The mei3 region of the Schizosaccharomyces pombe genome

Expression of the mei3 gene is sufficient to induce meiosis in the fission yeast Schizosaccharomyces pombe. The mei3 gene is located 0.64 Mb from the telomere of the left arm of Sz. pombe chromosome II. We have sequenced and analysed 107 kb of DNA from the mei3 genomic region. The sequence includes 14 known genes (bag1-B, csh3, dps1, gpt1, mei3, mfm3, pac1, prp31, rpl38-1, rpn3, rti1, spa1, spm1 and ubc4) and 26 other open reading frames (ORFs) longer than 100 codons: a density of one protein-coding gene per 2.7 kb. Twenty-one of the 40 ORFs (53%) have introns. In addition there is one lone Tf1 transposon long terminal repeat (LTR), tRNA(Trp) and tRNA(Ser) genes and a 5S rRNA gene. 14 of the novel ORFs show sequence similarities which suggest functions of their products, including a coatomer alpha-subunit, a catechol O-methyltransferase, protein kinase, asparagine synthetase, zinc metalloprotease, acetyltransferase, phosphatidylinositol 4-kinase, inositol polyphosphate phosphatase, GTPase-activating protein, permease, pre-mRNA splicing factor, 20S proteasome component and a thioredoxin-like protein. One predicted protein has similarity to the human Cockayne syndrome protein CSA and one with human GTPase XPA binding protein XAB1. Three ORFs are likely to code for proteins because they have sequence similarity with hypothetical proteins, three encode predicted coiled-coil proteins and four are sequence orphans.     

Hyperthermotolerant fission yeast mutations, sow1 and sow2, suppress the cell cycle defect and stress sensitivity of MAP kinase kinase wis1Delta

Wis1 is a mitogen-activated protein kinase kinase (MAPKK) that regulates mitosis and mediates stress responses in the fission yeast, Schizosaccharomyces pombe. wis1Delta strains are viable but stress-sensitive and show a mitotic delay. At high temperatures, wis1Delta cells cease division but cellular growth continues. Mutations that suppress the heat sensitivity of a wis1Delta strain were isolated and map to two apparently novel loci, sow1 (for suppressor of wis1Delta) and sow2. In addition to suppressing wis1Delta heat sensitivity, sow1 and sow2 can suppress wis1Delta osmosensitivity and cell cycle defects. sow1 and sow2 mutants in a wis1+ background were able to grow at higher temperatures than wild-type and sow1 showed a mitotic advance. The sow genes may therefore define a novel connection between stress tolerance and cell cycle control.     

Detection of a point mutation in FAS2 gene of sake yeast strains by allele-specific PCR amplification

To identify yeast mutants with a point mutation, detection of the specific mutant alleles is necessary. For this purpose, we applied allele-specific polymerase chain reaction (PCR) to detect the FAS2-1250S dominant mutant allele that encodes an altered fatty acid synthase in Japanese brewer's yeast strains. These strains are known to produce a higher amount of ethyl caproate in Japanese sake. The mutant strains were supposed to be diploid and to contain heterozygous alleles, including wild-type FAS2 and a dominant FAS2-1250S. A set of oligonucleotide primers was designed to contain different nucleotides at their 3' termini: one type was identical to the wild type and the other to the mutant FAS2. Another set of primers was designed to have an additional mismatch at the second nucleotide from their 3' termini. By testing with control strains, we established PCR conditions for specific amplification. Using these conditions and a simple template preparation procedure with SDS, the presence of the allele was detected in commercially used sake yeast strains. The method presented here will be useful for the identification of specific yeast strains.     

Isolation and characterization of Schizosaccharomyces pombe mutants lacking aminopeptidase activity

A mutant strain of the fission yeast Schizosaccharomyces pombe defective in aminopeptidase I was isolated by screening for lack of activity against the chromogenic substrate lysine-beta-naphthylamide in isolated colonies. Tetrad dissection of sporulated diploids heterozygous for the wild-type and mutant allele resulted in a 2:2 segregation of mutant and wild-type phenotype indicating a single chromosomal gene mutation. Gene dosage experiments indicated that the mutation might reside in the structural gene of aminopeptidase I. No vital consequences of aminopeptidase I deficiency on cell life and sporulation could be detected. However, the enzyme seems to be involved in protein degradation under conditions of nutrient deprivation.     

A fission yeast kinesin affects Golgi membrane recycling

We report here an in vivo study of kinesin heavy chain (KHC) functions in yeast. We have identified in Schizosaccharomyces pombe a kinesin motor gene, klp3(+), which has the highest homology to the Neurospora crassa KHC. Using indirect immunofluorescence, HA epitope-tagged Klp3 protein is cytoplasmic and appears as one to a few distinct patches that are coincident with microtubules. The klp3 null allele is viable. In klp3 deleted cells, ER, Golgi and mitochondrial distribution appear normal. Mitochondrial distribution in S. pombe is known to be microtubule-associated. We show that latrunculin A does not cause mitochondria to aggregate, suggesting that mitochondrial distribution in fission yeast, unlike budding yeast, is not dependent upon actin-based processes. Neither latrunculin A nor thiabendazole affects ER or Golgi distribution. We also used the vital dye FM4-64 to visualize the internalization of the dye and its transport to vacuoles in fission yeast in the presence and absence of Klp3. We observed no significant difference between the wild-type and Klp3 null cells in either the dynamics of endocytosis or the distribution and fusion of vacuoles. The drug brefeldin A causes Golgi-to-ER recycling in wild-type fission yeast cells. Although recycling of Golgi to ER after brefeldin A treatment occurs in klp3 null cells, recycling is defective and the distribution pattern we see is different from that observed in the wild-type strain. We conclude that Klp3 plays a role in BFA-induced membrane transport. The nucleotide sequence of S. pombe klp3(+) was submitted to GenBank under Accession No. AF154055.     

Characterization and behaviour of alpha-glucan synthase in Schizosaccharomyces pombe as revealed by electron microscopy

Alpha-1,3-Glucan is a cell wall component in Schizosaccharomyces pombe and is exclusive to budding yeast. We analysed the ultrastructure of the cell wall in the alpha-glucan synthase mutant mok1 and determined the role of alpha-1,3-glucan in cell wall formation of Sz. pombe. The mok1 mutant cell has an abnormal shape, with swelling at the tip or at the site of the septum. The cell wall is thicker and looser than that of wild-type cells, and the layered structure of the cell wall is broken. The glucan fibrils forming the protoplast retain a fine fibril structure, although their development into bundles is abnormal. We also report the localization of Mok1p by immunoelectron microscopy using high-pressure freeze substitution and SDS-digested freeze-fracture replica labelling methods. The Mok1p is localized on the cell membrane and moves from the cell tip to the medial region during the cell cycle. These results confirm that Mok1p plays an important role in the normal construction of the cell wall and in the primary step of glucan bundle formation, and that it is required for new cell wall synthesis during vegetative growth. These findings suggest that alpha-1,3-glucan is an essential component for cell wall formation in fission yeast.     

Isolation and study of KlLSM4, a Kluyveromyces lactis gene homologous to the essential gene LSM4 of Saccharomyces cerevisiae

We have isolated the KlLSM4 gene as a multicopy suppressor of a Kluyveromyces lactis mutant which shows a rag(-) phenotype (resistance to antimycin A on glucose). This gene is homologous to the ScLSM4 of Saccharomyces cerevisiae, which codes for an essential 187 amino acid protein containing Sm-like domains. These motifs are present in the evolutionarily conserved family of the Sm-like proteins, which are involved in a large number of cellular processes, including pre-mRNA splicing and mRNA decapping. We demonstrated that the first 72 amino acids of KlLsm4p, which contain the Sm-like domains, can restore cell viability in both K. lactis and S. cerevisiae cells lacking the wild-type protein. However, the absence of the carboxy-terminal region resulted in a remarkable loss of cell viability in the stationary phase. The KlLSM4 sequence has been deposited in the EMBL Data library under Accession No. AJ311719.     

Deletion of PDE2, the gene encoding the high-affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in Candida albicans

A role for the cAMP-dependent pathway in regulation of the cell wall in the model yeast Saccharomyces cerevisiae has recently been demonstrated. In this study we report the results of a phenotypic analysis of a Candida albicans mutant, characterized by a constitutive activation of the cAMP pathway due to deletion of PDE2, the gene encoding the high cAMP-affinity phosphodiesterase. Unlike wild-type strains, this mutant has an increased sensitivity to cell wall and membrane perturbing agents such as SDS and CFW, and antifungals such as amphotericin B and flucytosine. Moreover, the mutant is characterized by an altered sensitivity and a significantly reduced tolerance to fluconazole. The mutant's membrane has around 30% higher ergosterol content and the cell wall glucan was 22% lower than in the wild-type. These cell wall and membrane changes are manifested by a considerable reduction in the thickness of the cell wall, which in the mutant is on average 60-65 nm, compared to 80-85 nm in the wild-type strains as revealed by electron microscopy. These results suggest that constitutive activation of the cAMP pathway affects cell wall and membrane structure, and biosynthesis, not only in the model yeast S. cerevisiae but also in the human fungal pathogen C. albicans.     

Dosage rescue by UBC4 restores cell wall integrity in Saccharomyces cerevisiae lacking the myosin type II gene MYO1

Myosin II is important for normal cytokinesis and cell wall maintenance in yeast cells. Myosin II-deficient (myo1) strains of the budding yeast Saccharomyces cerevisiae are hypersensitive to nikkomycin Z (NZ), a competitive inhibitor of chitin synthase III (Chs3p), a phenotype that is consistent with compromised cell wall integrity in this mutant. To explain this observation, we hypothesized that the absence of myosin type II will alter the normal levels of proteins that regulate cell wall integrity and that this deficiency can be overcome by the overexpression of their corresponding genes. We further hypothesized that such genes would restore normal (wild-type) NZ resistance. A haploid myo1 strain was transformed with a yeast pRS316-GAL1-cDNA expression library and the cells were positively selected with an inhibitory dose of NZ. We found that high expression of the ubiquitin-conjugating protein cDNA, UBC4, restores NZ resistance to myo1 cells. Downregulation of the cell wall stress pathway and changes in cell wall properties in these cells suggested that changes in cell wall architecture were induced by overexpression of UBC4. UBC4-dependent resistance to NZ in myo1 cells was not prevented by the proteasome inhibitor clasto-lactacystin-beta-lactone and required the expression of the vacuolar protein sorting gene VPS4, suggesting that rescue of cell wall integrity involves sorting of ubiquitinated proteins to the PVC/LE-vacuole pathway. These results point to Ubc4p as an important enzyme in the process of cell wall remodelling in myo1 cells.     

The essential function of Rrs1 in ribosome biogenesis is conserved in budding and fission yeasts

The Rrs1 protein plays an essential role in the biogenesis of 60S ribosomal subunits in budding yeast (Saccharomyces cerevisiae). Here, we examined whether the fission yeast (Schizosaccharomyces pombe) homologue of Rrs1 also plays a role in ribosome biogenesis. To this end, we constructed two temperature‐sensitive fission yeast strains, rrs1‐D14/22G and rrs1‐L51P, which had amino acid substitutions corresponding to those of the previously characterized budding yeast rrs1‐84 (D22/30G) and rrs1‐124 (L61P) strains, respectively. The fission yeast mutants exhibited severe defects in growth and 60S ribosomal subunit biogenesis at high temperatures. In addition, expression of the Rrs1 protein of fission yeast suppressed the growth defects of the budding yeast rrs1 mutants at high temperatures. Yeast two‐hybrid analyses revealed that the interactions of Rrs1 with the Rfp2 and Ebp2 proteins were conserved in budding and fission yeasts. These results suggest that the essential function of Rrs1 in ribosome biogenesis may be conserved in budding and fission yeasts. Copyright © 2015 John Wiley & Sons, Ltd.     

Spatial controls for growth zone formation during the fission yeast cell cycle

Because of its regular shape, fission yeast is becoming an increasingly important organism in the study of cellular morphogenesis. Genetic experiments with mutants and drug treatment studies with wild-type cells have revealed the importance of microtubules in controlling new growth zone formation. It is believed that microtubules exert this role by delivering to cell ends a 'dynamic landmark' protein, tea1p, which promotes actin polymerization and growth zone formation. Here we present a simple model for fission yeast morphogenesis that describes the interplay between these two cytoskeletal elements. An essential assumption of the model is that actin polymerization is a self-reinforcing process: filamentous actin promotes its own formation from globular actin subunits via regulatory molecules. In our model, microtubules stimulate actin polymerization by delivering a component of the autocatalytic actin-assembly feedback loop (not by delivering a de novo inducer of actin polymerization). We show that the model captures all the characteristic features of polarized growth in fission yeast during normal mitotic cycles. We categorize the types of growth patterns that can exist in the model and show that they correspond to the major classes of morphogenetic mutants (monopolar, orb, banana and tea). Based on these results, we propose that fission yeast cells have specific size ranges in which they can exhibit two or more different stable patterns of growth.     

Mutations sensitizing yeast cells to the start inhibitor nalidixic acid

The regulatory step Start in the cell cycle of the budding yeast Saccharomyces cerevisiae is inhibited by nalidixic acid (Nal). To study this inhibition, mutations were identified that alter the sensitivity of yeast cells to Nal. Nal-sensitive mutations were sought because the inhibitory effects of Nal on wild-type cells are only transient, and wild-type cells naturally become refractory to Nal. Three complementation groups of Nal-sensitive mutations were found. Mutations in the first complementation group were shown to reside in the ARO7 gene, encoding chorismate mutase; tyrosine and phenylalanine synthesis was inhibited by Nal in these aro7 mutants, whereas wild-type chorismate mutase was unaffected. These aro7 alleles demonstrate ‘recruitment’, by mutation, of an innately indifferent protein to an inhibitor-sensitive form. The Nal-sensitive aro7 mutant cells were used to show that the resumption of Nal-inhibited nuclear activity and cell proliferation takes place while cytoplasmic Nal persists at concentrations inhibitory for the mutant chorismate mutase. Mutations in the second complementation group, nss2 (Nal-supersensitive), increased intracellular Nal concentrations, and may simply alter the permeability of cells to Nal. The third complementation group was found to be the ERG6 gene, previously suggested to encode the ergosterol biosynthetic enzyme sterol methyltransferase. Mutation or deletion of the ERG6 gene had little effect on the inhibition of Start by Nal, but prevented recovery from this inhibition. Mutation of ERG3, encoding another ergosterol biosynthetic enzyme, also caused Nal sensitivity, suggesting that plasma membrane sterol composition, and plasma membrane function, mediates recovery from Nal-mediated inhibition of Start.     

Increased endocytosis in the Saccharomyces cerevisiae fragile mutant VY1160

The VY1160 mutant is characterized by cell lysis in hypotonic solutions and generally increased permeability to substances for which Saccharomyces cerevisiae cells are not permeable. Two mutations, srb1 and ts1, have been identified in VY1160 mutant, and previous studies (Kozhina et al., 1979) have shown srb1 to be responsible for cell lysis. We now present evidence that the ts1 mutation leads to increased endocytosis in VY1160 cells. The internalization of lucifer yellow carbohydrazide in VY1160 cells is time-, temperature- and energy-dependent and consistent with a fluid-phase mechanism of endocytosis. The rate of steady-state accumulation of the dye at 37 degrees C is 145 ng/micrograms DNA per h for VY1160 mutant and 23 ng/micrograms DNA per h for S288C parental strain. Studies with isogenic strains having either the srb1 or the ts1 mutation, or SRB1 TS1 wild-type alleles have shown that only ts1 strains possess increased endocytosis. Quantitation of endocytosis in cells grown at 24 degrees C and shifted at 38 degrees C shows that ts1 strains, but not srb1 and wild-type strains, increase ten-fold the internalization of lucifer yellow 2 h after the shift at 38 degrees C. The analysis of ts1 x wild-type crosses provides evidence that the temperature-sensitive phenotype segregates together with the enhanced endocytosis. It is concluded that the increased endocytosis might explain the generally increased permeability of VY1160 mutant cells.     

Kluyveromyces lactis SSO1 and SEB1 genes are functional in Saccharomyces cerevisiae and enhance production of secreted proteins when overexpressed

The SEB1/SBH1 and the SSO genes encode components of the protein secretory machinery functioning at the opposite ends, ER translocation and exocytosis, respectively, of the secretory pathway in Saccharomyces cerevisiae. Overexpression of these genes can rescue temperature-sensitive (ts) growth defect of many sec mutants impaired in protein secretion. Furthermore, their overexpression in wild-type yeast enhances production of secreted proteins in S. cerevisiae, which suggests that they may be rate-limiting factors in this process. Here we report isolation of Kluyveromyces lactis homologues of these genes. KlSSO1 and KlSEB1 were isolated as clones capable of rescuing growth of ts sso2-1 and seb1Delta seb2Delta sem1Delta strains, respectively, at the restrictive temperature. The encoded Kluyveromyces proteins are up to 70% identical with the S. cerevisiae homologues at the amino acid level and can functionally replace them. Interestingly, KlSSO1 and KlSEB1 show similar enhancing effect on production of a secreted protein as the SSO and SEB1 genes of S. cerevisiae when overexpressed. In accordance with the high homology level of the secretory pathway proteins in different yeast species, the polyclonal antibodies raised against S. cerevisiae Seb1p, Sso2p and Sec4p can detect homologous proteins in cell lysates of K. lactis and Pichia pastoris, the latter also in Candida utilis. The GenBank Accession Nos are AF307983 (K. lactis SSO1) and AF318314 (K. lactis SEB1).     

Sequence analysis of two cosmids from Schizosaccharomyces pombe chromosome III

We report the complete sequence of two cosmids, SPCC895 (38457 bp insert, EMBL Accession No. AL035247) and SPCC1322 (42068 bp insert, EMBL Accession No. AL035259), localized on chromosome III of the Schizosaccharomyces pombe genome. Fourteen Coding DNA sequences (CDSs) were identified in SPCC895 and 17 in SPCC1322. Two known genes were found in each cosmid: map2 and gms1 on SPCC895, encoding the mating type P-factor precursor and an UDP-galactose transporter, respectively, and bub1 and ade6 in SPCC1322, encoding a protein kinase and a phosphoribosylaminoimidazole carboxylase, respectively. The fission yeast K RNA gene has been localized to SPCC895. Three ribosomal proteins have been predicted among these two cosmids. Nine CDSs similar to known proteins were found on SPCC895, and seven on SPCC1322. They include putative genes for an uridylate kinase, a proteasome catalytic component, an ion transporter, a checkpoint protein, a translation initiation protein, a SNARE complex protein, a protein involved in cytoskeletal organization, a spindle pole body-associating protein, pre-mRNA splicing factor RNA helicase, a 3'-5' exonuclease for RNA 3' ss-tail, an UTP-glucose-1-phosphate uridylyltransferase, a leukotriene A(4) hydrolase, a member of the RanBP7-importin beta-Cse1p superfamily, a Ca(++)-calmodulin-dependent serine/threonine protein kinase and a prohibitin antiproliferative protein. One CDS is predicted to be an integral membrane protein. One CDS from SPCC895 is similar to a CDS of unknown function from Saccharomyces cerevisiae and three from SPCC1322 are similar to CDSs of unknown function from Candida albicans, S. cerevisiae and Sz. pombe, respectively. Finally, one CDS of SPCC895 and three of SPCC1322 correspond to orphan genes.     

Targeted gene deletion in Zygosaccharomyces bailii

Yeasts of the genus Zygosaccharomyces are notable agents of large-scale food spoilage. Despite the economic importance of these organisms, little is known about the stress adaptations whereby they adapt to many of the more severe conditions of food preservation. In this study it was shown that genes of Z. bailii, a yeast notable for its high resistances to food preservatives and ethanol, can be isolated by complementation of the corresponding mutant strains of Saccharomyces cerevisiae. It was also discovered that the acquisition by S. cerevisiae of a single small Z. bailii gene (ZbYME2) was sufficient for the former yeast to acquire the ability to degrade two major food preservatives, benzoic acid and sorbic acid. Using DNA cassettes containing dominant selectable markers and methods originally developed for performing gene deletions in S. cerevisiae, the two copies of ZbYME2 in the Z. bailii genome were sequentially deleted. The resulting Zbyme2/Zbyme2 homozygous deletant strain had lost any ability to utilize benzoate as sole carbon source and was more sensitive to weak acid preservatives during growth on glucose. Thus, ZbYME2, probably the nuclear gene for a mitochondrial mono-oxygenase function, is essential for Z. bailii to degrade food preservatives. This ability to catabolize weak acid preservatives is a significant factor contributing to the preservative resistance of Z. bailii under aerobic conditions. This study is the first to demonstrate that it is possible to delete in Z. bailii genes that are suspected as being important for growth of this organism in preserved foods and beverages. With the construction of further mutant of Z. bailii strains, a clearer picture should emerge of how this yeast adapts to the conditions of food preservation. This information will, in turn, allow the design of new preservation strategies. GenBank Accession Nos: ZbURA3 (AF279259), ZbTIM9 (AF279260), ZbYME2 (AF279261), ZbTRP1 (AF279262), ZbHHT1(AF296170).     

Monitoring of Brewing Yeast Propagation Under Aerobic and Anaerobic Conditions Employing Flow Cytometry

The vitality and viability of industrial strains of Saccharomyces cerevisiae was monitored during pilot plant experiments simulating yeast propagation under aerobic and anaerobic conditions. Industrial wort of 12°P original gravity was used as a growth substrate for yeast propagation. The work was carried out with three widely used Czech lager yeast industrial strains: strains 2, 7 and 95. Cell cycle, cell size, granularity, glycogen content, DNA and protein content were analyzed by flow cytometry. Significantly higher specific growth rates, higher content of yeast glycogen, earlier G2/M phase cells maximum, and faster cell protein creation was observed under aerobic conditions compared to anaerobic. Strains 7 and 95 showed losses in flocculation ability after aerobic propagation compared to anaerobic propagation. Under either aerobic or strictly anaerobic conditions, only strain 2 did not show a significant loss in flocculation ability.     

Candida albicans Tpk1p and Tpk2p isoforms differentially regulate pseudohyphal development, biofilm structure, cell aggregation and adhesins expression

Candida albicans undergoes a reversible morphological transition from single yeast cells to pseudohyphal and hyphal filaments. In this organism, cAMP-dependent protein kinase (PKA), coded by two catalytic subunits (TPK1 and TPK2) and one regulatory subunit (BCY1), mediates basic cellular processes, such as the yeast-to-hypha transition and cell cycle regulation. It is known that both Tpk isoforms play positive roles in vegetative growth and filamentation, although distinct roles have been found in virulence, stress response and glycogen storage. However, little is known regarding the participation of Tpk1p and/or Tpk2p in pseudohyphal development. This point was addressed using several C. albicans PKA mutants having heterozygous or homozygous deletions of TPK1 and/or TPK2 in different BCY1 genetic backgrounds. We observed that under hypha-only inducing conditions, all BCY1 heterozygous strains shifted growth toward pseudohyphal morphology; however, the pseudohypha:hypha ratio was higher in strains devoid of TPK2. Under pseudohypha-only inducing conditions, strains lacking TPK2 were prone to develop short and branched pseudohyphae. In tpk2 Δ/tpk2 Δ strains, biofilm architecture was markedly less dense, composed of short pseudohyphae and blastospores with reduced adhesion ability to abiotic material, suggesting a significant defect in cell adherence. Immunolabelling assays showed a decreased expression of adhesins Als1p and Als3p only in the tpk2 Δ/tpk2 Δ strain. Complementation of this mutant with a wild-type copy of TPK2 restored all the altered functions: pseudohyphae elongation, biofilm composition, cell aggregation and adhesins expression. Our study suggests that the Tpk2p isoform may be part of a mechanism underlying not only polarized pseudohyphal morphogenesis but also cell adherence.     

A Lallzyme MMX‐based rapid method for fission yeast protoplast preparation

Fungal cells including yeasts are surrounded by cell wall that counteracts turgor pressure and prevents cell lysis. Many yeast experiments, including genetic manipulation of sterile strains, morphogenesis studies, nucleic acid isolation and many others, require mechanical breakage or enzymatic removal of the cell wall. Some of these experiments require the generation of live cells lacking cell walls, called protoplasts, that can be maintained in osmostabilized medium. Enzymatic digestion of cell wall proteoglycans is a commonly used method of protoplast preparation. Currently existing protocols for fission yeast cell wall digestion are time consuming and not very efficient. We developed a new rapid method for fission yeast protoplast preparation that relies on digesting cell walls with Lallzyme MMX commercial enzyme mix, which produces protoplasts from all cells in less than 10 min. We demonstrate that these protoplasts can be utilized in three commonly used fission yeast protocols. Thus, we provide the fission yeast community with a robust and efficient plasmid extraction method, a new protocol for diploid generation and an assay for protoplast recovery that should be useful for studies of morphogenesis. Our method is potentially applicable to other yeasts and fungi. Copyright © 2013 John Wiley & Sons, Ltd.