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Horizontal gene transfer, a process through which an organism acquires genes from other organisms, is a rare evolutionary event in yeasts. Artificial random gene transfer can emerge as a valuable tool in yeast bioengineering to investigate the background of complex phenotypes, such as heat tolerance. In this study, a cDNA library was constructed from the mRNA of a methylotrophic yeast, Ogataea polymorpha, and then introduced into Saccharomyces cerevisiae. Ogataea polymorpha was selected because it is one of the most heat-tolerant species among yeasts. Screening of S. cerevisiae populations expressing O. polymorpha genes at high temperatures identified 59 O. polymorpha genes that contribute to heat tolerance. Gene enrichment analysis indicated that certain S. cerevisiae functions, including protein synthesis, were highly temperature-sensitive. Additionally, the results confirmed that heat tolerance in yeast is a complex phenotype dependent on multiple quantitative loci. Random gene transfer would be a useful tool for future bioengineering studies on yeasts.

To achieve inorganic phosphate (Pi) homeostasis, cells must be able to sense intracellular and extracellular Pi concentrations. In the Pi signaling (PHO) pathway in Saccharomyces cerevisiae, high Pi represses genes involved in Pi uptake (e.g., PHO84) and Pi utilization (PHO5); conversely, the cyclin-dependent kinase inhibitor Pho81 inhibits the activity of the Pho80-Pho85 cyclin-cyclin dependent kinase complex in low-Pi conditions, leading to induction of these genes. However, how yeast senses Pi availability remains unresolved. To identify factors involved in Pi sensing upstream of the Pho81-Pho80-Pho85 complex, we generated and screened suppressor mutants of a Δpho84 strain that shows constitutive PHO5 expression. By a series of genetic tests, including dominance-recessiveness, complementation and tetrad analyses, three sef (suppressor of pho84 [pho eighty-four]) mutants (sef8, sef9 and sef10) were shown to contain a novel single mutation. The sef mutants suppressed the phenotype of constitutive PHO5 expression at the transcriptional level, but did not show restored Pi uptake capacity. An epistasis-hypostasis test revealed that the sef mutations were hypostatic to pho80 mutation, indicating that their gene products function upstream of the Pho81-Pho80-Pho85 complex in the PHO pathway. The sef mutations identified are associated with gene(s) that may be involved in the homeostasis of an intracellular Pi level-sensing mechanism in S. cerevisiae.

The methylotrophic yeast Ogataea polymorpha (syn. Hansenula polymorpha) is an attractive industrial non-conventional yeast showing high thermo-tolerance (up to 50°C) and xylose assimilation. However, genetic manipulation of O. polymorpha is often laborious and time-consuming because it has lower homologous recombination efficiency relative to Saccharomyces cerevisiae. To overcome this disadvantage, we applied the CRISPR/Cas9 system as a powerful genome editing tool in O. polymorpha. In this system, both single guide RNA (sgRNA) and endonuclease Cas9 were expressed by a single autonomously-replicable plasmid and the sgRNA portion could be easily changed by using PCR and In-Fusion cloning techniques. Because the mutation efficiency of the CRISPR/Cas9 system was relatively low when the sgRNA was expressed under the control of the OpSNR6 promoter, the tRNACUG gene was used for sgRNA expression. The editing efficiency of this system ranged from 17% to 71% of transformants in several target genes tested (ADE12, PHO1, PHO11, and PHO84). These findings indicate that genetic manipulation of O. polymorpha will be more convenient and accelerated by using this CRISPR/Cas9 system.

Farnesyl diphosphate synthase (FPS) is an essential enzyme in the biosynthesis of prenyl precursors for the production of primary and secondary metabolites, including sterols, dolichols, carotenoids and ubiquinones, and for the modification of proteins. Here we identified and characterized two FPSs (EuFPS1 and EuFPS2) from the plant Eucommia ulmoides. The EuFPSs had seven highly conserved prenyltransferase-specific domains that are critical for activity. Complementation and biochemical analyses using bacterially produced recombinant EuFPS isoforms showed that the EuFPSs had FPP synthesis activities both in vivo and in vitro. In addition to the typical reaction mechanisms of FPS, EuFPSs utilized farnesyl diphosphate (FPP) as an allylic substrate and participated in further elongation of the isoprenyl chain, resulting in the synthesis of geranylgeranyl diphosphate. However, despite the high amino acid similarities between the two EuFPS isozymes, their specific activities, substrate preferences, and final reaction products were different. The use of dimethylallyl diphosphate (DMAPP) as an allylic substrate highlighted the differences between the two enzymes: depending on the pH, the metal ion cofactor, and the cofactor concentration, EuFPS2 accumulated geranyl diphosphate as an intermediate product at a constant rate, whereas EuFPS1 synthesized little geranyl diphosphate. The reaction kinetics of the EuFPSs demonstrated that isopentenyl diphosphate and DMAPP were used both as substrates and as inhibitors of EuFPS activity. Taken together, the results indicate that the biosynthesis of FPP is highly regulated by various factors indispensable for EuFPS reactions in plants.

The methylotrophic yeast, Ogataea thermomethanolica TBRC656, is an attractive host organism for heterologous protein production owing to the availability of protein expression vectors and a genome-editing tool. In this study, we focused on mating-type switching and gene expression in order to elucidate its sexual life cycle and establish genetic approaches applicable for the strain. A putative mating-type gene cluster was identified in TBRC656 that is syntenic to the cluster in Ogataea parapolymorpha DL-1 (previously named Hansenula polymorpha). Like DL-1, TBRC656 possesses two mating loci, namely MATa and MATα, and also shows flip-flop mating-type switching. Interestingly, unlike any other methylotrophic yeast, TBRC656 robustly switched mating type during late growth in rich medium (YPD). Under nutrient depletion, mating-type switching was observed within one hour. Transcription from both MATa and MATα mating loci was detected during growth in YPD, and possibly induced upon nitrogen depletion. Gene expression from MATα was detected as a single co-transcript from a three-gene array (α2-α1-a1S). Deletion of a putative a1S ORF at the MATα locus had no observed effect on mating-type switching but demonstrated significant effect on mating-type gene expression at both MATa and MATα loci.

To achieve inorganic phosphate (Pi) homeostasis, cells must be able to sense intracellular and extracellular Pi concentrations. In the Pi signaling (PHO) pathway in Saccharomyces cerevisiae, high Pi represses genes involved in Pi uptake (e.g., PHO84) and Pi utilization (PHO5); conversely, the cyclin-dependent kinase inhibitor Pho81 inhibits the activity of the Pho80-Pho85 cyclin-cyclin dependent kinase complex in low-Pi conditions, leading to induction of these genes. However, how yeast senses Pi availability remains unresolved. To identify factors involved in Pi sensing upstream of the Pho81-Pho80-Pho85 complex, we generated and screened suppressor mutants of a Δpho84 strain that shows constitutive PHO5 expression. By a series of genetic tests, including dominance-recessiveness, complementation and tetrad analyses, three sef (suppressor of pho84 [pho eighty-four]) mutants (sef8, sef9 and sef10) were shown to contain a novel single mutation. The sef mutants suppressed the phenotype of constitutive PHO5 expression at the transcriptional level, but did not show restored Pi uptake capacity. An epistasis-hypostasis test revealed that the sef mutations were hypostatic to pho80 mutation, indicating that their gene products function upstream of the Pho81-Pho80-Pho85 complex in the PHO pathway. The sef mutations identified are associated with gene(s) that may be involved in the homeostasis of an intracellular Pi level-sensing mechanism in S. cerevisiae.

Some plant trans-1,4-prenyltransferases (TPTs) produce ultrahigh molecular weight trans-1,4-polyisoprene (TPI) with a molecular weight of over 1.0 million. Although plant-derived TPI has been utilized in various industries, its biosynthesis and physiological function(s) are unclear. Here, we identified three novel Eucommia ulmoides TPT isoforms—EuTPT1, 3, and 5, which synthesized TPI in vitro without other components. Crystal structure analysis of EuTPT3 revealed a dimeric architecture with a central hydrophobic tunnel. Mutation of Cys94 and Ala95 on the central hydrophobic tunnel no longer synthesizd TPI, indicating that Cys94 and Ala95 were essential for forming the dimeric architecture of ultralong-chain TPTs and TPI biosynthesis. A spatiotemporal analysis of the physiological function of TPI in E. ulmoides suggested that it is involved in seed development and maturation. Thus, our analysis provides functional and mechanistic insights into TPI biosynthesis and uncovers biological roles of TPI in plants., Kajiura and Yoshizawa et al. identify three new prenyltransferases in the tree Eucommia ulmoides that synthesize exceptionally high molecular weight trans-1,4-polyisoprene (TPI). Through crystal structure and mutational analyses, they identify key residues required for TPI synthesis and reveal its functional importance in seed development.

Saccharomyces cerevisiae and its closely related yeasts undergo mating type switching by replacing DNA sequences at the active mating type locus (MAT) with one of two silent mating type cassettes. Recently, a novel mode of mating type switching was reported in methylotrophic yeast, including Ogataea polymorpha, which utilizes chromosomal recombination between inverted-repeat sequences flanking two MAT loci. The inversion is highly regulated and occurs only when two requirements are met: haploidy and nutritional starvation. However, links between this information and the mechanism associated with mating type switching are not understood. Here we investigated the roles of transcription factors involved in yeast sexual development, such as mating type genes and the conserved zinc finger protein Rme1. We found that co-presence of mating type a1 and α2 genes was sufficient to prevent mating type switching, suggesting that ploidy information resides solely in the mating type locus. Additionally, RME1 deletion resulted in a reduced rate of switching, and ectopic expression of O. polymorpha RME1 overrode the requirement for starvation to induce MAT inversion. These results suggested that mating type switching in O. polymorpha is likely regulated by two distinct transcriptional programs that are linked to the ploidy and transmission of the starvation signal.

Cytoplasmic microtubules (cMT) control mitotic spindle positioning in many organisms, and are therefore pivotal for successful cell division. Despite its importance, the temporal control of cMT formation remains poorly understood. Here we show that unlike the best-studied yeast Saccharomyces cerevisiae, position of pre-anaphase nucleus is not strongly biased toward bud neck in Ogataea polymorpha and the regulation of spindle positioning becomes active only shortly before anaphase. This is likely due to the unstable property of cMTs compared to those in S. cerevisiae. Furthermore, we show that cMT nucleation/anchoring is restricted at the level of recruitment of the γ-tubulin complex receptor, Spc72, to spindle pole body (SPB), which is regulated by the polo-like kinase Cdc5. Additionally, electron microscopy revealed that the cytoplasmic side of SPB is structurally different between G1 and anaphase. Thus, polo-like kinase dependent recruitment of γ-tubulin receptor to SPBs determines the timing of spindle orientation in O. polymorpha., eLife digest Before a cell divides, it needs to duplicate its genetic material to provide the new daughter cell with a full set of genetic information. To do so, the cell forms a complex of proteins called the spindle apparatus, which is made up of string-like microtubules that divide the chromosomes evenly. In many organisms, the position of the spindle determines where in the cell this separation happens. However, in baker’s yeast, the location where the cell will divide is determined well before the spindle is formed. Unlike many other eukaryotic cells, these yeast cells divide asymmetrically and create buds that will form the new daughter cells. The position of this bud determines where the spindle should be located and where the chromosomes separate. The spindle itself is then organised by a structure called the spindle pole body, which connects to microtubules inside the cell nucleus and microtubules in the cell plasma. Several proteins control where and how the spindle forms, including a protein called the spindle pole component 72, or Spc72 for short, and an enzyme called Cdc5. However, until now it was unclear how spindle formation is timed and controlled in other yeast species. Now, Maekawa et al. have used fluorescent markers and time lapse microscopy to examine how the spindle forms in the yeast species Ogataea polymorpha, an important industrial yeast used to produce medicines and alcohol. The results show that in O. polymorpha, the positioning and orientation of the spindle only occurred very late in the cell cycle and the microtubules in the cell plasma remained unstable until the chromosomes were about to separate. This was linked to changes in the level of Spc72, which increased at the spindle pole body before the chromosomes separated and then dropped again. This was controlled by Cdc5. Understanding when and where microtubules are formed is an important step in understanding how cells divide. This is the first example of a budding yeast that creates new microtubules in the cell plasma every time the cell divides. Unravelling the molecular differences between yeast species could lead to new ways to optimise the use of industrial yeasts like O. polymorpha, or to combat disease-causing ones.

High-temperature ethanol fermentation has several benefits including a reduction in cooling cost, minimizing risk of bacterial contamination, and enabling simultaneous saccharification and fermentation. To achieve the efficient ethanol fermentation at high temperature, yeast strain that tolerates to not only high temperature but also the other stresses present during fermentation, e.g., ethanol, osmotic, and oxidative stresses, is indispensable. The C3253, C3751, and C4377 Saccharomyces cerevisiae strains, which have been previously isolated as thermotolerant yeasts, were found to be multiple stress-tolerant. In these strains, continuous expression of heat shock protein genes and intracellular trehalose accumulation were induced in response to stresses causing protein denaturation. Compared to the control strains, these multiple stress-tolerant strains displayed low intracellular reactive oxygen species levels and effective cell wall remodeling upon exposures to almost all stresses tested. In response to simultaneous multi-stress mimicking fermentation stress, cell wall remodeling and redox homeostasis seem to be the primary mechanisms required for protection against cell damage. Moreover, these strains showed better performances of ethanol production than the control strains at both optimal and high temperatures, suggesting their potential use in high-temperature ethanol fermentation.

Yeast (Saccharomyces cerevisiae) RNA is an important source of 5'-ribonucleotides that is used in both the food and pharmaceutical industries. Efficient transcription of rDNA is very important to construct yeast strains with high RNA content. The gene RRN10, which encodes, a component of the upstream activation factor, is essential to promote high-level transcription of rDNA. In our previous study, we isolated SupE strain as a dominant suppressor, which showed the ability to restore the severe growth defects and reduced RNA content caused by disruption of the RRN10 gene. SupE strain has multiple mutations which we designated collectively as SUPE. Further analysis on SUPE mutation indicated that RPL40A was responsible for suppression of defect caused by rrn10 disruption. However, there were no base changes in this gene as compared with the parental Δrrn10 strain, thus suggesting that an additional copy of RPL40A suppress the defects caused by Δrrn10 disruption, and that, in SupE strain, these defects are suppressed by increased transcription of RPL40A whose copy is doubled. When multiple copies of RPL40A were combined with SUPE mutation on an RRN10⁺ background, the resultant SupE strain had significantly higher RNA content than wild-type strain. In addition, increased transcription of RPL40B also showed significant effect to restore the growth defect and reduced RNA content caused by Δrrn10 disruption. We propose a model to explain how SUPE mutation increases the transcription of ribosomal protein genes such as RPL40A and RPL40B in SupE strain, resulting in an increase in RNA content.

Using chemical mutagenesis, mutants of Hansenula polymorpha that were defective in fatty acid synthesis were selected based on their growth requirements on saturated fatty acid mixtures. One mutant (S7) was incapable of synthesizing polyunsaturated fatty acids (PUFA), linoleic and α-linolenic acids. A genetic analysis demonstrated that the S7 strain had a double lesion affecting fatty acid synthesis and Δ(12)-desaturation. A segregant with a defect in PUFA synthesis (H69-2C) displayed normal growth characteristics in the temperature range of 20-42 °C through a modulation of the cellular fatty acid composition. Compared with the parental strain, this yeast mutant had increased sensitivity at low and high temperatures (15 and 48 °C, respectively) with an increased tolerance to oxidative stress. The responses to ethanol stress were similar for the parental and PUFA-defective strains. Myristic acid was also determined to play an essential role in the cell growth of H. polymorpha. These findings suggest that both the type of cellular fatty acids and the composition of fatty acids might be involved in the stress responsive mechanisms in this industrially important yeast.

Reversible phosphorylation is one of the key post-translational modifications for the regulation of many essential cellular processes. We have previously reported that the disruption of two protein phosphatase (PPase) genes, PTP2 and MSG5, causes calcium sensitivity indicating that functional redundancy exists between the two PPases in response to high extracellular calcium. In this paper, we found that the inactivation of calcineurin by the disruption of the calcineurin regulatory subunit, CNB1 or treatment with a calcineurin inhibitor, FK506, can suppress the calcium-sensitive phenotype of the ptp2Δmsg5Δ double disruptant. In the wake of a calcium-induced, calcineurin-driven signaling pathway activation, the calcium sensitivity of the ptp2Δmsg5Δ double disruptant can be suppressed by regulating the SLT2 pathway through the disruption of the major kinases in the SLT2 signal cascade that include BCK1, MKK1 and SLT2. Also, we show that PTP2 and MSG5 are key regulatory PPases that prevent over-activation of the calcium-induced signaling cascade under the parallel control of the SLT2 and calcineurin pathways.

The capability of multi-stress-tolerant Saccharomyces cerevisiae diploid strain TJ14 for the production of cellulosic bio-ethanol by semi-simultaneous saccharification and fermentation (SSSF) technology was evaluated under high-temperature conditions. At 39°C, the TJ14 produced 45 g/l ethanol by SSSF of 100 g (w/v)/l cellulose - a significantly higher concentration than reported in prevailing literature.

The simultaneous saccharification and fermentation process requires thermo-tolerant yeast to facilitate the enzymatic hydrolysis of cellulose. In this paper, we describe a Htg+ strain that exhibits confluent growth at high temperature (41 °C) and resistance to heat shock, ethanol, osmotic, oxidative and DNA damage stresses. HTG6, one of the six genes responsible for the thermotolerant phenotype was identified to be the gene RSP5 encoding a ubiquitin ligase. The RSP5 allele of the Htg+ strain, designated RSP5-C, possessed five, one and two base changes in the promoter, open reading frame and terminator region, respectively. The base changes in the promoter region of the RSP5-C allele were found to be responsible for the thermotolerant phenotype by strongly increasing transcription of the RSP5 gene and consequently causing a rise in the ubiquitination of cell proteins. Overexpression of the RSP5-BY allele present in the htg6 host strain (Htg-) conferred thermotolerance at 41°C, to this strain as in the case of RSP5-C allele. We also discovered that an Htg+ strain overexpressing the RSP5-C allele exhibits a more robust Htg+ phenotype against higher temperature (43 °C). The data presented here also suggest that overexpression of RSP5 could be applied to raise the upper limit of thermotolerance in S. cerevisiae strain used for industrial bioethanol production.

Yeast RNA is a good source of nucleotide seasoning, and more than half of yeast RNA consists of ribosomal RNA (rRNA). Previously, we reported the development of a Saccharomyces cerevisiae strain displaying a 1.4- to 2.3-times higher RNA content than the wild-type strain through the isolation of dominant suppressors (designated SupA to SupG strains) from a Δrrn10 disruptant showing decreased rRNA transcription. In the present study, the cloning of one of the genes responsible for the suppression was attempted using a genomic library from the SupD strain. NOP15, a gene involved in ribosome biogenesis, was found to be responsible for suppressing the growth defect of the Δrrn10 disruptant. The isolated NOP15 allele (designated NOP15(T-279C)) possessed a single T to C substitution at nucleotide position-279 of NOP15. The transcription level of NOP15(T-279C) in the originally isolated SupD strain was 2-fold higher than that in the Δrrn10 disruptant. Furthermore, a dose-dependent relationship between the transcription level of NOP15 and total amount of RNA in the Δrrn10 disruptant was observed: the enhanced transcription due to the NOP15(T-279C) allele is involved in the suppression mechanisms in the SupD strain. Introduction of the NOP15(T-279C) allele into the wild-type strain increased the total RNA content by 1.4-fold. These results indicate that the transcription level of NOP15 is an important determinant of the productivity of RNA and that its increased transcription provides an effective approach to obtain higher RNA yields in yeast.

To gain more insight into adaptation response to lactic-acid stress in yeast, a genome-wide screening for genes whose disruption caused hypersensitivity to 4.0% l-lactic acid (pH 2.8) was performed using the gene deletion collection of Saccharomyces cerevisiae. We identified 107 genes that contributed significantly to the ability of yeast cells to adapt lactic-acid stress. More than 30% of the genes identified in this screening were newly identified to be involved in mechanisms for adaptation response to lactic acid. We found that protein urmylation by Uba4 and N-terminal acetylation by Nat3 were involved in lactic acid adaptation mechanisms. Functional categorization of the genes followed by microscopic analysis revealed that a variety of cellular functions were involved in adaptation response to lactic acid and function associated with vacuolar transport played important roles in adaptation response to lactic acid. We also found that vacuole fragmented immediately upon exposure to lactic- and hydrochloric-acid stress. In addition, our analysis revealed that lactic-acid stress significantly reduced the amount of intracellular amino acids. Amino acid supplementation recovered the adaptation deficiency to lactic acid, suggesting that intracellular amino-acid homeostasis plays important roles in adaptation response to lactic-acid stress. These data suggest that enhancing vacuolar integrity, as well as maintaining intracellular amino-acid homeostasis may be an efficient approach to confer resistance to lactic-acid stress.

Use of super strains exhibiting tolerance to high temperature, acidity and ethanol is a promising way to make ethanol production economically feasible. We describe here the breeding and performance of such a multiple-tolerant strain of Saccharomyces cerevisiae generated by a spore-to-cell hybridization technique without recombinant DNA technology. A heterothallic strain showing a high-temperature (41°C) tolerant (Htg(+)) phenotype, a derivative from a strain isolated from nature, was crossed with a homothallic strain displaying high-ethanol productivity (Hep(+)), a stock culture at the Thailand Institute of Scientific and Technological Research. The resultant hybrid TJ14 displayed ability to rapidly utilize glucose, and produced ethanol (46.6g/l) from 10% glucose fermentation medium at high temperature (41°C). Not only ethanol productivity at 41°C but also acid tolerance (Acd(+)) was improved in TJ14 as compared with its parental strains, enabling TJ14 to grow in liquid medium even at pH 3. TJ14 maintained high ethanol productivity (46.0g/l) from 10% glucose when fermentation was done under multiple-stress conditions (41°C and pH 3.5). Furthermore, when TJ14 was subjected to a repeated-batch fermentation scheme, the growth and ethanol production of TJ14 were maintained at excellent levels over ten cycles of fermentation. Thus, the multiple-stress (Htg(+) Hep(+) Acd(+)) resistant strain TJ14 should be useful for cost-effective bioethanol production under high-temperature and acidic conditions.

Polylactic acid plastics are receiving increasing attention for the control of atmospheric CO2 emissions. Lactic acid, the building block for polylactic acid, is produced by fermentation technology from renewable carbon sources. The yeast Saccharomyces cerevisiae, harboring the lactate dehydrogenases gene (LDH), produces lactic acid at a large scale due to its strong acid resistance, to its simple nutritional requirements and to its ease of genetic engineering. Since improvement of lactic acid resistance is correlated with an increase of lactic acid production under non-neutralizing condition, we isolated a novel gene that enhances lactic acid resistance using a multi-copy yeast genomic DNA library. In this study, we identified the ESBP6 gene, which increases lactic acid resistance when overexpressed and which encodes a protein with similarity to monocarboxylate permeases. Although ESBP6 was not induced in response to lactic acid stress, it caused weak but reproducible sensitivity to lactic acid when disrupted. Furthermore, intracellular pH in the ESBP6 overexpressing strain was higher than that in the wild-type strain under lactic acid stressed condition, suggesting that Esbp6 plays some roles in lactic acid adaptation response. The ESBP6 overexpressing strain carrying the LDH gene induced 20% increase in lactic acid production compared with the wild-type strain carrying the LDH gene under non-neutralizing conditions. These results indicate that overexpression of ESBP6 provides a novel and useful tool to improve lactic acid resistance and lactic acid production in yeast.

The Saccharomyces cerevisiae Yvh1, a dual-specificity protein phosphatase involved in glycogen accumulation and sporulation, is required for normal vegetative growth. To further elucidate the role of Yvh1, we generated dominant mutants suppressing the slow growth caused by YVH1 disruption. One of the mutant alleles, designated as SVH1-1 (suppressor of Δyvh1 deletion), was identical to MRT4 (mRNA turnover) that contained a single-base substitution causing an amino acid change from Gly(68) to Asp. Mrt4(G68D) restored the deficiencies in growth and rRNA biogenesis that occurs in absence of Yvh1. Here, we report that the interaction between Mrt4 and Yvh1 is also essential for normal glycogen accumulation and mRNA decay as well as the induction of sporulation genes IME2, SPO13 and HOP1. The Mrt4(G68D) could restore the plethora of phenotypes we observed in absence of Yvh1. We found that Yvh1 is not essential for wild-type induction of the transcriptional regulator of these genes, IME1, suggesting that either translation or post-translational modification to activate Ime1 has been compromised. Since a defect in ribosome biogenesis in general can be related to other various defects, the ribosome biogenesis defect caused by absence of Yvh1 might be an indirect cause of observed phenotypes.

To save cost and input energy for bioethanol production, a consolidated continuous solid-state fermentation system composed of a rotating drum reactor, a humidifier, and a condenser was developed. Biomass, saccharifying enzymes, yeast, and a minimum amount of water are introduced into the system. Ethanol produced by simultaneous saccharification and fermentation is continuously recovered as vapor from the headspace of the reactor, while the humidifier compensates for the water loss. From raw corn starch as a biomass model, 95 +/- 3, 226 +/- 9, 458 +/- 26, and 509 +/- 64 g l(-1) of ethanol solutions were recovered continuously when the ethanol content in reactor was controlled at 10-20, 30-50, 50-70 and 75-85 g kg-mixture(-1), respectively. The residue showed a lesser volume and higher solid content than that obtained by conventional liquid fermentation. The cost and energy for intensive waste water treatment are decreased, and the continuous fermentation enabled the sustainability of enzyme activity and yeast in the system.

During fermentation, yeast cells are exposed to a number of stresses -- such as high alcohol concentration, high osmotic pressure, and temperature fluctuation - so some overlap of mechanisms involved in the response to these stresses has been suggested. To identify the genes required for tolerance to alcohol (ethanol, methanol, and 1-propanol), heat, osmotic stress, and oxidative stress, we performed genome-wide screening by using 4828 yeast deletion mutants. Our screens identified 95, 54, 125, 178, 42, and 30 deletion mutants sensitive to ethanol, methanol, 1-propanol, heat, NaCl, and H2O2, respectively. These deleted genes were then classified based on their cellular functions, and cross-sensitivities between stresses were determined. A large number of genes involved in vacuolar H(+)-ATPase (V-ATPase) function, cytoskeleton biogenesis, and cell wall integrity, were required for tolerance to alcohol, suggesting their protective role against alcohol stress. Our results revealed a partial overlap between genes required for alcohol tolerance and those required for thermotolerance. Genes involved in cell wall integrity and the actin cytoskeleton are required for both alcohol tolerance and thermotolerance, whereas the RNA polymerase II mediator complex seems to be specific to heat tolerance. However, no significant overlap of genes required for osmotic stress and oxidative stress with those required for other stresses was observed. Interestingly, although mitochondrial function is likely involved in tolerance to several stresses, it was found to be less important for thermotolerance. The genes identified in this study should be helpful for future research into the molecular mechanisms of stress response.

Saccharomyces cerevisiae Deltasiw14 disruptant exhibits caffeine sensitivity. To understand the function of Siw14, double disruptants for SIW14 and each of 102 viable protein kinases (PKase) genes were constructed and examined for suppression of caffeine sensitivity based on the premise that the sensitivity was caused either by accumulation of an unknown phosphorylated Siw14 substrate(s) or by depletion of an unphosphorylated substrate(s) of Siw14 in the Deltasiw14 disruptant. Among 102 pkase disruptions, only one, Deltanpr1, suppressed the caffeine sensitivity of the Deltasiw14 disruptant. Because Gln3 (a phosphorylated transcriptional activator)-dependent transcription is induced by disruption of NPR1, we further examined the effect of disruption and overexpression of GLN3 on the caffeine sensitivity of the Deltasiw14 disruptant. Disruption of GLN3 was found to partially suppress the caffeine sensitivity of the Deltasiw14 disruptant, while overexpression of GLN3 in wild-type cells caused caffeine sensitivity, providing the first evidence that Siw14 functions in the Gln3 regulatory network. We also found that, unlike in a wild-type background, Gln3 accumulates in the nucleus whether cells are exposed or not to caffeine in the Deltasiw14 disruptant, and that this nuclear localization was abolished by disruption of NPR1. Interestingly, the level of Gln3 phosphorylation in both the Deltasiw14 and Deltanpr1 disruptants decreased relative to wild type, independent of exposure to caffeine. We conclude that Siw14 controls the intracellular localization of Gln3 in combination with Npr1, and one of the causes for the caffeine sensitivity of the Deltasiw14 disruptant was an accumulation of dephosphorylated Gln3 in the nucleus.

We describe the cloning and functional characterization of the fatty acid elongase gene HpELO2, a homologue of the HpELO1 gene required for the production of C24:0 in the yeast Hansenula polymorpha. The open reading frame (ORF) of HpELO2 consists of 1,035 bp, encoding 344 amino acids, sharing about 65% identity with that of Saccharomyces cerevisiae Elo2. Expression of HpELO2 rescued the lethality of the S. cerevisiae elo2Delta elo3Delta double disruptant. An accumulation of C18:0 and a significant increase and decrease in the levels of C24:0 and C26:0, respectively, were observed in the Hpelo2Delta disruptant. These results supported an idea that HpELO2 encodes a fatty acid elongase involved in the elongation of C18:0 to very long-chain fatty acids. The Hpelo1Delta Hpelo2Delta double disruption was nonviable, suggesting that HpELO1 and HpELO2 are the only two genes necessary for the biosynthesis in H. polymorpha. Interestingly, transcription of HpELO2 and HpELO1 were found to be transiently up-regulated by exogenous long-chain fatty acids; however, this up-regulation was not observed with HpELO1 and HpELO2 genes driven by the constitutively expressed promoter of the HpACT gene, suggesting that exogenous fatty acids specifically trigger the transcriptional induction of HpELO1 and HpELO2 through their promoter regions.

To understand the biosynthetic network of fatty acids in the methylotrophic yeast Hansenula polymorpha, which is able to produce poly-unsaturated fatty acids, we have attempted to identify genes encoding fatty acid elongase. Here we have characterized HpELO1, a fatty acid elongase gene encoding a 319-amino-acid protein containing five predicted membrane-spanning regions that is conserved throughout the yeast Elo protein family. Phylogenetic analysis of the deduced amino acid sequence suggests that HpELO1 is an ortholog of the Saccharomyces cerevisiae ELO3 gene that is involved in the elongation of very long-chain fatty acids (VLCFAs). In the fatty acid profile of the Hpelo1Delta disruptant by gas chromatography/mass spectrometry, the amount of C24:0 and C26:0 decreased to undetectable levels, whereas there was a large accumulation of C22:0, suggesting that the HpELO1 is involved in the elongation of VLCFAs and is essential for the production of C24:0. Expression of HpELO1 suppressed the lethality of the S. cerevisiae elo2Delta elo3Delta double disruptant and recovered the synthesis of VLCFAs. Similar to the S. cerevisiae elo3Delta strain, the Hpelo1Delta disruptant exhibited the extraordinary growth sensitivity to fumonisin B(1), a ceramide synthase inhibitor. Furthermore, cells of the Hpelo1Delta disruptant were more sensitive to Zymolyase and more flocculent than the wild-type cells, clumping together and falling rapidly out of suspension, suggesting that the Hpelo1Delta mutation causes changes in cell wall composition and structure.

Saccharomyces cerevisiae, for centuries the yeast that has been the workhorse for the fermentative production of ethanol, is now also a model system for biological research. The recent development of chromosome-splitting techniques has enabled the manipulation of the yeast genome on a large scale, and this has allowed us to explore questions with both biological and industrial relevance, the number of genes required for growth and the genome organization responsible for the ethanol production. To approach these questions, we successively deleted portions of the yeast genome and constructed a mutant that had lost about 5% of the genome and that gave an increased yield of ethanol and glycerol while showing levels of resistance to various stresses nearly equivalent to those of the parental strain. Further systematic deletion could lead to the formation of a eukaryotic cell with a minimum set of genes exhibiting appropriately altered regulation for enhanced metabolite production.

Norfloxacin belongs to the group of fluoroquinolone antibiotics which has been approved for treatment in animals. However, its residues in animal products can pose adverse side effects to consumer. Therefore, detection of the residue in different food matrices must be concerned. In this study, a single chain variable fragment (scFv) that recognizes norfloxacin antibiotic was constructed. The cDNA was synthesized from total RNA of hybridoma cells against norfloxacin. Genes encoding VH and VL regions of monoclonal antibody against norfloxacin (Nor155) were amplified and size of VH and VL fragments was 402 bp and 363 bp, respectively. The scFv of Nor155 was constructed by an addition of (Gly4Ser)3 as a linker between VH and VL regions and subcloned into pPICZαA, an expression vector of Pichia pastoris. The sequence of scFv Nor155 (GenBank No. AJG06891.1) was confirmed by sequencing analysis. The complementarity determining regions (CDR) I, II, and III of VH and VL were specified by Kabat method. The obtained recombinant plasmid will be useful for production of scFv antibody against norfloxacin in P. pastoris and further engineer scFv antibody against fluoroquinolone antibiotics.

Segmental aneuploidy can play an important role in environmental adaptation. However, study of segmental aneuploids is severely hampered by the difficulty of creating them in a designed fashion. Here, we describe a PCR-mediated chromosome duplication (PCDup) technology that enables the generation of segmental aneuploidy at any desired chromosomal region in Saccharomyces cerevisiae. We constructed multiple strains harboring 100 kb to 200 kb segmental duplications covering the whole of the S. cerevisiae genome. Interestingly, some segmental aneuploidies confer stress tolerance, such as to high temperature, ethanol and strong acids, while others induce cell lethality and stress sensitivity, presumably as result of the simultaneous increases in dosages of multiple genes. We suggest that our PCDup technology will accelerate studies into the phenotypic changes resulting from alteration of gene dosage balance of multiple genes and will provide new insights into the adaptive molecular mechanisms in the genome in segmental aneuploidy-derived human diseases.

We isolated a strain carrying a recessive constitutive mutation (thi81) for the expression of thiamine metabolism in Saccharomyces cerevisiae. The thi81 mutant exhibits significant thiamine transport, thiamine-repressible acid phosphatase (T-rAPase) activities and significant activities of enzymes involved in thiamine biosynthesis which are repressed in the wild-type strain in medium supplemented with thiamine (2 x 10(-7) M). The thi81 mutant exhibited the same level of thiamine pyrophosphokinase activity and intracellular thiamine pyrophosphate concentration as the wild-type strain in medium supplemented with exogenous thiamine. The mutant strain constitutively produced PHO3 mRNA encoding T-rAPase in medium supplemented with thiamine. These results suggest that the thi81 mutant lacks a negative factor involved in the regulation of the genes encoding proteins involved in yeast thiamine metabolism.

Yeast mating type is determined by the genotype at the mating type locus (MAT). In homothallic (self-fertile) Saccharomycotina such as Saccharomyces cerevisiae and Kluveromyces lactis, high-efficiency switching between a and α mating types enables mating. Two silent mating type cassettes, in addition to an active MAT locus, are essential components of the mating type switching mechanism. In this study, we investigated the structure and functions of mating type genes in H. polymorpha (also designated as Ogataea polymorpha). The H. polymorpha genome was found to harbor two MAT loci, MAT1 and MAT2, that are ∼18 kb apart on the same chromosome. MAT1-encoded α1 specifies α cell identity, whereas none of the mating type genes were required for a identity and mating. MAT1-encoded α2 and MAT2-encoded a1 were, however, essential for meiosis. When present in the location next to SLA2 and SUI1 genes, MAT1 or MAT2 was transcriptionally active, while the other was repressed. An inversion of the MAT intervening region was induced by nutrient limitation, resulting in the swapping of the chromosomal locations of two MAT loci, and hence switching of mating type identity. Inversion-deficient mutants exhibited severe defects only in mating with each other, suggesting that this inversion is the mechanism of mating type switching and homothallism. This chromosomal inversion-based mechanism represents a novel form of mating type switching that requires only two MAT loci., Author Summary The mating system of Saccharomycotina has evolved from the ancestral heterothallic system as seen in Yarrowia lipolytica to homothallism as seen in Saccharomyces cerevisiae. The acquisition of silent cassettes was an important step towards homothallism. However, some Saccharomycotina species that diverged from the common ancestor before the acquisition of silent cassettes are also homothallic, including Hansenula polymorpha. We investigated the structure and functions of the mating type locus (MAT) in H. polymorpha, and found two MAT loci, MAT1 and MAT2. Although MAT1 contains both a and α information, the results suggest that it functions as MATα. MAT a is represented by MAT2, which is located at a distance of 18 kb from MAT1. The functional repression of MAT1 or MAT2 was required to establish a or α mating type identity in individual cells. The chromosomal location of MAT1 and MAT2 was found to influence their transcriptional status, with only one locus maintained in an active state. An inversion of the MAT intervening region resulted in the switching of the two MAT loci and hence of mating type identity, which was required for homothallism. This chromosomal inversion-based mechanism represents a novel form of mating type switching that requires two MAT loci, of which only one is expressed.

In Saccharomyces cerevisiae, the phosphate signal transduction PHO pathway is involved in regulating several phosphate-responsive genes such as PHO5, which encodes repressible acid phosphatase. In this pathway, a cyclin-dependent kinase inhibitor (Pho81p) regulates the kinase activity of the cyclin-cyclin-dependent kinase complex Pho80p-Pho85p, which phosphorylates the transcription factor Pho4p in response to intracellular phosphate levels. However, how cells sense phosphate availability and transduce the phosphate signal to Pho81p remains unknown. To identify additional components of the PHO pathway, we have screened a collection of yeast deletion strains. We found that disruptants of PLC1, ARG82, and KCS1, which are involved in the synthesis of inositol polyphosphate, and ADK1, which encodes adenylate kinase, constitutively express PHO5. Each of these factors functions upstream of Pho81p and negatively regulates the PHO pathway independently of intracellular orthophosphate levels. Overexpression of KCS1, but not of the other genes, suppressed PHO5 expression in the wild-type strain under low phosphate conditions. These results raise the possibility that diphosphoinositol tetrakisphosphate and/or bisdiphosphoinositol triphosphate may be essential for regulation of the PHO pathway. Furthermore, the Deltaplc1, Deltaarg82, and Deltakcs1 deletion strains, but not the Deltaipk1 deletion strain, had significantly reduced intracellular polyphosphate levels, suggesting that enzymes involved in inositol pyrophosphate synthesis are also required for polyphosphate accumulation.

Hydrogen induced disproportionation and reproportionation for Zr2Fe, Zr2Co and Zr2Ni were studied by means of volumetric measurements of hydrogen absorption and desorption, and X-ray diffraction analyses of the product phases. These alloys formed respective hydrides of Zr2MH5 (M=Fe, Co and Ni) type at room temperature only under conditions of very slow hydrogen absorption, otherwise the alloys disproportionated. Heating Zr2MH5 to 1073 K gave Zr2Ni and Zr2Co through decomposition, disproportionation and reproportionation. On the other hand, heating Zr2FeH5 to 1073 K yielded ZrFe2 and Zr3Fe. At the elevated temperature of 773 K, each alloy disproportionated very fast, within several tens of seconds, to ZrH2 and Zr-deficient alloys such as ZrCo, ZrNi and ZrFe2 for Zr2Co, Zr2Ni and Zr2Fe, respectively. ZrCo and ZrNi further disproportionated to ZrCo2 and Zr7Ni10 at this temperature, but the rates were very slow. These observations showed that the stability to hydrogen induced disproportionation is in the order of Zr2Ni>Zr2Co>Zr2Fe. This order is also valid for the ease of reproportionation, although the behavior of Zr2Fe was completely different from the others.

Chromosome engineering techniques that can manipulate a large segment of chromosomal DNA are useful not only for studying the organization of eukaryotic genomes but also for the improvement of industrially important strains. Toward the development of techniques that can efficiently manipulate a large segment of chromosome, we have previously reported a one-step chromosome splitting technique in a haploid Saccharomyces cerevisiae cell, with which we could successfully split yeast chromosome 11, XIII, or XI into two halves to create a haploid strain having 17 chromosomes. We have now constructed chromosome splitting vectors bearing ADE2, HIS3, LEU2, or TRP1 marker, and by using these vectors, we could successively split yeast chromosomes to create a novel yeast haploid strain having up to 21 chromosomes. The specific growth rates of yeast strains carrying more than 16 chromosomes up to 21 did not differ significantly, suggesting that yeast cells can harbor more chromosomes than they do in their natural state, that is, 16 chromosomes, without serious effects on their growth.

VDE is a homing endonuclease gene originally discovered as an intervening element in VMA1s of Saccharomyces cerevisiae. There have been two independent subfamilies of VDE, one from S. cerevisiae strain X2180-1A and the other from Saccharomyces sp. DH1-1A in the host VMA1 gene, and they share the identity of 96.3%. In order to search the occurrence, intra/interspecies transfer and molecular degeneration of VDE, complete sequences of VMA1 in 10 strains of S. cerevisiae, eight species of saccharomycete yeasts, Candida glabrata and Kluyveromyces lactis were determined. We found that six of 10 S. cerevisiae strains contain VDEs 99.7–100% identical to that of the strain X2180-1A, one has no VDE, whereas the other three harbour VDEs 100% identical to that of the strain DH1-1A. S. carlsbergensis has two VMA1s, one being 99.8% identical to that of the strain X2180-1A with VDE 100% identical to that of the strain DH1-1A and the other containing the same VMA1 in S. pastorianus with no VDE. This and other evidence indicates that intra/interspecies transmissions of VDEs have occurred among saccharomycete yeasts. Phylogenetic analyses of VMA1 and VDE suggest that the S. cerevisiae VDEs had branched earlier than other VDEs from an ancestral VDE and had invaded into the host loci as relatively late events. The two VDEs seemed to degenerate in individual host loci, retaining their splicing capacity intact. The degeneration of the endonuclease domains was distinct and, if compared, its apparent rate was much faster than that of the protein-splicing domains. The VMA1 gene sequences determined in this study have been deposited in the GenBank data library under the Accession Nos shown in Table 1. Copyright © 2003 John Wiley & Sons, Ltd.

Saccharomyces sensu stricto is a species complex that includes most of the yeast strains relevant in the fermentation industry as well as in basic science. The taxonomy of these yeasts has always been controversial, particularly at species level. Over the years, the grouping of Saccharomyces sensu stricto yeasts has undergone changes in accordance with the system employed in classifying yeast cultures. Names of species and single isolates have also undergone changes that have caused confusion for yeast scientists and fermentation technologists. Recent findings have demonstrated that Saccharomyces hayanus and S. pastorianus are not homogeneous and do not seem to be natural groups. The present work examines the current trends in systematics studies, evidences the importance and mechanism of genetic variation and explores the most recent evolutionary theories as a way to elucidate the mechanism of speciation and achieve a more natural grouping of Saccharomyces sensu stricto species.

Thirty-two protein phosphatase (PPase) genes were identified in Saccharomyces cerevisiae based on the nucleotide sequences of the entire genome. In an effort to understand the role of PPases and their functional redundancy in the cellular physiology of one of the reference eukaryotic organisms, a series of single and double PPase gene disruptants were constructed in the W303 strain background. Two single disruptants for the CDC14 and GLC7 genes were lethal. Double disruptants for 30 non-essential PPase genes were constructed in all possible 435 combinations. No double disruptant showed synthetic lethality. Several phenotypes of the viable 30 single and 435 double disruptants were examined; temperature-sensitive growth, utilization of carbon sources and sensitivity to cations and drugs. Four double disruptants exhibited synthetic phenotypes in addition to eight single ones: the pph21pph22 double disruptant showed slow growth on complete medium, as did the sit4 and yvh1 single ones. In addition to the ptc1, ynr022c and ycr079w single disruptants, the ppz1ppz2 double disruptant showed temperature-sensitive slow growth. The msg5 ptp2 double disruptant, like the ynr022c single one, did not grow on complete medium containing 0.3 M CaCl2. The double msg5 ptc2 disruptant failed to grow on medium containing 1.0 M NaCl and, like the ynr022c single deletion, also could not grow on medium containing 0.3 M CaCl2. The synthetic phenotypes in the two latter cases where each of the PPases is categorized in a different phosphatase family led us to discuss the novel mechanism involved in the functional redundancy of the PPases. Copyright © 2002 John Wiley & Sons, Ltd.

Chromosome engineering enables large-scale genome manipulation and can be used as a novel technology for breeding of yeasts. PCR-mediated chromosome splitting (PCS) offers a powerful tool for chromosome engineering by enabling a yeast chromosome to be split at any desired site. By applying PCS, a huge variety of chromosome combinations can be created and the best strain under specific conditions can be selected-a technology that we have called genome reorganization. Once the optimal strain is obtained, chromosome constitutions need to be maintained stably; however, mini-chromosomes of less than 50 kb are at relatively high frequency lost during cultivation. To overcome this problem, in this study we screened for multicopy suppressors of the high loss of mini-chromosomes by using a multicopy genomic library of Saccharomyces cerevisiae. We identified a novel gene, YCR041W, that stabilizes mini-chromosomes. The translational product of YCR041W was suggested to play an important role in increasing stability for mini-chromosome maintenance, probably by decreasing the rate of loss during mitotic cell division. The stabilization of mini-chromosomes conferred by YCR041W overexpression was completely dependent on the silencing protein Sir4, suggesting that a process related to telomere function might be involved in mini-chromosome stabilization. Overexpression of YCR041W stabilized not only a yeast artificial chromosome vector, but also a mini-chromosome derived from a natural chromosome. Taking these results together, we propose that YCR041W overexpression can be used as a novel chromosome engineering tool for controlling mini-chromosome maintenance and loss.

Improvement of the lactic acid resistance of the yeast Saccharomyces cerevisiae is important for the application of the yeast in industrial production of lactic acid from renewable resources. However, we still do not know the precise mechanisms of the lactic acid adaptation response in yeast and, consequently, lack effective approaches for improving its lactic acid tolerance. To enhance our understanding of the adaptation response, we screened for S. cerevisiae genes that confer enhanced lactic acid resistance when present in multiple copies and identified the transcriptional factor Haa1 as conferring resistance to toxic levels of lactic acid when overexpressed. The enhanced tolerance probably results from increased expression of its target genes. When cells that expressed Haa1 only from the endogenous promoter were exposed to lactic acid stress, the main subcellular localization of Haa1 changed from the cytoplasm to the nucleus within 5 min. This nuclear accumulation induced upregulation of the Haa1 target genes YGP1 , GPG1 , and SPI1 , while the degree of Haa1 phosphorylation observed under lactic acid-free conditions decreased. Disruption of the exportin gene MSN5 led to accumulation of Haa1 in the nucleus even when no lactic acid was present. Since Msn5 was reported to interact with Haa1 and preferentially exports phosphorylated cargo proteins, our results suggest that regulation of the subcellular localization of Haa1, together with alteration of its phosphorylation status, mediates the adaptation to lactic acid stress in yeast.

Fatty acid desaturation catalyzed by fatty acid desaturases requires molecular oxygen (O 2 ). Saccharomyces cerevisiae cells derepress expression of OLE1 encoding Δ9 fatty acid desaturase under hypoxic conditions to allow more-efficient use of limited O 2 . It has been proposed that aerobic conditions lead to repression of OLE1 by well-established O 2 -responsive repressor Rox1p, since putative binding sequences for Rox1p are present in the promoter of OLE1 . However, we revealed in this study that disruption of ROX1 unexpectedly did not affect the O 2 repression of OLE1 , indicating that a Rox1p-independent novel mechanism operates for this repression. We identified by promoter deletion analysis the 50-bp O 2 -regulated (O2R) element in the OLE1 promoter approximately 360 bp upstream of the start codon. Site-directed mutagenesis of the O2R element showed that the putative binding motif (5′-GATAA-3′) for the GATA family of transcriptional factors is important for O 2 repression. Anaerobic derepression of OLE1 transcription was repressed by unsaturated fatty acids (UFAs), and interestingly the O2R element was responsible for this UFA repression despite not being included within the fatty acid-regulated (FAR) element previously reported. The fact that such a short 50-bp O2R element responds to both O 2 and UFA signals implies that O 2 and UFA signals merge in the ultimate step of the pathways. We discuss the differential roles of FAR and O2R elements in the transcriptional regulation of OLE1.

Modern biological scientists employ numerous approaches for solving their problems. Among these approaches, the gene fusion is surely one of the well-established valuable tools in various fields of biological sciences. A wide range of applications have been developed to analyze a variety of biological phenomena such as transcriptional regulation, pre-mRNA processing, mRNA decay, translation, protein localization and even protein transport in both prokaryotic and eukaryotic organisms. Gene fusions were also used for the study of protein purification, protein structure, protein folding, protein-protein interaction and protein-DNA interaction. Here, we describe applications of gene fusion technology using the Saccharomyces cerevisiae PHO5 gene encoding repressible acid phosphatase to molecular genetics and biotechnology in S. cerevisiae. Using the PHO5 gene fusion as a reporter, we have identified several cis- and trans-acting genes of S. cerevisiae which are involved in splicing of pre-mRNA, biosynthesis of amino acids, ubiquitin-dependent protein degradation, signal transduction of oxygen and unsaturated fatty acid, regulation of transcription by the nucleosome and chromatin. The PHO5 gene fusions exhibiting the mating-type specific expression were also generated to develop a breeding technique for industrial yeast. It is concluded that the PHO5 gene fusion is extremely useful and should be further exploited to investigate various cellular steps of the eukaryotic gene expression.