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www.impactjournals.com/oncotarget/ Oncotarget, Vol. 6, No. 31
Genome-wide array-CGH analysis reveals YRF1 gene copy
number variation that modulates genetic stability in distillery
yeasts
Anna Deregowska1,*, Marek Skoneczny2,*, Jagoda Adamczyk1, Aleksandra
Kwiatkowska1, Ewa Rawska1, Adrianna Skoneczna3, Anna Lewinska4,** and Maciej
Wnuk1,**
1 Department of Genetics, University of Rzeszow, Rzeszow, Poland
2 Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
3 Laboratory of Mutagenesis and DNA Repair, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw,
Poland
4 Department of Biochemistry and Cell Biology, University of Rzeszow, Poland
* These authors have contributed equally as rst authors
** These authors have contributed equally as last authors
Correspondence to: Anna Lewinska, email: alewinska@o2.pl
Correspondence to: Maciej Wnuk, email: mawnuk@gmail.com
Keywords: distillery yeasts, genome, array-CGH, chromosomes, genetic instability, Chromosome Section
Received: July 19, 2015 Accepted: August 24, 2015 Published: September 10, 2015
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.
ABSTRACT
Industrial yeasts, economically important microorganisms, are widely used in diverse
biotechnological processes including brewing, winemaking and distilling. In contrast to a well-
established genome of brewer’s and wine yeast strains, the comprehensive evaluation of
genomic features of distillery strains is lacking. In the present study, twenty two distillery yeast
strains were subjected to electrophoretic karyotyping and array-based comparative genomic
hybridization (array-CGH). The strains analyzed were assigned to the Saccharomyces sensu
stricto complex and grouped into four species categories: S. bayanus, S. paradoxus, S. cerevisiae
and S. kudriavzevii. The genomic diversity was mainly revealed within subtelomeric regions and
the losses and/or gains of fragments of chromosomes I, III, VI and IX were the most frequently
observed. Statistically signicant differences in the gene copy number were documented in six
functional gene categories: 1) telomere maintenance via recombination, DNA helicase activity
or DNA binding, 2) maltose metabolism process, glucose transmembrane transporter activity; 3)
asparagine catabolism, cellular response to nitrogen starvation, localized in cell wall-bounded
periplasmic space, 4) siderophore transport, 5) response to copper ion, cadmium ion binding
and 6) L-iditol 2- dehydrogenase activity. The losses of YRF1 genes (Y’ element ATP-dependent
helicase) were accompanied by decreased level of Y’ sequences and an increase in DNA double
and single strand breaks, and oxidative DNA damage in the S. paradoxus group compared to
the
S. bayanus
group. We postulate that naturally occurring diversity in the YRF1 gene copy
number may promote genetic stability in the S. bayanus group of distillery yeast strains.
INTRODUCTION
The budding Saccharomyces cerevisiae is the most
scientically and industrially exploited species among the
Saccharomyces sensu stricto complex as it is widely used
as a model organism and in the fermentation processes
such as the production of food and alcoholic beverages
[1, 2]. There are at least seven natural Saccharomyces
sensu stricto species (S. cerevisiae, S. paradoxus, S.
mikatae, S. kudriavzevii, S. arboricola, S. eubayanus
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and S. uvarum) and numerous related industrial hybrids
of a biotechnological interest (e.g., S. cerevisiae x S.
kudriavzevii, S. pastorianus, S. bayanus, S. cerevisiae x S.
mikatae) [1, 3-11]. More recently, S. paradoxus has been
also established as a main yeast component in Croatian
wines that may suggest a potentially important enological
characteristics for this species [12].
The domestication within the Saccharomyces
sensu stricto complex has led to the evolution of special
phenotypic features via hybridization, polyploidization,
gene duplication and gene transfer [2]. The best example
of how fermentative conditions can shape the yeast
genome is the acquiring SSU1-R allele-based resistance
to sulte by wine yeasts [13]. This adaptation is a result
of a reciprocal translocation between chromosomes
VIII and XVI due to unequal crossing-over mediated
by microhomology between very short sequences on
the 5’ upstream regions of the SSU1 and ECM34 genes
that provokes the induction of the SSU1 transporter and
increases the ability of yeast cells to expulse sulte from
the cytoplasm [13]. This genetic change can be found in
50% of the wine strains, whereas it has not been observed
among wild strains suggesting that the use for millennia
of sulte as a preservative in wine production could have
favored its selection [14].
In contrast to the best studied genomes of wine
and brewing yeast strains, the information on genetic
and genomic diversity of yeast isolates involved in the
production of distilled spirits is limited. In the present
study, array-CGH-based genome-wide analysis of
twenty two commercially available distillery yeasts was
conducted. We have revealed four groups with different
pattern of the gene copy number variants that in the case
of the YRF1 gene dosage diversity may provoke changes
in genetic stability.
RESULTS
Electrophoretic karyotyping of distillery yeasts
reveals four species categories
As there are limited number of published data on
genomic and genetic characteristics of distillery yeasts
[15, 16], the karyotype and the genome of, commercially
available and widely used in food industry, twenty two
distillery yeast strains were comprehensively investigated
(Table 1).
On the basis of PFGE separation (electrophoretic
karyotyping), one can conclude that all yeasts examined
belonging to the Saccharomyces sensu stricto complex
[17]. In general, the chromosome number of analyzed
yeasts is 16 (Figure 1). However, an additional band was
Figure 1: Electrophoretic karyotyping of twenty two distillery yeast strains (A, lanes from 1 to 22). The yeast S. cerevisiae
chromosome marker YNN295 (BIORAD) is shown (A., lane M). The dendrogram of chromosome band-based similarity is also presented
B. The species classication within the Saccharomyces sensu stricto complex is provided.
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observed between chromosomes IV and VII in strains
from 1 to 6 and strain 16 and between chromosomes I and
VI in strain 19 (Figure 1). In almost all strains examined,
chromosomes IV and XII migrated together (Figure 1).
The strains from 1 to 6 and strain 16 had the S.
bayanus-like chromosome pattern, whereas strains 7, 8,
10, 11, 12, 13, 14, 15, 17, 18, 20, 21 and 22 were classied
as S. paradoxus, strain 9 as S. cerevisiae and strain 19 as
S. kudriavzevii (Figure 1). A chromosomal band of about
1300 kb (between chromosomes IV and VII) observed in
strains from 1 to 6 and strain 16 is a characteristic feature
of S. bayanus karyotype [18]. Chromosome similarity
between analyzed strains was also further evaluated
using UPGMA clustering (Figure 1). Strains from 2 to 6
were the most similar within assigned S. bayanus group,
whereas strains 1 and 16 differed from other S. bayanus
Figure 2: The ploidy analysis. Fluorescence-activated cell sorting (FACS)-based analysis of DNA content of distillery strains B.
Haploid, diploid, triploid and tetraploid reference strains are also shown A.
Table 1: Distillery yeast strains used in this study.
No. Trade name Company
1Samogon turbo CBF Drinkit
2Superyeast T48 Dual Use CBF Drinkit”
3Spiritferm Extreme 8 kg Turbo Spiritferm
4 Spiritferm T3 Spiritferm
5Spiritferm turbo fruit Spiritferm
6Spiritferm Moskva style Spiritferm
7Coobra 24 Snabbsats CBF Drinkit
8 Coobra 6 Magnum Snabbsats CBF Drinkit
9 Coobra 8 Snabbsats CBF Drinkit
10 Coobra 48 Turbo Yeast CBF Drinkit
11 Coobra RUM YEAST CBF Drinkit
12 Double Snake Turbo Yeast C3 Extra Hambleton Bard Ltd.
13 Alcotec Pure Turbo Super Yeast 48 Hambleton Bard Ltd.
14 Drożdże gorzelnicze Turbo 72h BIOWIN
15 Black Bull Turbo Yeast Avedore Trading
16 Gozdawa 1410 Turbo Gozdawa
17 Superyeast T Vodka Star CBF Drinkit
18 Alcotec Vodka Star Turbo Yeast Hambleton Bard Ltd.
19 Alcotec Single Strain Whisky with Amyloglucosidase Alcotec
20 Fermiol drożdże gorzelnicze BIOWIN/FERMIOL
21 BIOWIN Turbo Super Yeast 48h BIOWIN
22 Alcotec Pure Turbo Super Yeast 24h Hambleton Bard Ltd.
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Figure 3: Comparison of the gene copy number between analyzed distillery yeasts using array-CGH. A. The strains with
similar array-CGH proles were grouped together. Each grey dot represents the value of the log2 ratio for an individual gene. Blue lines
were provided to emphasize the most accented differences (DNA losses and gains). B. The relatedness of distillery strains as determined by
cluster analysis. Similarity tree is shown (see Materials and Methods section for the details).
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strains (Figure 1). Similarly, strains 21 and 22 were more
distant from other S. paradoxus strains (Figure 1).
Distillery yeasts are diploid
The ploidy of distillery strains was then analyzed
using uorescence-activated cell sorting (FACS) (Figure
2).
We found that all strains used were diploid when
compared to reference laboratory yeast cells with known
ploidy (haploid, diploid, triploid and tetraploid cells)
(Figure 2).
The diversity of gene copy number and loci-
specic gains and losses involve mainly the
subtelomeric regions
After electrophoretic karyotyping, the genome of
distillery strains was characterized using array-based
comparative genomic hybridization (array-CGH) (Figure
3).
The analysis of array-CGH proles revealed
Figure 4: The divergence of relative abundance of genes as determined by array-CGH analysis represented by
standard deviation (SD) of log2 ratio values for each gene in all analyzed strains. A. The summary plot for the whole genome.
B. Individual plots for each chromosome. Blue dots indicate the SD values for individual genes, the red line denotes the smoother trend
calculated by moving average of SD values to expose the genome regions of higher log2 ratio divergence and green triangles indicate
centromere position.
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variabilities in the gene copy number exclusively within
the subtelomeric regions of all analyzed chromosomes and
two short intrachromosomal regions of chromosomes IV
and XII (Figures 3A and 4).
The differences between strains were more
accented including the losses and/or gains of fragments
of chromosomes I, III, VI and IX, and in the case of
strain 5 also the changes within chromosome XII (Figure
3A). The gain of chromosomes I and VI in strains 3, 5
and 17, and the loss of chromosomes I and VI in strains
7, 9, 11, 12 and 15 were revealed (Figure 3A). The gain
of chromosome III in strains 3, 5 and 17, and the loss of
chromosome III in strains 7, 9, 11, 12, 14, 15 and 21 were
observed (Figure 3A). The most variable chromosome
was chromosome IX. The gains of chromosome IX were
shown in strains 1, 2, 3, 4, 5, 6 and 16, whereas the losses
of chromosome IX were documented in strains 7, 9, 11,
12, 15 and 19 (Figure 3A). The gains of chromosome XII
was exclusively reported in strain 5. Interestingly, small
chromosomes were frequently affected and changes in one
small chromosome were accompanied by changes in other
small chromosomes. However, these gains and losses were
too small to be interpreted as duplications or deletions of
chromosomal regions or whole chromosome aneuploidy
events within the whole population of particular strain.
Perhaps, the chromosome variations may suggest the
cellular heterogeneity within a population. Additionally,
array-CGH proles were used to estimate the level of
similarity (relatedness) between distillery strains on
the basis of observed diversity in subtelomeric regions
and chromosome IX (Figure 3B). Array-CGH-based
relationships between analyzed strains were comparable
with electrophoretic karyotyping-based relationships
(Figures 1 and 3B). The strains from 1 to 6 and strain 16
already classied as S. bayanus (Figure 1) were clustered
together (Figure 3B). According to both similarity analyses
used, strains 2, 4 and 6, and strains 1 and 16 were closely
located (Figures 1 and 3B). The strains belonging to S.
paradoxus species (Figure 1), were grouped into several
categories using array-CGH-based analysis, namely the
group of the strains 7, 8 and 12; 10 and 21; 13, 14 and
15; 11, 17, 18 and 20 (Figure 3B). The most variable was
strain 22 (S. paradoxus species, Figure 1) with its own
category (Figure 3B).
Gene ontology overrepresentation proles are
species-specic
As the observed differences in the gene copy number
and loci-specic gains and losses may affect the functional
Figure 5: A heat map generated from array-CGH data. Functional categories overrepresented in the group of genes that were the
most divergent among analyzed strains are shown. The strains were ordered according to the result of clustering analysis (Figure 3B) and
the selected genes were grouped according to their functional assignment. Positive and negative log2 ratio values represent higher and lower
than average abundance of the gene, as determined by array-CGH analysis (see Materials and Methods section for the details).
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properties of distillery strains, the genes that were most
divergent according to array-CGH-based analysis were
then subjected to gene ontology overrepresentation
analysis (Figure 5).
The selected gene-set consisted of 257 genes,
for which, in at least one strain, the log2 ratio value
was greater than four standard deviations of log2 ratio
calculated for all genes in all strains. Six functional
categories overrepresented in the group of selected genes
were revealed, namely 1) telomere maintenance via
recombination, DNA helicase activity or DNA binding;
2) maltose metabolism process, glucose transmembrane
transporter activity; 3) asparagine catabolism, cellular
response to nitrogen starvation, localized in cell wall-
bounded periplasmic space; 4) siderophore transport;
5) response to copper ion, cadmium ion binding and 6)
L-iditol 2- dehydrogenase activity (p < 0.05) and are
presented as a heat map in Figure 5. Species-dependent
variability in the gene copy number within functional
categories of selected genes were revealed, e.g., similar
genetic features were observed among strains belonging
to S. bayanus species that differed from genetic features in
the strains of S. paradoxus species (Figure 5). Moreover,
strains 9 (S. cerevisiae) and 19 (S. kudriavzevii) had their
own overrepresentation proles (Figure 5). Interestingly,
within functional category of genes involved in the
telomere maintenance via recombination, DNA helicase
activity or DNA binding, the gains of YRF1 genes
(helicases encoded by the Y’ element of subtelomeric
regions) were exclusively shown in the S. bayanus strain
group and strain 19 (S. kudriavzevii), whereas the losses
of YRF1 genes were observed in the S. paradoxus strain
group (Figure 5). A heat map generated from array-CGH
data reecting the variability in the gene copy number of
the whole genome of all analyzed distillery strains is also
presented in Supplementary File 1.
The YRF1 gene copy number corresponds to the
presence of Y’ telomeric sequences
Since array-CGH-based analysis revealed that
the majority of genomic differences can be found in
Figure 6: The presence of Y’ telomeric sequences in twenty two distillery yeasts (categories from 1 to 22, lane gDNA:
chromosome pattern of an individual strain, lane Y’ seq: Y’ telomeric sequences) detected using Southern blot using
Y’ telomeric sequence probes.
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subtelomeric regions of the genome of distillery strains
and Y’ element ATP-dependent helicase activity may be
affected in the opposite direction in the S. bayanus and
S. paradoxus strain groups (Figures 3, 4 and 5), we then
evaluated the presence of Y’ telomeric sequences in all
examined strains (Figure 6).
Y’ telomeric sequences were the most accented in
the S. bayanus strain group, whereas they were marginally
noticeable in the S. paradoxus strain group (Figure 6).
Southern blot data using Y’ telomeric probes are in
agreement with array-CGH results (Figure 5). The same
relationship was observed for strain 19 (S. kudriavzevii)
with the highest log
2
ratios of YRF1 genes (Figure 5) and
rich in Y’ telomeric sequences (Figure 6).
The YRF1 gene copy number modulates genetic
stability
We hypothesized that altered Y’ telomeric sequence-
dependent helicase activity may modulate genetic stability
in distillery strains. Thus, we also evaluated the strain-
Figure 7: The susceptibility to DNA double strand breaks (DSBs) A. and DNA single strand breaks (SSBs) B. DSBs and SSBs
were assessed using neutral and alkaline comet assay, respectively. The strains belonging to the same species were grouped together and
the data were marked in different colors (S. bayanus: green, S. kudriavzevii: blue, S. cerevisiae: red and S. paradoxus: yellow). As a DNA
damage marker, the % tail DNA was used. The bars indicate SD, n = 150, ***p < 0.001 compared to the S. paradoxus group (ANOVA and
Tukey’s a posteriori test). C. The typical micrographs are shown. DNA was visualized using YOYO-1 staining (green).
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dependent susceptibility to DNA damage (Figures 7 and
8).
Indeed, the S. bayanus strain group (p < 0.001) and
strains 9 (S. cerevisiae) (p < 0.001) and 19 (S. kudriavzevii)
with the abundance of Y’ telomeric sequences and
higher number of YRF1 gene copies were less affected
by DNA double strand breaks (DSBs) and DNA single
strand breaks (SSBs) than the S. paradoxus strain group
(Figure 7). Moreover, the level of oxidative DNA damage
(8-hydroxy-2’-deoxyguanosine, 8-oxo-dG, content) was
increased in the S. paradoxus group compared to the
S. bayanus group (Figure 8). However, the effect was
statistically insignicant. The intracellular production of
reactive oxygen species (ROS) was also elevated in the S.
paradoxus group (p < 0.001) but no clear-cut relationship
between ROS production and the 8-oxo-dG level was
observed in this group, e.g., strains 21 and 22 with the
most imbalanced redox equilibrium were characterized by
relatively low level of 8-oxo-dG (Figure 8). Thus, it might
not be concluded that the elevation in 8-oxo-dG level was
a result of increased ROS production in the S. paradoxus
group.
DISCUSSION
This is the rst report on detailed evaluation of
genomic features of twenty two distillery yeast strains
used in food industry to produce distilled spirits such
as vodka and whisky. To date, one paper has been
published on molecular genetic characteristics of thirty
six distillery yeast strains belonging to the S. cerevisiae
species [15]. The authors performed PCR-RFLP analysis
of rDNA 5.8S-ITS fragment, molecular karyotyping
(PFGE separation), and Southern blot-based detection
of MAL, SUC and MEL genes [15]. Analyzed strains
were aneuploid and rich in polymeric genes SUC and
MAL important for sucrose and maltose fermentation,
respectively [15]. As we have purchased the strains
from multiple suppliers, we are aware that our analyzed
“distillery group” may be more heterogeneous. Indeed, the
strains examined in the present study were more diverse
and belonged to four species of the Saccharomyces sensu
Figure 8: The intracellular reactive oxygen species (ROS) production A. and the level of oxidative DNA damage (8-hydroxy-
2’-deoxyguanosine, 8-oxo-dG, level) B. ROS production was assessed using H2DCF-DA uorogenic probe and the results are presented as
relative uorescence units per minute (RFU/min). The level of 8-oxo-dG was analyzed using ELISA-based assay. The strains belonging to
the same species were grouped together and the data were marked in different colors (S. bayanus: green, S. kudriavzevii: blue, S. cerevisiae:
red and S. paradoxus: yellow). The bars indicate SD, n = 5, ***p < 0.001, **p < 0.01 compared to the S. paradoxus group (ANOVA and
Tukey’s a posteriori test).
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stricto complex, namely S. bayanus (n = 7), S. paradoxus
(n = 13), S. cerevisiae (n = 1) and S. kudriavzevii (n
= 1) according to electrophoretic karyotyping. The
obtained species-specic chromosome patterns were in
agreement with previously reported data on karyotypic
characteristics of reference yeast strains [9, 17-19].
Similar chromosome proles were observed within the S.
bayanus group (strains 1 to 6 and strain 16). However,
one should remember that some karyotypic variants may
also occur within the yeast species. This is particularly
true for the S. bayanus group [6, 19]. S. bayanus var.
uvarum isolates are typically characterized by only two
small chromosomal bands in the range of 245-370 kb
(between chromosomes I and III) instead of three or more
in S. bayanus var. bayanus [6, 19]. The strains assigned
to the S. bayanus group (this study) exhibited karyotypic
features of the S. bayanus var. bayanus. In general, the
analyzed strains were diploid but aneuploid events (the
presence of some additional chromosome bands) were
also observed. It is widely accepted the industrially
relevant yeast strains, e.g., brewer’s and wine yeasts, are
aneuploid with disomies, trisomies and tetrasomies [20,
21]. Alloploidy is also a common phenomenon [1]. The
best and most well-known example of industrial hybrid
is the lager yeast S. pastorianus (syn. S. carlsbergensis),
which is the cold-adapted S. cerevisiae x S. eubayanus
allotetraploid [22]. Under certain conditions, e.g., during
fermentation-associated biotic and abiotic stresses,
aneuploidy events and changes in the ploidy may be
adaptive and advantageous by increasing the number
of copies of benecial genes or by protecting the yeasts
against recessive lethal or deleterious mutations that may
confer resistance to low temperature or high ethanol levels
[20, 23].
Genome-wide array-CGH analysis reveals
variations in the gene copy number almost exclusively in
the subtelomeric regions of the genome of distillery yeasts,
and the most affected chromosomes were the chromosome
I, III, VI and IX. It is worthwhile to note that the strain
relatedness based on array-CGH data was comparable
with electrophoretic karyotyping-based similarities among
strains. Statistically signicant differences in the gene
dosage were observed in six functional gene categories,
namely 1) telomere maintenance via recombination, DNA
helicase activity or DNA binding, 2) maltose metabolism
process, glucose transmembrane transporter activity;
3) asparagine catabolism, cellular response to nitrogen
starvation, localized in cell wall-bounded periplasmic
space, 4) siderophore transport, 5) response to copper ion,
cadmium ion binding and 6) L-iditol 2- dehydrogenase
activity. The effects were species-dependent that may
suggest that strains within distillery group analyzed may
differently respond to changing environments and may
have diverse adaptation strategies. Surprisingly, in almost
all gene categories, the effects observed in the S. bayanus
and S. paradoxus groups were opposite, e.g., increased
and decreased copy number of YRF1 genes (YRF1-1 to
YRF1-7) in the S. bayanus and S. paradoxus group was
shown, respectively. The YRF1 genes (YRF1-1 to YRF1-
7) are localized on different yeast chromosomes within
the Y’ element of subtelomeric regions and encoded Y’
element ATP-dependent helicase (Y’-Help1, Y’-HELicase
Protein 1) implicated in telomerase-independent telomere
maintenance [24]. In laboratory yeasts, Y’-Help1 is highly
induced in the survivors of telomerase decient cells
[24]. It has been speculated that Y’-Help1 may enhance
homologous DNA recombination among Y’ elements and,
as a consequence, may induce Y’ amplication to prevent
chromosomal loss and cell death [24]. We hypothesized
that altered YRF1 gene copy number and the presence
of Y’ elements may affect genetic stability in distillery
strains. Indeed, the strains from the S. paradoxus group
with decreased YRF1 gene dosage and the lack of Y’
sequences were more prone to DNA double and single
strand breaks and oxidative DNA damage than the S.
bayanus group that may inuence the biotechnological
processes using distillery strains. The opposite effect,
namely increased copy number of MEC3 gene encoded a
DNA damage and meiotic pachytene checkpoint protein
[25, 26] was observed in the S. paradoxus group that
may have implications for DNA damage response and
adaptations to DNA-damaging conditions.
The other genes with affected copy number
were mainly involved in carbohydrate and amino acid
metabolism, and ion transport that may also modulate a
biotechnological process. The dosage of numerous genes
implicated in maltose metabolism was affected (e.g.,
MAL11, MAL13, MAL31, MAL33, MPH2 and MPH3).
The MAL gene family of Saccharomyces is comprised
of ve multigene complexes, MAL1, MAL2, MAL3,
MAL4 and MAL6, located at or near the telomere of a
different chromosome, any one of which is sufcient
for yeast to metabolize the disaccharide maltose and
encodes maltose permease (GENE l), maltase (GENE
2) and the trans-acting MAL-activator (GENE 3) [27].
MAL11 and MAL13 are part of the MAL1 complex
locus located on chromosome VII and encode high-
afnity maltose transporter (α-glucoside transporter) and
MAL-activator protein, respectively, whereas MAL31
and MAL33 are part of the MAL3 complex located on
chromosome II and encode maltose permease and MAL-
activator protein, respectively [28, 29]. It has been
suggested that the MAL loci have been translocated to
different chromosomes via a mechanism that involved
the rearrangement(s) of chromosome termini [30]. MPH2
and MPH3 genes (maltose permease homologs) encode
α-glucoside permeases that transport maltose, maltotriose,
α-methylglucoside, and turanose [31].
The distillery strains also differed in the copy
number of ASP3 genes, especially highly elevated
ASP3 gene copy number was revealed in strain 19 (S.
kudriavzevii). ASP3 contains a gene cluster located on
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chromosome XII comprised of four identical genes,
ASP3-1, ASP3-2, ASP3-3, and ASP3-4, which encode
for cell wall-associated L-asparaginase II that catalyzes
the conversion of L-asparagine to aspartate and ammonia
[32]. Asp3p is induced in response to nitrogen starvation
and regulated by Gln3p/Ure2p [33]. More recently, the
ASP3 locus has been shown to be originated by horizontal
gene transfer from Wickerhamomyces [34]. It has been
speculated that ASP3 acquisition may have aided yeast
adaptation to articial environments and may further
highlight the importance of gene sharing between yeasts in
the evolution of their remarkable metabolic diversity [34].
The most accented differences were observed in
the copy number of SOR1 and SOR2 genes. The SOR1
gene encode a NAD-dependent sorbitol dehydrogenase
that is a member of the polyol dehydrogenase branch
of the medium-chain dehydrogenase/reductase (MDR)
superfamily of enzymes [35]. It has been reported that
the expression of SOR1 gene is elevated in the presence
of sorbitol or xylose, though S. cerevisiae is a non-
xylose-utilizing microorganism [35, 36]. Similarly, high
variability in the gene copy number of genes involved
in the siderophore transport, namely ENB1, FRE3,
FRE5, FIT2 and FIT3, was observed. They represent
two genetically separable systems for the uptake of
siderophore-bound iron in S. cerevisiae. One system
is based on family of transporters that is expressed as
part of the AFT1 regulon and are termed ARN1, ARN2
(TAF1), ARN3 (SIT1) and ARN4 (ENB1) [37, 38]. These
transporters are expressed in intracellular vesicles [39].
The second system relies on the high afnity ferrous
iron transport complex, which is encoded by FET3 and
FTR1 and is located on the plasma membrane [40, 41].
Ferric reductases encoded by FRE genes take part in iron
uptake by the reduction of siderophore-bound iron prior
to uptake by transporters [42, 43]. There are also three
cell wall mannoproteins (Fit1, Fit2, Fit3) that facilitate
the uptake of iron [44]. Low iron levels stimulate the
expression of components of both systems [45]. Perhaps,
increased copy number of genes involved in the transport
of siderophore-bound iron in the S. paradoxus group
may be advantageous in the certain growth conditions,
e.g., during iron deprivation. Additionally, in all groups
analyzed, the metallothionein gene dosage CUP1-1 and
CUP1-2 was increased that was the most accented in
strain 9 (S. cerevisiae). This may be also benecial as may
confer resistance to copper and cadmium [46].
In conclusion, we have provided for the rst
time array-CGH-based comprehensive genomic
characterization of commercially available twenty two
distillery yeast strains. We have documented the naturally
occurring diversity in the gene copy number within six
functional gene categories and revealed that the variations
in the YRF1 gene copies may be accompanied by altered
genetic stability in the analyzed yeast groups. Our
genomic data may be helpful for better understanding of
the fermentative environment-mediated changes in the
yeast genome and accompanying phenotypic features.
Thus, the knowledge on genetic diversity of distillery
strains may be further exploited in economically important
biotechnological processes.
MATERIALS AND METHODS
Reagents
All reagents, if not otherwise mentioned, were
purchased from Sigma (Poland) and were of analytical
grade.
Yeast strains and growth conditions
All distillery yeast strains used in this study are
listed in Table 1. Yeast from one single colony was grown
either on liquid YPD medium (1% w/v Difco Yeast
Extract, 2% w/v Difco Yeast Bacto-Peptone, 2% w/v
dextrose) or on solid YPD medium containing 2% w/v
Difco Bacto-agar, at 28 °C.
Pulsed-eld gel electrophoresis (PFGE)
Preparation of agarose-embedded yeast DNA
and PFGE separation of yeast DNA were conducted
as described elsewhere [47]. The dendrogram of
chromosomal DNA-based similarity was created using
Free-Tree software [48] using UPGMA (Unweighted Pair
Group Method with Arithmetic Mean) algorithm, Jaccard
similarity coefcient and Java TreeView 1.1.6.r2 (http://
jtreeview.sourceforge.net/).
FACS-based ploidy analysis
The DNA content was measured via ow cytometry
as previously described [49] except that a total of 3x104
cells were counted in a single assay.
Array-based comparative genomic hybridization
(array-CGH)
Genomic DNA (0.5 μg) was labeled with SureTag
DNA Labeling Kit and either Cy3- or Cy5-dUTP. Equal
amounts of labeled DNA of tested and of the reference
strain (BY4741) were combined and hybridized to
Yeast (V2) Gene Expression Microarray, 8x15K using
Oligo aCGH Hybridization Kit. All components were
supplied by Agilent Technologies Inc. (Santa Clara, CA,
USA) and all steps of the experiment were performed
according to manufacturer’s protocols. Following
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hybridization and washing, the slides were scanned using
Axon GenePix 4000B. Feature extraction was conducted
using GenePix Pro 6.1 and normalization using Acuity
4.0 (Molecular Devices, Sunnyvale, CA, USA). CGH
proles with superimposed piecewise regression plots
to highlight aneuploidies, were generated using CGH-
Explorer v3.2 [50]. The original CGH proles obtained
after the comparison of analyzed strains to BY4741 gave
consistently high noise due most probably to genomic
DNA sequence differences between BY4741 and the
industrial strains, which inuenced the hybridization
strength of individual probes. Therefore to obtain nal
CGH proles, the data for each strain were compared to
the average of all industrial strains used in the experiment.
Gene analysis after array-CGH
The analysis of over-representation of functional
categories was performed using Cytoscape v. 2.8.2 with
BiNGO v. 2.44 plug-in and hypergeometric test using
Benjamini and Hochberg False Discovery Rate (FDR)
correction and signicance level of 0.05.
Cluster analysis
The array-CGH data for all strains were subjected
to complete linkage clustering with Cluster 3.0 software
using Euclidean distance similarity metrics [51]. To obtain
the tree graph of similarity, the clustering output was
visualized using Java TreeView 1.1.6.r2 (http://jtreeview.
sourceforge.net/).
Detection of telomeric Y’ sequences
Y’ element telomeric probe was obtained according
to [52] with minor modications. After standard
PFGE separation, Y’ sequences within particular yeast
chromosomes were detected using digoxigenin labeling,
anti-digoxigenin antibody and phosphate alkaline-based
chemiluminescence [53].
Comet assay
Yeast spheroplasts were obtained [47] and DNA
double-strand breaks (DSBs) and DNA single-strand
breaks (SSBs) were assessed by neutral and alkaline
single-cell microgel electrophoresis (comet assay),
respectively, as described elsewhere [54]. The percentage
of tail DNA was used as a parameter of DNA damage.
Oxidative stress parameters
Intracellular reactive oxygen species
(ROS) production was measured using
2’,7’-dichlorodihydrouorescein diacetate (H2DCF-DA)
as described elsewhere [53]. Oxidative DNA damage as a
level of 8-hydroxy-2’-deoxyguanosine (8-OHdG, 8-oxo-
dG) was measured using Epigentek EpiQuik 8-OHdG
DNA Damage Quantication Direct Kit (Gentaur,
Poland) using the standard protocol according to the
manufacturer’s instructions.
Statistical analysis
The results represent the mean ± SD from at least
three independent experiments. Statistical signicance was
assessed by 1-way ANOVA using GraphPad Prism 5, and
with the Tukey’s multiple comparison test.
ACKNOWLEDGMENTS
We are indebted to Prof. Martin Kupiec and Dr
Yaniv Harari (Tel Aviv University, Israel) for sharing with
us the protocol on the construction of Y’ telomeric probes.
CONFLICTS OF INTEREST
No potential conicts of interest were disclosed.
GRANT SUPPORT
This work was supported by European Union,
within Regional Operational Programme of Subcarpathia
Voivodeship (2007-2013), Priority 1: Competitive and
Innovative Economy, Action 1.3, Regional Innovation
System, grant WND-RPPK-01.03.00-18-038/13.
Authors’ contributions
Conceived and designed the experiments: MW.
Performed the experiments: AD MS JA AK ER AS
AL MW. Analyzed the data: MS AL MW. Contributed
reagents/materials/analysis tools: AL MW. Wrote the
paper: AL MW.
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