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Rate of spontaneous polyploidization in haploid yeast Saccharomyces cerevisiae
Yulia Andreychuk1, Anna Zhuk2, Elena Tarakhovskaya13, Sergei Inge-Vechtomov14, and Elena Stepchenkova14
1Vavilov Institute of General Genetics, Russian Academy of Sciences, ul. Gubkina, 3, Moscow, 117971, Russian Federation
2ITMO University, Kronverkskiy pr., 49, Saint Petersburg, 197101, Russian Federation 3Department of Plant Physiology and Biochemistry, Faculty of Biology, Saint Petersburg State University, Universitetskaya nab., 7-9, Saint Petersburg, 199034, Russian Federation
4Department of Genetics and Biotechnology, Faculty of Biology,
Saint Petersburg State University, Universitetskaya nab., 7-9, Saint Petersburg,
199034, Russian Federation
Address correspondence and requests for materials to Yulia Andreychuk, yulianndreychuk@gmail.com
Abstract
Citation: Andreychuk,Yu., Zhuk, A., Tarakhovskaya, E., Inge-Vechtomov, S., and Stepchenkova, E. 2022. Rate of spontaneous polyploidization in haploid yeast Saccharomyces cerevisiae. Bio. Comm. 67(2): 88-96. https://doi.org/10.21638/ spbu03.2022.202
Authors' information: Yulia Andreychuk, PhD Student, orcid.org/0000-0003-0957-9840; Anna Zhuk, PhD, Researcher, orcid.org/0000-0001-8683-9533; Elena Tarakhovskaya, PhD, Associate Professor, orcid.org/0000-0002-5341-2813; Sergei Inge-Vechtomov, RAS Academician, Dr. of Sci. in Biology, Professor, orcid.org/0000-0002-2832-6825; Elena Stepchenkova, PhD, Head of Laboratory, orcid.org/0000-0002-5854-8701
Manuscript Editor: Anna Malashicheva, Laboratory of Regenerative Biomedicine, Institute of Cytology, Russian Academy of Sciences, Saint Petersburg, Russia; Laboratory of Molecular Cardiology, Almazov National Medical Research Centre, Saint Petersburg, Russia
Received: September 29, 2021;
Revised: January 31, 2022;
Accepted: February 3, 2022.
Copyright: © 2022 Andreychuk et al. This is an open-access article distributed under the terms of the License Agreement with Saint Petersburg State University, which permits to the authors unrestricted distribution, and self-archiving free of charge.
Funding: The reported study was funded by the Russian Foundation for Basic Research according to the research project No. 20-34-90040. AZ was supported by the Government of the Russian Federation through the ITMO Fellowship and Professorship Program.
Ethics statement: This paper does not contain any studies involving human participants or animals performed by any of the authors.
Competing interests: The authors have declared that no competing interests exist.
Polyploidization is involved in a variety of biological processes. It is one of the principal mechanisms of new species formation since it provides material for gene diversification and subsequent selection. Multiple cases of polyploidization were registered in different branches of the evolutionary tree of eukary-otes. Besides its role in evolution, polyploidization affects gene expression in living cells: pathological genome duplications often happen in cancer cells. The mechanisms and consequences of polyploidization are being studied extensively. However, quantitative determination of the polyploidization rate is challenging due to its low frequency and the absence of selective genetic markers that would phenotypically distinguish between haploids and polyploids. Our work describes a robust and straightforward method for discriminating haploid and polyploid states in the yeast Saccharomyces cerevisiae, a model organism for studying different aspects of polyploidization. The measurement of polyploidization rate showed that in yeast cells this process is mainly caused by autodiploidization rather than mating-type switching followed by hybridization. Keywords: Saccharomyces cerevisiae, polyploidization, haploid, diploid, whole-genome duplication
Introduction
Polyploidization occurs during rounds of duplication of the entire set of chromosomes; thus, polyploid organisms have additional copies of the original genome. Individuals with two copies of the genome are called diploids; with three copies — triploids, etc. Hugo de Vries first described polyploidization as a genomic mutation and suggested the theory of mutations based on polyploid mutants in Oenothera (DeVries, 1915). Polyploidy is common in nature — up to 35 % of vascular plants are polyploids (Meyers and Levin, 2006; Wood et al., 2009), and poly-ploid species were found among all major taxa of animals, in particular, among fishes (Leggatt and Iwama, 2003) and amphibians (Schmid et al., 2015). Ploidy variation is also typical for many yeast strains, including Saccharomyces cerevisiae (Zhu et al., 2016). Polyploidization plays a key role in evolution as a mechanism of new species formation; it provides a source of new genomes and material for gene specification (Ohno, 1970; Taylor, Van de Peer and Meyer, 2001; Crow and Wagner, 2006). In somatic tissues, the increased number of all chromosomes, caused by polyploidization, results in the enhancement of gene expression. Some extreme examples of somatic genome amplification are polythene chromosomes
of insects (Zhimulev et al., 2004). Polyploid cells have been revealed in higher vertebrates in hepatocytes, cardiac myocytes, arterial smooth muscle cells, lactating mammary glands, and decidual cells in the uterus during embryo implantation. Besides contributing to normal physiological processes, polyploidization is involved in some pathological situations: ploidy of cells increases during chronic inflammation, wound healing, or viral infection. Polyploidization also contributes to tumor progression (Sroga, Ma and Das, 2012; Orr-Weaver, 2015; Rios et al., 2016; Matsumoto et al., 2021). Since the increase in chromosome number leads to the increase of both the nucleus and whole-cell size, polyploid organisms are typically larger than their lower-ploidy ancestors. This phenotype is widely exploited in plant breeding, and most modern cultivated plants are polyploids (Feng et al., 2021; Ferrao et al., 2018).
One of the mechanisms of polyploidization is whole-genome duplication (WGD), or autodiploidiza-tion, when the replicated chromosomes fail to separate during cell division, resulting in polyploid progeny. New WGD rounds of a polyploid genome lead to further increase of ploidy. Autopolyploidization is supposed to have happened repeatedly in the evolutionary history of vertebrates and plants (Meyer and Shartl, 1999; Soltis, Marchant, Van de Peer and Soltis, 2015). Another source of polyploidy is interspecies hybridization, occasionally occurring in animals, plants, and microorganisms such as yeast (Whitney et al., 2010; Morales and Dujon, 2012; Cahill et al., 2015; Zhung et al., 2016; Figueiro et al., 2017). Most interspecies hybrids are initially sterile because they produce chromosome-imbalanced gametes, but after further genome autoduplication fertile individuals emerge, which can evolve into a new species (Kar-pechenko, 1928; Charron et al., 2019). Allopolyploidi-zation accompanied by WGD is supposed to be one of the main mechanisms of speciation among eukaryotes (Villanea and Schraiber, 2019; Marcet-Houben and Ga-baldon, 2015).
The yeast S. cerevisiae is a relevant model organism to investigate mechanisms of polyploidization because haploid, diploid, and polyploid forms represent natural, industrial, and laboratory S. cerevisiae strains. The S. cerevisiae life cycle includes the alternation of haploid and diploid stages: haploid cells of the opposite mating types (a and a) form non-mating (a/a) diploid hybrid cells, which further undergo meiosis and thus restore the haploid state. Typically, meiosis is stimulated by starvation (Kerr et al., 2012).
There are homo- and heterothallic strains of S. cerevisiae, and they differ in the relative duration of the hap-loid and diploid stages. Homothallic strains are diploids for almost the entire life cycle, with the haploid state represented mainly by ascospores. Haploid cells originating from ascospores in homothallic strains can easily
switch their mating type to the opposite, and then mate to form diploid cells. The mating type of S. cerevisiae is controlled by the MAT locus mapped in the right arm of chromosome III. The MAT locus contains one of two alternative sequences — MATa or MATa determining a and a mating types, respectively (Lee and Haber, 2015). Besides the MAT locus, chromosome III contains two silent cassettes (HMLa and HMRa) also bearing information about the mating type. Cells of the homothallic yeast strains possess endonuclease HO, which makes a double-strand break in a specific site of the MAT locus. During the subsequent programmed gene conversion between the active MAT locus and one of two silent cassettes, HMLa or HMRa, genetic material in the MAT locus is replaced by one of the two cassettes containing information about the opposite mating type (Haber, 1992; Klar, 2010). As a result, the cells undergo mating type switching and become able to mate with neighboring cells of the opposite mating type. Unlike homothallic strains, heterothallic ones lack endonuclease HO activity and thus can maintain their haploid state for an unlimited time. This capacity makes them a suitable model for studying the mechanisms of polyploidization unrelated to the normal life cycle of yeast.
Since the cells of heterothallic strains are haploid, the simplest and the most frequent variant of polyploidization for them is diploidization. It is known that yeast heterothallic haploid cells can diploidize by two mechanisms — hybridization and WGD (Fig. 1) (Edgar and Orr-Weaver, 2001; Lee and Haber, 2015).
The mechanism of heterothallic strains hybridization is based on the mating type switching, which in the absence of HO activity has a very low frequency (Meiron, Nahon and Raveh et al., 1995). This rare event can be detected due to the appearance of "illegitimate" hybrids during the crossing of the two strains with the same mating type. It can happen due to various genetic changes in the MAT locus (Repnevskaya, 1987; Inge-Vechtomov and Repnevskaya, 1989). a^a switching occurs when the MATa locus is lost, inactivated due to mutations or temporary lesions, or replaced with the HMRa cassette — in all these cases a-specific genes are expressed constitutively. a^a switching occurs when MATa is replaced by the silent cassette HMLa. The frequencies of different genetic events leading to mating type switching a^a were studied previously in the a-test (Kochenova et al., 2011). According to our previous results, the frequency of spontaneous "illegitimate" hybridization caused by the mating type switching a^a in heterothallic strains is approximately 10-6 (Inge-Vechtomov et al., 1986; Kochenova et al., 2011; Stepchenkova et al., 2011; Zhuk et al., 2020).
The frequency and mechanism of WGD remains unclear. Establishing the polyploidization rate in S. cerevisiae requires screening of thousands of individual colonies,
Fig. 1. The mechanisms of diploidization in haploid yeast. (a) Normal cell cycle. During the normal cell cycle, the chromosomes are replicated during the S-phase, and then doublet chromosomes equally separate into the daughter cells in mitosis. (b) Hybridization. One of the maternal cells switches its mating type due to different genetic events related to the MAT locus, copulates with a neighbor cell, and produces a diploid hybrid. As a result, the hybrid contains two copies of genomes, heterozygous at the MAT locus. (c) Autopolyploidization. The chromosomes are replicated during the S-phase but fail to separate during cell division. During WGD, the maternal haploid cell produces diploid progeny, which is the absolute homozygote.
which is challenging because there are no direct selective markers of polyploids. Though several methods of ploidy identification have been described, not all of them are relevant for measuring the polyploidization rate (Table 1). Recently, Harari et al. suggested an indirect statistical approach and estimated the polyploidization rate in yeast in a long-term in-lab evolutionary experiment by monitoring the genome size of cells in several cultures during 100 generations using flow cytometry (FC) (2018). The value of the WGD rate varied from 5.3 x 10-5 to 9 x 10-5 in MATa and MATa strains, correspondingly. The
disadvantage of this approach is that the polyploids in yeast culture could be revealed only when their proportion was high enough to be detected by FC (a minimum of several percent). Another problem is the duration of the experiments. The relatively high ratio of polyploids in the culture might mean that polyploids had an adaptive advantage over haploids, or it could be the result of their appearance at an early stage of cultivation and the resulting production of numerous progeny. Therefore, the value of the polyploidization rate obtained in long-term evolutionary experiments may be modified by indirect factors.
Table 1. Methods of identification of ploidy states in S. cerevisiae
Method of polyploid detection Selective criteria Frequency or rate of polyploidization Reference
Flow cytometry Genome size =10-4 Harari et al., 2018
Sensitivity to y-radiation The efficiency of DNA-strand breaks repair by homologous recombination ND Mortimer, 1958; Lobachevskii, Cherevatenko and Mishonova, 1988
Mutation accumulation test Decrease in survival after mutagen exposure due to accumulation of recessive mutations in the genome ND Sharp, Sandell and Otto, 2018
Colony color essay Red/Pink/ White selection The degree of colony pigmentation in a strain containing the ade3 null mutation in its genome and the ADE3 allele on a centromere plasmid depends on the number of plasmid copies in relation to the number of genomic copies of the ade2. ND Baum, Yip, Goetsch and Byers, 1988
Loss of heterozygosity Frequency of mutant colonies' appearance due to loss of heterozygosity 0-75 % during plasmid transformation Karpova et al., 1984
Canavanine papillation ploidy test Frequency of appearance of recessive canavanine-resistant colonies on a selective media after UV irradiation ND Schield, Ananthaswamy and Mortimer, 1981
Genetic analysis Ability to produce viable spores ND Schield, Ananthaswamy and Mortimer, 1981
ND — not determined.
Here we suggest a simple, rapid, and straightforward method for the identification of polyploids in a culture of a haploid S. cerevisiae strain of the a mating type, building on the approach of Shield et al. (1981). This method undeniably discriminates between hap-loids and polyploids by the phenotype of yeast colonies. It is based on the difference in the frequency of recessive UV-induced Canavanine-resistant mutants in haploid and polyploid strains bearing the wild-type CAN1 gene. In our work, we have estimated the rate of polyploidization in overnight S. cerevisiae cultures and concluded that most diploids originated as a result of autodiploidi-zation since the frequency of "illegitimate" hybridization was dramatically lower.
Materials and methods
Strains. All the strains used in this work were from the Peterhof yeast collection. Strain K5-35V-D924-ade1-14 (MATa lys5::KanMX ura3A leu2A met15A ade1-14 cyhr [P7N+]) was used to measure polyploidization rate; strains 2G-P2345 (MATa his5) and 78А-P2345 (MATa his5) were used as mating-type testers. Strain D926 (MATa//MATa ade1A//ADE1 lys2A//lys2A his3Allhis3 Aura3A//ura3A leu2A//leu2A thr4A//thr4A) was used as a diploid control.
Media. Yeast strains were incubated in YEPD, rich broth (1 % yeast extract, 2 % Peptone, 2 % glucose). Solid
YEPD media contained 2 % agar. The selective media for discrimination between haploid and polyploid cells was minimal media (MD) (Zakharov et al., 1984) containing L-canavanine (40 mg/L) and uracil, adenine, lysine, leucine, methionine at corresponding final concentrations 20 mg/L, 20 mg/L, 30 mg/L, 60 mg/L, 150 mg/L.
Identification of polyploid strains. The analyzed cultures were grown overnight at 30 °C, then diluted to the appropriate concentration and placed on YEPD plates to obtain individual colonies. Each colony was then transferred to a fresh YEPD plate at a square about 0.5 cm2 and grown overnight. Then the plates were replicated on the selective media containing L-canavanine and exposed to UV at a dose of 40 J/m2. After five days of incubation at 30 °C, colonies unable to form canavan-ine-resistant (Canr) colonies (non-mutable phenotype) were selected (see Fig. 2). Later, the ploidy of the selected colonies was confirmed by the FC method.
Measuring polyploidization rate. The rates of polyploidization were measured by the fluctuation test in experiments with sets of six independent cultures. The polyploidization rate was calculated from polyploidiza-tion frequency (the ratio of the polyploids to the total amount of living cells in each culture) using the Drake equation (Drake, 1991). Data from six independent cultures were used to calculate the median and 95 % confidence intervals for the median as described (Dixon and Massey, 1969).
Fig. 2. The scheme of the experiment. The upper part: A suspension of cells (overnight culture) was diluted and plated on a YEPD media to obtain individual colonies, which were then plated as small patches on a fresh YEPD plate. After one day of incubation at 30 °C, the plates were replicated on the selective media containing canavanine and exposed to UV irradiation. In five days, haploid colonies form Can-resistant colonies at the replica, while no resistant colonies could be seen in polyploids. The lower part: the appearance of Canr colonies after UV irradiation. Patches of haploid clones have multiple Canr papillae, potential di- or polyploid clone does not produce recessive Canr mutations (circulated by the yellow line).
Flow cytometry. FC analysis was performed in the Saint Petersburg State University Research Park in the Center of Molecular and Cell Technologies using a flow cytometer and sorter BD FACS Arialll. The cells were collected and fixed in 70 % ethanol overnight at 4 °C. Fixed cells were washed with water and then treated by RNase for 6-15 hours at 37 °C and Proteinase K for 1 hour at 50 °C. The pellet cells were diluted in 50 mM Tris buffer pH = 7.5 to an OD 600 nm of 0.1-0.2, sonicated for 2-5 min at 40 kHz, and stained by Prop-idium Iodide (PI) at a final concentration of 5 ^g/ml. Samples were stored in the dark at 8 °C until analysis. The 100,000 cells per sample were analyzed with a laser at 488 nm using filter 616/23 nm to detect the PI fluorescence. The Flow Jo software was used to create the histograms of fluorescence intensity.
Results and discussion
The method we used in this study to evaluate the dip-loidization rate in yeast cells is based on a qualitative difference in the frequency of recessive Canr mutations after UV-irradiation in clones of haploid and polyploid strains of S. cerevisiae. The CAN1 locus determines sensitivity to the toxic arginine analog L-canavanine. This
genetic marker is a widespread standard reporter gene in mutational tests. The haploid strains with a single wild-type allele of the CAN1 gene form Canr colonies with low frequency (~10-7). The presence of the second wild-type copy of the CAN1 gene in homozygous dip-loids drastically decreases the frequency of Canr colony formation, because it demands two steps of inactivation of both CAN1 copies. The first step is the inactivation of one of the two copies of CAN1 due to mutation (base substitutions or frameshift mutations). Inactivation of the second copy of CAN1 usually happens through allel-ic crossover, gene conversion, or chromosome loss with the rate of about 10-4, so the predicted rate of appearance of mutation in diploids is ~10-11 (Gordenin and Inge-Vechtomov, 1981; Pavlov et al., 1988; Ohnishi et al., 2004; Lada et al., 2013). The frequency of Canr mutations in polyploid strains bearing three and more copies of the wild-type CAN1 is even lower. Thus, the mutant Canr colonies derive more frequently from haploid cells than from polyploids. The difference between haploid and diploid strains becomes more visible after UV irradiation: in haploids the frequency UV-induced Canr mutations increases by several orders of magnitude, while diploid and polyploid colonies produce almost no Canr mutants.
ing the weakness of long-term experiments discussed in the introduction. The few non-mutable colonies which turned out to be haploids might have appeared due to duplication of the CAN1 locus or mutation in the genes encoding for the mutasome components (Halas et al., 2021). Thus, though the suggested method looks robust enough for rapid routine analysis of ploidy, it should be used in combination with FC when high accuracy of polyploid identification is needed.
Two processes may lead to the appearance of poly-ploid cells in a haploid yeast culture: WGD and mating-type switching followed by hybridization with a neighboring cell. During WGD, polyploids contain identical genome copies, including the MAT locus. Consequently, such polyploids have the same mating type as maternal haploid cells (MATa//MATa or MATa//MATa). "Illegitimate" hybridization demands preceding mating-type switching, so the hybrids that appeared in the yeast culture of a mating type may have either a- (MATa//mata and MATa//mataA) or non-mating phenotype (MATa// MATa). To assess the relative contribution of WDG and hybridization to polyploid formation, we identified the mating type of selected non-mutable colonies (Table 2).
Table 2. Mating type of selected non-mutable clones of
different ploidy
Fig. 3. FC profiles of yeast strains. Histogram of DNA content in cells stained by propidium iodide (PI-A). The histogram reflects the total number of cells in a sample with PI fluorescence corresponding to 1c, 2c, 4c or 8c DNA content.
Figure 2 represents the scheme of the experiment revealing the ploidy of yeast cells in the overnight culture. Using this technique, we analyzed approximately 1500 colonies from each of 6 independent cultures (9266 colonies total) of the haploid strain K5-35B-D924-ade1-14 and selected 48 UV non-mutable colonies. The ploidy of the selected colonies was checked by FC, and 39 of them turned out to be diploids, 1 — tetraploid, and 8 — haploids; i.e., polyploids were detected in 83 % of immutable clones. Considering only confirmed diploids and one tetraploid, we calculated the spontaneous rate of polyploidization in each of the six cultures and estimated that the median and 95 % confidence interval of diploidization rate was 6.2 x 10-5 (5.1 x 10-5 — 36.4 x 10-5). The FC profiles of two polyploids obtained in the experiment, diploid and haploid controls, are presented in Fig. 3.
The rate of polyploidization measured in our study is very close to the value obtained by Harari et al. (2018). The method used in our work considers the phenotypes of colonies that originated from single cells, thus exclud-
^^^^^^mating type ploidy a a n/m
haploid 3 5 0
diploid 38 0 1
tetraploid 1 0 0
Most selected diploids, as well as the tetraploid, had an a mating type. Thus, they could appear as a result of autoduplication or "illegitimate" hybridization. Moreover, only one diploid was non-mating, originating due to heritable mating type switching. Among the selected non-mutable haploids there were cells with a-mating type. They might have appeared due to the mating type switching and incomplete mating. According to our previous results (Inge-Vechtomov et al., 1986; Kochenova et al., 2011; Stepchenkova et al., 2011), the frequency of "illegitimate" hybridization in the same strain is approximately 10-6, which is about 100 times lower than the polyploidization rate. Thus, we can assume that mating type switching and successive hybridization is a much rarer event than genome autoduplication. The results shown in Table 2 do not contradict this assumption. Ha-rari et al. (2018) made the same conclusion. Considering two different mechanisms of the origin of diploids, they analyzed the sequences of the MAT locus from isolated diploid cells to estimate the rate of both autoduplication and mating type switching and subsequent hybridization. The estimated values were «10-4 and «10-7, respec-
tively, and the authors reasonably concluded that poly-ploidization occurred more likely via autoduplication.
Multiple environmental factors can influence the polyploidization rate in a yeast culture. Haploids have a selective advantage over polyploids in the presence of caffeine and hydroxyurea (Harari et al., 2018). Ethanol is a well-known factor provoking polyploidization, and many industrial yeast strains, selected to produce beverages with up to 12 % ethanol, are polyploids (Turanli-Yildiz et al., 2017). The other polyploidy inducers are high hydrostatic pressure and KCl (Hamada et al., 1992; Harari et al., 2018). It is known that stressful factors can trigger polyploidization in the hepatocytes of mammals (Gentric and Disdouets, 2014). The mechanism and the consequences of stress-induced polyploidization are still not well understood. Some researchers suppose that polyploidization can protect cells from genotoxic damaging factors (Lin et al., 2020), while others suggest that proliferating polyploid cells can provoke cancer development (Davoli and de Lange, 2011; Matsumoto et al., 2021).
As all organisms using sexual reproduction have an alternation of haploid and diploid phases in their life cycle, ploidy is under strict genetic control. A robust and straightforward method of polyploid identification, which we have used in our work, can help to reveal the genes controlling ploidy and to study the molecular mechanisms of polyploidization. The genetic changes leading to the increased rate of polyploidization should be associated with the genes controlling different steps of cell division. To date, 16 genes contributing to the increase of polyploidization level have been found in the S. cerevisiae genome. BBP1, CDC31, ILP1, KAR1, MPS1, MPS2, MPS3, and NDC1 encode for proteins participating in the control of spindle polar body duplication (Shield et al., 1981; Rose and Fink, 1987; Winey et al., 1991; Chan and Botstein, 1993; Xue et al., 1996; Chen et al., 2000; Jaspersen et al., 2002; Jaspersen et al., 2006); MOB1 is required for cytokinesis and cell separation (Luca and Winey, 1998); BEM2 is involved in the control of cytoskeleton organization (Chan and Botstein, 1993); WHI3 is involved in the cell-cycle control (Shladebeck and Mosch, 2013); ESP1 and BIR1 regulate chromosome segregation (Baum et al., 1988; Li et al., 2000). The other genes encode for proteins indirectly participating in chromosome duplication and segregation: DEF1 is RNAPII degradation factor (Stepchenkova et al., 2018), KAP123 mediates nuclear import of ribosomal proteins and import of histones H3 and H4 (Ptak et al., 2009), and BFR1 is necessary for nuclear secretion (Xue et al., 1996).
The molecular mechanisms controlling the genome size are still unclear and require new methods for further investigation. Establishing a direct, simple method for polyploid identification in the S. cerevisiae model
organism may contribute considerably to elucidating the mechanisms of cancer development, drug resistance of pathogens, and the evolution of species.
Acknowledgments
We thank the Research Park of St Petersburg State University for assistance with flow cytometry assays. We also thank Dr. Youri I. Pavlov (UNMC, USA) for his critical reading of the manuscript.
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