Search

In this paper, we prove the presence of limit cycles of given multiplicity, together with a complete unfolding, in families of (singularly perturbed) polynomial Liénard equations. The obtained limit cycles are relaxation oscillations. Both classical Liénard equations and generalized Liénard equations are treated. [ABSTRACT FROM AUTHOR]

Background: Acetic acid is one of the major inhibitors in lignocellulose hydrolysates used for the production of second-generation bioethanol. Although several genes have been identified in laboratory yeast strains that are required for tolerance to acetic acid, the genetic basis of the high acetic acid tolerance naturally present in some Saccharomyces cerevisiae strains is unknown. Identification of its polygenic basis may allow improvement of acetic acid tolerance in yeast strains used for second-generation bioethanol production by precise genome editing, minimizing the risk of negatively affecting other industrially important properties of the yeast., Results: Haploid segregants of a strain with unusually high acetic acid tolerance and a reference industrial strain were used as superior and inferior parent strain, respectively. After crossing of the parent strains, QTL mapping using the SNP variant frequency determined by pooled-segregant whole-genome sequence analysis revealed two major QTLs. All F1 segregants were then submitted to multiple rounds of random inbreeding and the superior F7 segregants were submitted to the same analysis, further refined by sequencing of individual segregants and bioinformatics analysis taking into account the relative acetic acid tolerance of the segregants. This resulted in disappearance in the QTL mapping with the F7 segregants of a major F1 QTL, in which we identified HAA1, a known regulator of high acetic acid tolerance, as a true causative allele. Novel genes determining high acetic acid tolerance, GLO1, DOT5, CUP2, and a previously identified component, VMA7, were identified as causative alleles in the second major F1 QTL and in three newly appearing F7 QTLs, respectively. The superior HAA1 allele contained a unique single point mutation that significantly improved acetic acid tolerance under industrially relevant conditions when inserted into an industrial yeast strain for second-generation bioethanol production., Conclusions: This work reveals the polygenic basis of high acetic acid tolerance in S. cerevisiae in unprecedented detail. It also shows for the first time that a single strain can harbor different sets of causative genes able to establish the same polygenic trait. The superior alleles identified can be used successfully for improvement of acetic acid tolerance in industrial yeast strains.

Very high ethanol tolerance is a distinctive trait of the yeast Saccharomyces cerevisiae with notable ecological and industrial importance. Although many genes have been shown to be required for moderate ethanol tolerance (i.e., 6 to 12%) in laboratory strains, little is known of the much higher ethanol tolerance (i.e., 16 to 20%) in natural and industrial strains. We have analyzed the genetic basis of very high ethanol tolerance in a Brazilian bioethanol production strain by genetic mapping with laboratory strains containing artificially inserted oligonucleotide markers. The first locus contained the ura3Δ0 mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage for LYS2, LEU2, HIS3, and MET15. Tolerance to only very high ethanol concentrations was reduced by auxotrophies, while the effect was reversed at lower concentrations. Evaluation of other stress conditions showed that the link with auxotrophy is dependent on the type of stress and the type of auxotrophy. When the concentration of the auxotrophic nutrient is close to that limiting growth, more stress factors can inhibit growth of an auxotrophic strain. We show that very high ethanol concentrations inhibit the uptake of leucine more than that of uracil, but the 500-fold-lower uracil uptake activity may explain the strong linkage between uracil auxotrophy and ethanol sensitivity compared to leucine auxotrophy. Since very high concentrations of ethanol inhibit the uptake of auxotrophic nutrients, the active uptake of scarce nutrients may be a major limiting factor for growth under conditions of ethanol stress., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Circular DNA elements are involved in genome plasticity, particularly of tandem repeats. However, amplifications of DNA segments in Saccharomyces cerevisiae reported so far involve pre-existing repetitive sequences such as ribosomal DNA, Ty elements and Long Terminal Repeats (LTRs). Here, we report the generation of an eccDNA, (extrachromosomal circular DNA element) in a region without any repetitive sequences during an adaptive evolution experiment. We performed whole genome sequence comparison between an efficient D-xylose fermenting yeast strain developed by metabolic and evolutionary engineering, and its parent industrial strain. We found that the heterologous gene XylA that had been inserted close to an ARS sequence in the parent strain has been amplified about 9 fold in both alleles of the chromosomal locus of the evolved strain compared to its parent. Analysis of the amplification process during the adaptive evolution revealed formation of a XylA-carrying eccDNA, pXI2-6, followed by chromosomal integration in tandem arrays over the course of the evolutionary adaptation. Formation of the eccDNA occurred in the absence of any repetitive DNA elements, probably using a micro-homology sequence of 8 nucleotides flanking the amplified sequence. We isolated the pXI2-6 eccDNA from an intermediate strain of the evolutionary adaptation process, sequenced it completely and showed that it confers high xylose fermentation capacity when it is transferred to a new strain. In this way, we have provided clear evidence that gene amplification can occur through generation of eccDNA without the presence of flanking repetitive sequences and can serve as a rapid means of adaptation to selection pressure.

Background: In addition to efficient pentose utilization, high inhibitor tolerance is a key trait required in any organism used for economically viable industrial bioethanol production with lignocellulose biomass. Although recent work has succeeded in establishing efficient xylose fermentation in robust industrial Saccharomyces cerevisiae strains, the resulting strains still lacked sufficient inhibitor tolerance for efficient sugar fermentation in lignocellulose hydrolysates. The aim of the present work was to combine high xylose fermentation activity and high inhibitor tolerance in a single industrial yeast strain., Results: We have screened 580 yeast strains for high inhibitor tolerance using undetoxified acid-pretreated spruce hydrolysate and identified a triploid industrial baker's yeast strain as having the highest inhibitor tolerance. From this strain, a mating competent diploid segregant with even higher inhibitor tolerance was obtained. It was crossed with the recently developed D-xylose fermenting diploid industrial strain GS1.11-26, with the Ethanol Red genetic background. Screening of 819 diploid segregants from the tetraploid hybrid resulted in two strains, GSF335 and GSF767, combining high inhibitor tolerance and efficient xylose fermentation. In a parallel approach, meiotic recombination of GS1.11-26 with a haploid segregant of Ethanol Red and screening of 104 segregants resulted in a similar inhibitor tolerant diploid strain, GSE16. The three superior strains exhibited significantly improved tolerance to inhibitors in spruce hydrolysate, higher glucose consumption rates, higher aerobic growth rates and higher maximal ethanol accumulation capacity in very-high gravity fermentation, compared to GS1.11-26. In complex medium, the D-xylose utilization rate by the three superior strains ranged from 0.36 to 0.67 g/g DW/h, which was lower than that of GS1.11-26 (1.10 g/g DW/h). On the other hand, in batch fermentation of undetoxified acid-pretreated spruce hydrolysate, the three superior strains showed comparable D-xylose utilization rates as GS1.11-26, probably because of their higher inhibitor tolerance. They produced up to 23% more ethanol compared to Ethanol Red., Conclusions: We have successfully constructed three superior industrial S. cerevisiae strains that combine efficient D-xylose utilization with high inhibitor tolerance. Since the background strain Ethanol Red has a proven record of successful industrial application, the three new superior strains have strong potential for direct application in industrial bioethanol production.

Background: The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production., Results: An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate., Conclusions: An industrial yeast strain for bioethanol production with lignocellulose hydrolysates has been developed in the genetic background of a strain widely used for commercial bioethanol production. The strain uses glucose and D-xylose with high consumption rates and partial cofermentation in various lignocellulose hydrolysates with very high ethanol yield. The GS1.11-26 strain shows highly promising potential for further development of an all-round robust yeast strain for efficient fermentation of various lignocellulose hydrolysates.

The yeast Saccharomyces cerevisiae is able to accumulate ≥17% ethanol (v/v) by fermentation in the absence of cell proliferation. The genetic basis of this unique capacity is unknown. Up to now, all research has focused on tolerance of yeast cell proliferation to high ethanol levels. Comparison of maximal ethanol accumulation capacity and ethanol tolerance of cell proliferation in 68 yeast strains showed a poor correlation, but higher ethanol tolerance of cell proliferation clearly increased the likelihood of superior maximal ethanol accumulation capacity. We have applied pooled-segregant whole-genome sequence analysis to identify the polygenic basis of these two complex traits using segregants from a cross of a haploid derivative of the sake strain CBS1585 and the lab strain BY. From a total of 301 segregants, 22 superior segregants accumulating ≥17% ethanol in small-scale fermentations and 32 superior segregants growing in the presence of 18% ethanol, were separately pooled and sequenced. Plotting SNP variant frequency against chromosomal position revealed eleven and eight Quantitative Trait Loci (QTLs) for the two traits, respectively, and showed that the genetic basis of the two traits is partially different. Fine-mapping and Reciprocal Hemizygosity Analysis identified ADE1, URA3, and KIN3, encoding a protein kinase involved in DNA damage repair, as specific causative genes for maximal ethanol accumulation capacity. These genes, as well as the previously identified MKT1 gene, were not linked in this genetic background to tolerance of cell proliferation to high ethanol levels. The superior KIN3 allele contained two SNPs, which are absent in all yeast strains sequenced up to now. This work provides the first insight in the genetic basis of maximal ethanol accumulation capacity in yeast and reveals for the first time the importance of DNA damage repair in yeast ethanol tolerance., Competing Interests: The authors have declared that no competing interests exist.

Revealing QTLs with a minor effect in complex traits remains difficult. Initial strategies had limited success because of interference by major QTLs and epistasis. New strategies focused on eliminating major QTLs in subsequent mapping experiments. Since genetic analysis of superior segregants from natural diploid strains usually also reveals QTLs linked to the inferior parent, we have extended this strategy for minor QTL identification by eliminating QTLs in both parent strains and repeating the QTL mapping with pooled-segregant whole-genome sequence analysis. We first mapped multiple QTLs responsible for high thermotolerance in a natural yeast strain, MUCL28177, compared to the laboratory strain, BY4742. Using single and bulk reciprocal hemizygosity analysis we identified MKT1 and PRP42 as causative genes in QTLs linked to the superior and inferior parent, respectively. We subsequently downgraded both parents by replacing their superior allele with the inferior allele of the other parent. QTL mapping using pooled-segregant whole-genome sequence analysis with the segregants from the cross of the downgraded parents, revealed several new QTLs. We validated the two most-strongly linked new QTLs by identifying NCS2 and SMD2 as causative genes linked to the superior downgraded parent and we found an allele-specific epistatic interaction between PRP42 and SMD2. Interestingly, the related function of PRP42 and SMD2 suggests an important role for RNA processing in high thermotolerance and underscores the relevance of analyzing minor QTLs. Our results show that identification of minor QTLs involved in complex traits can be successfully accomplished by crossing parent strains that have both been downgraded for a single QTL. This novel approach has the advantage of maintaining all relevant genetic diversity as well as enough phenotypic difference between the parent strains for the trait-of-interest and thus maximizes the chances of successfully identifying additional minor QTLs that are relevant for the phenotypic difference between the original parents., Competing Interests: The authors declare that they have no competing interests. The VIB has submitted a patent application on the use of the mutant alleles to improve thermotolerance in industrial yeast strains.

High ethanol tolerance is an exquisite characteristic of the yeast Saccharomyces cerevisiae, which enables this microorganism to dominate in natural and industrial fermentations. Up to now, ethanol tolerance has only been analyzed in laboratory yeast strains with moderate ethanol tolerance. The genetic basis of the much higher ethanol tolerance in natural and industrial yeast strains is unknown. We have applied pooled-segregant whole-genome sequence analysis to map all quantitative trait loci (QTL) determining high ethanol tolerance. We crossed a highly ethanol-tolerant segregant of a Brazilian bioethanol production strain with a laboratory strain with moderate ethanol tolerance. Out of 5974 segregants, we pooled 136 segregants tolerant to at least 16% ethanol and 31 segregants tolerant to at least 17%. Scoring of SNPs using whole-genome sequence analysis of DNA from the two pools and parents revealed three major loci and additional minor loci. The latter were more pronounced or only present in the 17% pool compared to the 16% pool. In the locus with the strongest linkage, we identified three closely located genes affecting ethanol tolerance: MKT1, SWS2, and APJ1, with SWS2 being a negative allele located in between two positive alleles. SWS2 and APJ1 probably contained significant polymorphisms only outside the ORF, and lower expression of APJ1 may be linked to higher ethanol tolerance. This work has identified the first causative genes involved in high ethanol tolerance of yeast. It also reveals the strong potential of pooled-segregant sequence analysis using relatively small numbers of selected segregants for identifying QTL on a genome-wide scale.

Background: Controlling nuclear maturation during oocyte culture might improve nuclear-cytoplasmic maturation synchrony. In the present study, the quality of mouse and human cumulus-enclosed oocytes (CEOs) was examined after a two-step culture consisting of a three-dimensional prematuration culture (3D-PMC), followed by in vitro maturation (IVM)., Methods: Mouse and human CEOs were embedded in an extracellular matrix (collagen-gel Type I). The gels containing the CEOs were cultured in medium with a phosphodiesterase 3-inhibitor (PDE3-I; cilostamide 1 microM) for 24 h. Afterwards, CEOs were removed from the gel and washed away from inhibitor then underwent IVM. The optimal concentration of collagen (diluted 1:2 versus not-diluted) was first determined in the mouse model. Cytoplasmic maturation after IVM of human and mouse oocytes was assessed in relation to fertilization and embryonic developmental capacity., Results: The diluted form of collagen was better for supporting the structure of the expanding CEOs and meiotic competence of the oocytes. Electron microscopy in combination with Lucifer Yellow dye coupling assay revealed that oocyte-cumulus cell connections could be preserved during 3D-PMC. Percentages of mouse 2-cell embryos after IVF were higher in the 3D-PMC group compared with in vitro controls and 2D-PMC oocytes, but lower compared with in vivo controls. In the human model, percentages of polar body-extruded oocytes were significantly higher in the 3D-PMC group compared with conventionally matured oocytes. The 3D-PMC also had a beneficial effect on embryonic development on Day 3 post-ICSI., Conclusions: Applying a 3D-PMC in the presence of a PDE3-I preserves oocyte-cumulus cell connections and influences oocyte developmental capacity.

To save energy, space, and time, today's breweries make use of high-gravity brewing in which concentrated medium (wort) is fermented, resulting in a product with higher ethanol content. After fermentation, the product is diluted to obtain beer with the desired alcohol content. While economically desirable, the use of wort with an even higher sugar concentration is limited by the inability of brewer's yeast (Saccharomyces pastorianus) to efficiently ferment such concentrated medium. Here, we describe a successful strategy to obtain yeast variants with significantly improved fermentation capacity under high-gravity conditions. We isolated better-performing variants of the industrial lager strain CMBS33 by subjecting a pool of UV-induced variants to consecutive rounds of fermentation in very-high-gravity wort (>22 degrees Plato). Two variants (GT336 and GT344) showing faster fermentation rates and/or more-complete attenuation as well as improved viability under high ethanol conditions were identified. The variants displayed the same advantages in a pilot-scale stirred fermenter under high-gravity conditions at 11 degrees C. Microarray analysis identified several genes whose altered expression may be responsible for the superior performance of the variants. The role of some of these candidate genes was confirmed by genetic transformation. Our study shows that proper selection conditions allow the isolation of variants of commercial brewer's yeast with superior fermentation characteristics. Moreover, it is the first study to identify genes that affect fermentation performance under high-gravity conditions. The results are of interest to the beer and bioethanol industries, where the use of more-concentrated medium is economically advantageous.

Little information is available about the precise mechanisms and determinants of freeze resistance in baker's yeast, Saccharomyces cerevisiae. Genomewide gene expression analysis and Northern analysis of different freeze-resistant and freeze-sensitive strains have now revealed a correlation between freeze resistance and the aquaporin genes AQY1 and AQY2. Deletion of these genes in a laboratory strain rendered yeast cells more sensitive to freezing, while overexpression of the respective genes, as well as heterologous expression of the human aquaporin gene hAQP1, improved freeze tolerance. These findings support a role for plasma membrane water transport activity in determination of freeze tolerance in yeast. This appears to be the first clear physiological function identified for microbial aquaporins. We suggest that a rapid, osmotically driven efflux of water during the freezing process reduces intracellular ice crystal formation and resulting cell damage. Aquaporin overexpression also improved maintenance of the viability of industrial yeast strains, both in cell suspensions and in small doughs stored frozen or submitted to freeze-thaw cycles. Furthermore, an aquaporin overexpression transformant could be selected based on its improved freeze-thaw resistance without the need for a selectable marker gene. Since aquaporin overexpression does not seem to affect the growth and fermentation characteristics of yeast, these results open new perspectives for the successful development of freeze-resistant baker's yeast strains for use in frozen dough applications.

The routine production and storage of frozen doughs are still problematic. Although commercial baker's yeast is highly resistant to environmental stress conditions, it rapidly loses stress resistance during dough preparation due to the initiation of fermentation. As a result, the yeast loses gassing power significantly during storage of frozen doughs. We obtained freeze-tolerant mutants of polyploid industrial strains following screening for survival in doughs prepared with UV-mutagenized yeast and subjected to 200 freeze-thaw cycles. Two strains in the S47 background with a normal growth rate and the best freeze tolerance under laboratory conditions were selected for production in a 20-liter pilot fermentor. Before frozen storage, the AT25 mutant produced on the 20-liter pilot scale had a 10% higher gassing power capacity than the S47 strain, while the opposite was observed for cells produced under laboratory conditions. AT25 also retained more freeze tolerance during the initiation of fermentation in liquid cultures and more gassing power during storage of frozen doughs. Other industrially important properties (yield, growth rate, nitrogen assimilation, and phosphorus content) were very similar. AT25 had only half of the DNA content of S47, and its cell size was much smaller. Several diploid segregants of S47 had freeze tolerances similar to that of AT25 but inferior performance for other properties, while an AT25-derived tetraploid, TAT25, showed only slightly improved freeze tolerance compared to S47. When AT25 was cultured in a 20,000-liter fermentor under industrial conditions, it retained its superior performance and thus appears to be promising for use in frozen dough production. Our results also show that a diploid strain can perform at least as well as a tetraploid strain for commercial baker's yeast production and usage.

The Saccharomyces cerevisiae gene YPA1 encodes a protein homologous to the phosphotyrosyl phosphatase activator, PTPA, of the mammalian protein phosphatase type 2A (PP2A). In order to examine the biological role of PTPA, we disrupted YPA1 and characterised the phenotype of the ypa1Delta mutant. Comparison of the growth rate of the wild-type strain and the ypa1Delta mutant on glucose-rich medium after nutrient depletion showed that the ypa1Delta mutant traversed the lag period more rapidly. This accelerated progression through "Start" was also observed after release from alpha-factor-induced G1 arrest as evidenced by a higher number of budding cells, a faster increase in CLN2 mRNA expression and a more rapid reactivation of Cdc28 kinase activity. This phenotype was specific for deletion of YPA1 since it was not observed when YPA2, the second PTPA gene in budding yeast was deleted. Reintroduction of YPA1 or the human PTPA cDNA in the ypa1Delta mutant suppressed this phenotype as opposed to overexpression of YPA2. Disruption of both YPA genes is lethal, since sporulation of heterozygous diploids resulted in at most three viable spores, none of them with a ypa1Delta ypa2Delta genotype. This observation indicates that YPA1 and YPA2 share some essential functions. We compared the ypa1Delta mutant phenotype with a PP2A double deletion mutant and a PP2A temperature-sensitive mutant. The PP2A-deficient yeast strain also showed accelerated progression through the G1 phase. In addition, both PP2A and ypa1Delta mutants show similar aberrant bud morphology. This would support the notion that YPA1 may act as a positive regulator of PP2A in vivo., (Copyright 2000 Academic Press.)

Yeast cells growing in the presence of glucose or a related rapidly-fermented sugar differ strongly in a variety of physiological properties compared to cells growing in the absence of glucose. Part of these differences appear to be caused by the protein kinase A (PKA) and related signal transduction pathways. Addition of glucose to cells previously deprived of glucose triggers cAMP accumulation, which is apparently mediated by the Gpr1-Gpa2 G-protein coupled receptor system. However, the resulting effect on PKA-controlled properties is only transient when there is no complete growth medium present. When an essential nutrient is lacking, the cells arrest in the stationary phase G0. At the same time they acquire all characteristics of cells with low PKA activity, even if there is ample glucose present. When the essential nutrient is added again, a similar PKA-dependent protein phosphorylation cascade is triggered as observed after addition of glucose to glucose-deprived cells, but which is not cAMP-mediated. Because the pathway involved requires a fermentable carbon source and a complete growth medium, at least for its sustained activation, it has been called "fermentable growth medium (FGM)-induced pathway."

In frozen dough applications a prefermentation period during the preparation of the dough is unavoidable and might also be important to obtain bread with a good texture. A major disadvantage of the prefermentation period is that it is associated with a rapid loss of the freeze resistance of the yeast cells. A major goal for the development of new baker's yeast strains for use in frozen dough applications is the availability of strains that maintain a better freeze resistance during the prefermentation period. We have isolated mutants that retain a better stress resistance during the initiation of fermentation. Some of these showed the same growth rate and fermentation capacity as the wild type cells. These mutants are called 'fil', for deficient infermentation induced loss of stress resistance. First we used laboratory strains and heat stress treatment, given shortly after the initiation of fermentation, as the selection protocol. The first two mutants isolated in this way were affected in the glucose-activation mechanism of the Ras-cAMP pathway. The fil1 mutant had a partially inactivating point mutation in CYR1, the gene encoding adenylate cyclase, while fil2 contained a nonsense mutation in GPR1. GPR1 encodes a member of the G-protein coupled receptor family which acts as a putative glucose receptor for activation of the Ras-cAMP pathway. In a next step we isolated fil mutants directly in industrial strains using repetitive freeze treatment of doughs as selection protocol. Surviving yeast strains were tested individually for maintenance of fermentation capacity after freeze treatment in laboratory conditions and also for the best performing strains in frozen doughs prepared with yeast cultivated on a pilot scale. The most promising mutant, AT25, displayed under all conditions a better maintenance of gassing power during freeze-storage. It was not affected in other commercially important properties and will now be characterised extensively at the biochemical and molecular level.

The cAMP-protein kinase A (PKA) pathway in the yeast Saccharomyces cerevisiae plays a major role in the control of metabolism, proliferation and stress resistance. Derepressed cells show a rapid increase in the cAMP level (within 1 min) after addition of glucose or after intracellular acidification. A specific mutation in adenylate cyclase, the enzyme that catalyzes the synthesis in cAMP, largely prevents both cAMP responses. The responsible mutation was originally called lcr1 (for lack of cAMP responses); lcr1 was later identified as allelic with CYR1/CDC35. The mutation was introduced into the CYR1 gene of a W303-1A wild type strain, which resulted in a large decrease in cAMP signalling. Furthermore, there was a strong reduction in GTP/Mg2+-stimulated but not in Mn2+-stimulated adenylate cyclase activity in isolated plasma membranes, which is consistent with the absence of signalling through adenylate cyclase in vivo. Glucose-induced activation of trehalase was reduced and mobilization of trehalose and glycogen and loss of stress resistance were delayed in the lcr1 mutant. Because of the absence of cAMP signalling during exponential growth on glucose, it was concluded that glucose-induced cAMP signalling is restricted to the transition from gluconeogenic/respiratory to fermentative growth. Activation of the PKA pathway is mediated by a G protein (either Ras1/Ras2 or Gpa2). Constitutive activation of the pathway by Ras2val19 or Gpa2val132 has a negative effect on glycogen and trehalose accumulation and heat shock survival. The lcr1 mutation partially suppresses this effect indicating that the target sites of the two G-proteins on adenylate cyclase might have at least a part in common.

When glucose is added to Saccharomyces cerevisiae cells grown into stationary phase or on non-fermentable carbon sources a rapid loss of heat stress resistance occurs. Mutants that retain high stress resistance after addition of glucose are called 'fil', for deficient in fermentation induced loss of stress resistance. Transformation of the fil1 mutant, which harbours a point mutation in adenylate cyclase, with a yeast gene library on a single copy plasmid resulted in transformants that were again stress-sensitive. One of the genes isolated in this way was a gene of previously unknown function. We have called it SFI1, for suppressor of fil1. SFI1 is an essential gene. Combination of Sfi1 and cAMP pathway mutations indicates that Sfi1 itself is not involved in the cAMP pathway. Conditional sfi1 mutants did not show enhanced heat resistance under the restrictive condition, whereas overexpression of SFI1 rendered cells heat-sensitive. Sfi1 may be a downstream target of the protein kinase A pathway, but its precise relationship with heat resistance remains unclear. Further analysis showed that Sfi1 is required for cell cycle progression, more specifically for progression through G(2)-M transition. Cells expressing SFI1 under the control of a galactose-inducible promoter arrest after addition of glucose as doublets of undivided mother and daughter cells. These doublets contain a single nucleus and lack mitotic spindles. Sfi1 shares homology with Xenopus laevis XCAP-C, a protein required for chromosome assembly. The conserved residues between these two proteins show a strong bias for charged amino acids. Hence, Sfi1 might be required for correct mitotic spindle assembly and its precise role might be in chromosome condensation. In conclusion, we have identified an essential function in the G(2)-M transition of the cell cycle for a yeast gene of previously unknown function., (Copyright 1999 John Wiley & Sons, Ltd.)