to investigate
each individual ABC transporter mutation and then double
or multiple mutations for their combined effects on various
drug-induced toxicity. It is known that a large number of ABC
transporter genes are under the control of two master
transcriptional activators, Pdr1 and Pdr3, which confer distinct
as well as overlapping functions (Bauer et al., 1999). Since
most of the stress-induced ABC transporter gene expression is
virtually abolished in the pdr1 pdr3 double mutant (Mahe
et al., 1996), we decided to measure the RNR3-lacZ expression
in the above double mutant instead of individual transporter
gene mutants. To our surprise, the RNR3-lacZ induction by all
three testing compounds was significantly reduced in the pdr1
pdr3 double mutant (Fig. 3). This phenomenon is reminiscent
of our previous finding that Pdr3 is a positive regulator of two
DNA damage–inducible genes, MAG1 and DDI1, and that
deletion of PDR3, but not PDR1, abolished the MMS-induced
expression of these two genes (Zhu and Xiao, 2004). To ask
whether RNR3-lacZ induction is also affected by PDR3 but not
PDR1, we measured the RNR3-lacZ induction by the three test
compounds in the pdr1 and pdr3 single mutants and compared
with wild type and the double mutant. As shown in Figures
3A–C, in all cases, deletion of PDR1 alone has no effect on the
RNR3-lacZ expression, whereas deletion of PDR3 has the same
effect as that of the double mutation. Hence, it is apparent that
only Pdr3 is involved in the transcriptional regulation of RNR3
and that inactivation of these PDR transactivators is unsuitable
for the enhancement of the RNR3-lacZ sensitivity. Interest-
ingly, while deletion of either PDR1 or PDR3 did not enhance
cellular sensitivity to the testing chemicals, the simultaneous
inactivation of both genes increased cellular toxicity of 4-NQO
and phleomycin but not MMS (Figs. 3D–F).
Combined Deletion of CWP and PDR Genes Enhances the
RNR3-lacZ Sensitivity
The above results demonstrate that inactivation of ABC
transporters, especially the pdr5 snq2 double mutation, has
a pronounced effect on cell permeability and hence enhances
the sensitivity of the RNR3-lacZ genotoxicity testing. In a
previous study, we reported that inactivation of CWP genes
enhances the RNR3-lacZ sensitivity as well (Zhang et al.,
2008). Since PDR and CWP appear to protect cells by different
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FIG. 3. Effects of pdr1 and pdr3 mutations on DNA damage responses. (A–C) RNR3-lacZ assays; (D–F) cell killing experiments. (A and D) 4-NQO
treatment, (B and E) phleomycin treatment, and (C and F) MMS treatment. Results are the average of at least three independent experiments with SDs; *p < 0.01
and **p < 0.0001. Yeast strains used: (d) FY1679-28C (wild type), (s) naD1 (pdr1D), (;) naD3 (pdr3D), and (D) naD1D3 (pdr1D pdr3D).

mechanisms, we hypothesized that the combined inactivation
of PDR and CWP genes would have a synergistic effect on the
cellular sensitivity to certain toxic agents. To test this hypothesis,
we made a cwp1 cwp2 pdr5 snq2 quadruple deletion strain to
simultaneously disarm two major yeast protection mechanisms,
namely cell wall and the efflux pumps.
The quadruple mutant displayed extreme sensitivity to test
chemicals compared with either of the double mutants (Fig. 4).
For example, a greater than twofold enhancement in the RNR3-
lacZ induction can be observed in the quadruple mutant at the
lowest 4-NQO dose tested (0.031 lg/ml), whereas the RNR3-
lacZ induction was not detected in the cwp1 cwp2 double mutant
by 0.125 lg/ml or in snq2 pdr5 double mutant by 0.0675 lg/ml
4-NQO (Fig. 4A). It was also observed that the snq2 pdr5
double mutant is more sensitive to 4-NQO than the cwp1 cwp2
double mutant (p < 0.01). A linear regression analysis using
low-dose data indicates that the quadruple mutant (y ¼ 35.1x þ
2.327) enhances the RNR3-lacZ sensitivity by 11.2-fold over
wild type (y ¼ 3.13x þ 1.273), by 6.13-fold over the cwp1 cwp2
double mutant (y ¼ 5.72x þ 1.273), and by 2.7-fold over the
snq2 pdr5 double mutant (y ¼ 13.2x þ 1.125). The quadruple
mutation also further enhances the cytotoxicity of the test
compounds (p < 0.001), although the combined effect may not
be as impressive as that of the RNR3-lacZ assay (Fig. 4D).
The quadruple mutant also displayed synergistic sensitivity
to phleomycin-induced RNR3-lacZ expression (Fig. 4B). At
a phleomycin concentration of 2.5 lg/ml, the quadruple mutant
displayed a 2.7-fold induction, whereas the induction in the
snq2 pdr5 and cwp1 cwp2 double mutants was barely
detectable. After treatment with 10 lg/ml phleomycin, the
snq2 pdr5 double mutant showed 2.4-fold induction and the
cwp1 cwp2 double mutant showed 2.9-fold induction, while the
quadruple mutant showed over 6-fold induction. A linear
regression analysis indicates that the detection sensitivity was
increased by 27.5-fold in the quadruple mutant (y ¼ 0.753x þ
1.700) compared with wild type (y ¼ 0.0274x þ 1.276), by
3.1-fold over the cwp1 cwp2 double mutant (y ¼ 0.246x þ
1.022), and by 4.1-fold over the snq2 pdr5 double mutant (y ¼
0.183x þ 1.121). The quadruple mutant was also more
sensitive to killing by phleomycin than by snq2 pdr5 and
cwp1 cwp2 double mutants (p < 0.01), and the effect appears to
be additive (Fig. 4E).
Even for small–molecular weight compounds like MMS, the
quadruple mutant still displayed an increased sensitivity
compared with the corresponding double mutants (p <
0.001). With both RNR3-lacZ induction and cell survival
experiments, the effects of pdr and cwp mutations are additive
(Figs. 4C and 4F). At an MMS concentration of 6.25 ppm, the
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FIG. 4. Sensitivity of pdr and cwp mutants to DNA-damaging agents. (A–C) RNR3-lacZ assays; (D–F) cell killing experiments. (A and D) 4-NQO treatment,
(B and E) phleomycin treatment, and (C and F) MMS treatment. Results are the average of at least three independent experiments with standard deviations; *p <
0.01 and **p < 0.0001. Yeast strains used: (d) FY1679-28C (wild type), (=) WXY2373 (cwp1D cwp2D), (n) FYMK 26/8-10B (snq2-D1 pdr5-D1), and (e)
WXY2375 (cwp1D cwp2D snq2-D1 pdr5-D1).

quadruple mutant displayed a twofold induction, whereas the
induction in the snq2 pdr5 and cwp1 cwp2 double mutants was
barely detectable (Fig. 4C). A linear regression analysis
showed that the detection sensitivity was increased by twofold
in the quadruple mutant (y ¼ 0.6x þ 0.79) over wild-type
cells (y ¼ 0.3x þ 0.33) and the sensitivity in cwp1 cwp2
double mutant (y ¼ 0.4x þ 0.45) or snq2 pdr5 double mutant
(y ¼ 0.42x þ 0.29) is only slight higher than in the wild type. A
similar additive effect was also observed for cell survival after
MMS treatment (Fig. 4F).
Pdr and Cwp Mutations Enhance Sensitivity to Cancer
Chemotherapeutic Agents
As an initial attempt to demonstrate the general applicability
of the above genetic modifications, we examined the effects of
cwp and pdr mutations on the cellular sensitivity to chlor-
ambucil and cisplatin, which are DNA-damaging agents used as
chemotherapeutic drugs. Chlorambucil is a bifunctional alkylat-
ing agent similar to nitrogen mustard and has been mainly used
in the treatment of chronic lymphocytic leukemia (Bank, 1992).
Cisplatin is a platinum-based chemotherapeutic agent used to
treat various types of cancers by forming cisplatin-DNA ad-
ducts (Fichtinger-Schepman et al., 1985). It is noted that while
each pdr or cwp double mutation barely enhanced sensitivity to
chlorambucil (Fig. 5A) and has a moderate effect on cisplatin
(Fig. 5B)-induced RNR3-lacZ expression, the effect of quadru-
ple mutation was much more obvious. A linear regression
analysis showed that the sensitivity to chlorambucil was
increased by 16.6-fold in the quadruple mutant (y ¼ 0.46x þ
0.7914) over wild type (y ¼ 0.02768x þ 1.084), 6.2-fold over
the cwp1 cwp2 double mutant (y ¼ 0.07432x þ 0.8218), and
8.6-fold over the snq2 pdr5 double mutant (y ¼ 0.05353x þ
0.8603). The cisplatin detection sensitivity was increased by
2.6-fold in the quadruple mutant (y ¼ 0.1666x þ 2.601) over
the wild type (y ¼ 0.06423x þ 0.9265), 2.0-fold over the cwp1
cwp2 double mutant (y ¼ 0.08186x þ 1.954), and 2.3-fold over
the snq2 pdr5 double mutant (y ¼ 0.07176x þ 2.2112).
Compared to cwp or pdr double mutant, the quadruple mutant
also displayed enhanced sensitivity to killing by chlorambucil
(p < 0.01, Fig. 5C) or cisplatin (p < 0.01, Fig. 5D), although in
each case, the double mutants already displayed noticeable
sensitivity.
The Enhanced Sensitivity Is at the Cell Permeability Level
If the enhanced sensitivity of the RNR3-lacZ assay by
inactivation of PDR and CWP genes is indeed due to increased
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FIG. 5. Sensitivity of pdr and cwp mutants to chlorambucil and cisplatin. (A and B) RNR3-lacZ assays; (C and D) cell killing experiments. (A and C)
chlorambucil treatment; (B and D) cisplatin treatment. Results are the average of at least three independent experiments with SDs; *p < 0.01 and **p < 0.0001.
Yeast strains used: (d) FY1679-28C (wild type), (=) cwp1D cwp2D double mutant, (n) snq2D pdr5D double mutant, and (e) cwp1D cwp2D snq2D pdr5D
quadruple mutant.

cell permeability instead of some other unknown factors, one
would argue that pdr and cwp mutations should not affect the
RNR3-lacZ induction by DNA damage that does not involve
cell permeability. To test this prediction, we treated wild-type
and various mutant cells with UV irradiation at a dose known
to induce RNR3-lacZ. As shown in Figure 6, inactivation of
PDR (pdr5 snq2), CWP (cwp1 cwp2), or both pathways had no
obvious effect on the RNR3-lacZ expression.
Specificity of Enhanced RNR3-lacZ Sensitivity to Toxic
Compounds by Cell Permeability Mutations
Since we have previously shown that the RNR3-lacZ assay
only detects genotoxicants and does not respond to toxic
compounds that kill yeast cells by means other than dam-
aging DNA (Jia et al., 2002), one would predict that the
enhanced cell permeability by cwp and pdr mutations should
not affect the RNR3-lacZ response to those nongenotoxic
chemicals. We tested three such well-characterized antibiotics,
namely L-canavanine, tetracycline, and kanamycin. None of
the three antibiotics induced RNR3-lacZ to a noticeable level,
and most importantly, the cwp pdr double and the quadruple
mutants did not display enhanced RNR3-lacZ activity after
treatment with these antibiotics at any concentrations exam-
ined (Figs. 7A–C). Interestingly, the cell permeability muta-
tions variably increased the cellular sensitivity to killing by
L-canavanine (Fig. 7D) and tetracycline (Fig. 7E), but their
effects on kanamycin-induced killing is not obvious (Fig. 7F).
For example, at the highest L-canavanine dose tested, each
double mutant showed ~50% survival compared with 90%
survival of wild-type cells, whereas the survival rate in the
quadruple mutant was only 30% (Fig. 7D), suggesting an
additive effect of the two separate cell permeability pathway
mutations on the bioavailability of intracellular tested agents.
408 ZHANG ET AL.

combined enhancement, as judged by the minimal dose of

testing agents capable of two fold induction in the RNR3-lacZ
assay, achieved 1.6-fold for MMS, 14-fold for 4-NQO, 24-fold
for phleomycin, 11-fold for chlorambucil, and 3.4-fold for
cisplatin (Table 1). The improvement of yeast cell permeability
alone makes the RNR3-lacZ assay ninefold more sensitive to
MMS and chlorambucil and fivefold more sensitive to 4-NQO
than the Ames test (Table 1). There are no Ames test data
available on phleomycin in the database. We performed Ames
test with both TA98 and TA100 strains and found that

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FIG. 6. The effect of UV irradiation on RNR3-lacZ expression in wild
type, pdr, and cwp mutants. Results of b-gal assays are the average of at least
three independent experiments with SDs and expressed in Miller units. Open
boxes, untreated cells; solid boxes, cells were exposed to 50 J/m2 UV
irradiation.
Comparison of the Detection Limit between Yeast RNR3-lacZ
Assay and Ames Test
The pdr5 snq2 cwp1 cwp2 quadruple mutation significantly
improved the RNR3-lacZ sensitivity to detecting genotoxic
compounds examined. Quantitative analysis indicates that the
phleomycin doses up to 33 lg/plate (a lethal dose) did not
result in apparent increase in mutagenesis and that addition of
the S9 liver extract did not make a difference (data not shown).
DISCUSSION
The current study investigated the effect of the PDR pathway
on the sensitivity of the RNR3-lacZ testing system to detect
genotoxicants in S. cerevisiae. PDR is known to affect cellular
transport and sensitivity to various toxic chemicals. Since
genes involved in PDR pathways can be modified genetically
to alter cellular permeability, we hypothesized that inactivation

FIG. 7. Sensitivity of pdr and cwp mutants to nongenotoxic agents. (A–C) RNR3-lacZ assays; (D–F) cell killing experiments. (A and D) L-canavanine treatment;
(B and E) tetracycline treatment; (C and F) kanamycin treatment. Results are the average of at least three independent experiments with SDs; *p < 0.01 and **p <
0.0001. Yeast strains used: (d) FY1679-28C (wild type), (=) cwp1D cwp2D double mutant, (n) snq2D pdr5D double mutant, and (e) cwp1D cwp2D snq2D pdr5D
quadruple mutant.
 YEAST MUTANT WITH ENHANCED PERMEABILITY 409

TABLE 1 4-NQO and phleomycin, it is conceivable that Pdr3 is required

Minimum Detection Limits by RNR3-lacZ and Comparison for the transcriptional regulation of RNR3 in response to DNA
With the Ames Test damage and that this effect is specific to transcription-based
assays, such as RNR3-lacZ. Hence, results obtained from
Wild cwp1 snq2 cwp1 cwp2 killing experiments suggest that the pdr1 pdr3 double mutant
Chemicals type cwp2 pdr5 snq2 pdr5 Ames testa
has increased intracellular bioavailability of tested toxic agents
MMS 17.5b 17.5 17 11 100c (TA100) and hence remains an attractive strain to explore other toxicity-
4-NQO 0.34 0.22 0.11 0.024 0.125 (TA98) based assays.
Phleomycin 42.2 6.44 7.72 1.78 Not foundd (5)e Perhaps the most exciting observation in this study is the
Chlorambucil 44.7 24.6 31.2 3.97 333 (TA100) synergistic enhancement of the RNR3-lacZ assay by simulta-

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33 (TA1535)
Cisplatin 13.9 7.96 7.46 4.13 1 (TA100)
1 (TA98)
a
The Ames test data are obtained from the National Toxicology Program
genotoxicity database: http://ntp-apps.niehs.nih.gov/ntp_tox/index.cfm?
fuseaction¼ntpsearch.searchhome and GENE-TOX database: http://toxnet.
nlm.nih.gov/cgi-bin/sis/htmlgen?GENETOX
b
Minimum chemical dose (in micrograms per milliliter) required to cause
a twofold increase in the RNR3-lacZ assay as calculated by linear regression
analyses.
c
Ames test values are based on the chemical dose (micrograms per plate)
capable of inducing twofold increase in Hisþ revertants.
d pathway resulted in barely noticeable enhancement of the RNR3-
Not available from all major databases.
e
Based on an Escherichia coli Trpþ reversion assay (Hall, 1985).
of certain PDR genes would be able to enhance the RNR3-lacZ
sensitivity to selected genotoxic agents, which is indeed the
case. Furthermore, several conclusions can be drawn from this
study. First, individual deletion of major ABC transporter genes
had minimal effect on cell survival and RNR3-lacZ induction
with the exception of the effect of snq2 mutation on DNA
damage induced by 4-NQO. Second, simultaneous deletion of
two PDR genes could have an additive effect, even though
deletion of either gene may not cause an enhanced sensitivity.
This observation indicates overlapping functions of PDR genes
in the protection of cells against drug entry. Third, it appears
that the increased RNR3-lacZ activity can be detected at doses
lower than those that would induce an obvious killing effect,
suggesting that the RNR3-lacZ assay often is more sensitive
than the cell survival–based assays. Finally, the enhanced sen-
sitivity is deemed to be due to cell permeability since deletion of
PDR genes does not affect RNR3-lacZ expression induced by
UV irradiation, which does not require cell permeability.
To our surprise, deletion of the two PDR transcriptional
regulators PDR1 and PDR3, known to be required for the
activation of a large number of PDR genes, has different effects
on the RNR3-lacZ assay, while the deletion of PDR3
compromises RNR3-lacZ induction by all three tested chem-
icals, deletion of PDR1 has no effect on the RNR3-lacZ assay,
regardless of wild type or pdr3 background. The fact that
deletion of PDR3 affects the induction of different DNA
damage–inducible genes including RNR3, MAG1, and DDI1
suggests that it is involved in an SOS-like transcriptional
regulation (Fu et al., 2008). Nevertheless, since the pdr1 pdr3
double mutant displayed an increased sensitivity to killing by
neous inactivation of both PDR and CWP genes. This
investigation is based on the hypothesis that cellular protections
provided by efflux pumps and CWPs are two distinct
mechanisms. Confirmation of the above hypothesis through this
study is not only of great significance in theory but also offers an
excellent hyperpermeable yeast strain to enhance the detection of
genotoxic chemicals when existing in low abundance. Further-
more, the overlapping functions of PDR and CWP pathways in
preventing entry of genotoxic compounds into cells is best
illustrated by our observation that while the inactivation of either
lacZ activity by chlorambucil, simultaneous inactivation of both
pathways caused a 28-fold increase in the RNR3-lacZ induction
by the same compound.
Recently, several yeast transcription–based reporter systems
have been developed (Afanassiev et al., 2000; Benton et al.,
2007; Jia et al., 2002) and the recent focus was to improve the
sensitivity of these assays through genetic modifications of the
host cells, including inactivation of DNA repair pathways
(Benton et al., 2008; Jia and Xiao, 2003), metabolic activation,
and compound solubility (Walsh et al., 2005). These
manipulations are expected to apply to narrowly selected
compounds. In contrast, improvement of cell permeability is
anticipated to have a broad range of applications. Of great
significance is the fact that multidrug resistance (MDR) genes
in mammalian cells also encode membrane transporters (Gros
et al., 1986) homologous to the yeast PDR genes (Raymond
et al., 1992), making this study potentially applicable to public
health. It is rather exciting to notice that genetic modifications
of the yeast cell permeability alone make the RNR3-lacZ assay
more sensitive than Ames test in detecting most genotoxic
agents included in this study. An extreme case is phleomycin in
which inactivation of both CWP and PDR pathways results in a
stunning 24-fold enhancement of the RNR3-lacZ assay
sensitivity so that it only requires less than 2 lg/ml phleomycin
to induce more than twofold RNR3-lacZ activity. In contrast,
phleomycin is unable to induce mutagenesis in the Ames test
under all experimental conditions despite its strong cytotoxic
effect on the host cell. However, we noticed a report (Hall,
1985) in which 5 lg/plate phleomycin was able to cause an up
to twofold increase in an Escherichia coli Trpþ reversion assay
in the presence of pKM101.
Our observation that yeast cell permeability mutations do not
enhance the RNR3-lacZ activity in response to nongenotoxic
410 ZHANG ET AL.
compounds is consistent with the predicted RNR3-lacZ
specificity. Meanwhile, the cell permeability mutations do
enhance cytotoxicity of these nongenotoxic agents, suggesting
that the hyperpermeable quadruple mutant created in this study
is applicable to detect not only genotoxic agents but also
nongenotoxic chemicals.
SUPPLEMENTARY DATA
Supplementary data are available online at http://toxsci.
oxfordjournals.org/.
FUNDING
Natural Science Foundation of China (30440420603) to
H.D. and W.X.; Natural Sciences and Engineering Research
Council of Canada (138338) to W.X.; Chinese National Key
Laboratory FEBT 2008FB002.
ACKNOWLEDGMENTS
The authors wish to thank Drs A. Kolaczkowska and
A. Delahodde for yeast strains and Dr S. Elledge for the RNR3-
lacZ reporter plasmid.
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