SnRK1/TOR/T6P: three musketeers guarding energy for root growth.

Sugars derived from photosynthesis, specifically sucrose, are the primary source of plant energy. Sucrose is produced in leaves and transported to the roots through the phloem, serving as a vital energy source. Environmental conditions can result in higher or lower photosynthesis, promoting anabolism or catabolism, respectively, thereby influencing the sucrose budget available for roots. Plants can adjust their root system to optimize the search for soil resources and to ensure the plant's adaptability to diverse environmental conditions. Recently, emerging research indicates that SNF1-RELATED PROTEIN KINASE 1 (SnRK1), trehalose 6-phosphate (T6P), and TARGET OF RAPAMYCIN (TOR) collectively serve as fundamental regulators of root development, together forming a signaling module to interpret the nutritional status of the plant and translate this to growth adjustments in the below ground parts.

p-Coumaric acid loaded into liquid crystalline systems as a novel strategy to the treatment of vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC) is an extremely common type of vaginal infection, which is mainly caused by Candida albicans. However, non-albicans Candida species are frequently more resistant to conventional antifungal agents and can represent up to 30% of cases. Due to side effects and increasing antifungal resistance presented by standard therapies, phenolic compounds, such as p-coumaric acid (p-CA), have been studied as molecules from natural sources with potential antifungal activity. p-CA is a poorly water-soluble compound, thus loading it into liquid crystals (LCs) may increase its solubility and effectiveness on the vaginal mucosa. Thereby, here we propose the development of mucoadhesive liquid crystalline systems with controlled release of p-CA, for the local treatment of VVC. Developed LCs consisted of fixed oily and aqueous phases (oleic acid and cholesterol (5:1) and poloxamer dispersion 16%, respectively), changing only the surfactant phase components (triethanolamine oleate (TEA-Oleate) or triethanolamine (TEA), the latter producing TEA-Oleate molecules when mixed with oleic acid). Systems were also diluted in artificial vaginal mucus (1:1 ratio) to mimic the vaginal environment and verify possible structural changes on formulations upon exposure to the mucosa. From the characterization assays, p-CA loaded TEA-Oleate systems presented mucoadhesive profile, liquid crystalline mesophases, well-organized structures and pseudoplastic behaviour, which are desirable parameters for topical formulations. Moreover, they were able to control the release of p-CA throughout the 12 h assay, as well as decrease its permeation into the vaginal mucosa. p-CA showed antifungal activity in vitro against reference strains of C. albicans (SC5314), C. glabrata (ATCC 2001) and C. krusei (ATCC 6258), and exhibited higher eradication of mature biofilms than amphotericin B and fluconazole. In vivo experiments demonstrated that the formulations reduced the presence of filamentous forms in the vaginal lavages and provided an improvement in swelling and redness present in the mice vaginal regions. Altogether, here we demonstrated the potential and feasibility of using p-CA loaded liquid crystalline systems as a mucoadhesive drug delivery system for topical treatment of VVC.

THE UNKNOWN SHADOWS OF TREHALASE.

Each year brings new facts concerning multiple roles of sugar pathways in plant metabolism. One of them--the trehalose pathway--has been shown to play a role in stress signalling. The last enzyme of this pathway--trehalase--has been proven to be strongly expressed in guard cells. Modifications of its abundance cause changes in stomatal closure and response to abscisic acid. Our phenotypical studies of different mutants of Arabidopsis thaliana and Musa have enabled us to propose a new function of trehalase. It might play a role in the feedback of sucrose as a closing signal for stomata in reaction to an efficient photosynthesis. To characterize the phenotype we measured: the dynamic cumulative water loss, the dynamic leaf surface temperature, and the stomatal conductance. Based on the obtained results we have determined the time points for a proteomics study. The exact role of trehalase and related proteins in the proposed mechanism will be defined with multiple analysis including mass spectrophotometry and enzymatic activities. The samples will be collected from a wide type of plants including model organism (Arabidopsis--wild type, trehalase mutant plants) and crops (banana). The final results will shed light on the complete role of trehalase and the feedback pathway.

Parameters affecting ethyl ester production by Saccharomyces cerevisiae during fermentation.

Volatile esters are responsible for the fruity character of fermented beverages and thus constitute a vital group of aromatic compounds in beer and wine. Many fermentation parameters are known to affect volatile ester production. In order to obtain insight into the production of ethyl esters during fermentation, we investigated the influence of several fermentation variables. A higher level of unsaturated fatty acids in the fermentation medium resulted in a general decrease in ethyl ester production. On the other hand, a higher fermentation temperature resulted in greater ethyl octanoate and decanoate production, while a higher carbon or nitrogen content of the fermentation medium resulted in only moderate changes in ethyl ester production. Analysis of the expression of the ethyl ester biosynthesis genes EEB1 and EHT1 after addition of medium-chain fatty acid precursors suggested that the expression level is not the limiting factor for ethyl ester production, as opposed to acetate ester production. Together with the previous demonstration that provision of medium-chain fatty acids, which are the substrates for ethyl ester formation, to the fermentation medium causes a strong increase in the formation of the corresponding ethyl esters, this result further supports the hypothesis that precursor availability has an important role in ethyl ester production. We concluded that, at least in our fermentation conditions and with our yeast strain, the fatty acid precursor level rather than the activity of the biosynthetic enzymes is the major limiting factor for ethyl ester production. The expression level and activity of the fatty acid biosynthetic enzymes therefore appear to be prime targets for flavor modification by alteration of process parameters or through strain selection.

Attachment of MAL32-encoded maltase on the outside of yeast cells improves maltotriose utilization.

The fermentation of maltotriose, the second most abundant fermentable sugar in wort, is often incomplete during high-gravity brewing. Poor maltotriose consumption is due to environmental stress conditions during high-gravity fermentation and especially to a low uptake of this sugar by some industrial strains. In this study we investigated whether the use of strains with an alpha-glucosidase attached to the outside of the cell might be a possible way to reduce residual maltotriose. To this end, the N-terminal leader sequence of Kre1 and the carboxy-terminal anchoring domain of either Cwp2 or Flo1 were used to target maltase encoded by MAL32 to the cell surface. We showed that Mal32 displayed on the cell surface of Saccharomyces cerevisiae laboratory strains was capable of hydrolysis of alpha-1,4-linkages, and that it increased the ability of a strain lacking a functional maltose permease to grow on maltotriose. Moreover, the enzyme was also expressed and found to be active in an industrial strain. These data show that expressing a suitable maltase on the cell surface might provide a means of modifying yeast for more complete maltotriose utilization in brewing and other fermentation applications.

Immunogold localization of trehalose-6-phosphate synthase in leaf segments of wild-type and transgenic tobacco plants expressing the AtTPS1 gene from Arabidopsis thaliana.

Following the establishment of a transgenic line of tobacco (B5H) expressing the trehalose-6-phosphate synthase (TPS) gene from Arabidopsis thaliana, a preliminary immunolocalization study was conducted using leaves of adequately watered B5H and wild-type plants. Immunocytochemical staining, followed by electron microscopy showed that the enzyme could be detected in both B5H and wild-type plants at two different levels. Quantification showed the signal to be two to three times higher in transgenic plants than in the wild type. This enzyme was markedly present in the vacuoles and the cell wall, and to a lesser extent in the cytosol. Moreover, a high profusion of gold particles was detected in adjacent cells and in the sieve elements. Occasional spots were also detected in chloroplasts and the nucleus, especially in the transgenic B5H line. No labeling signal was detected in mitochondria. Protein localization seems to confirm the important role of TPS in sugar metabolism and transport through the plant, which could explain its role in plant stress tolerance. Finally, it can be expected that TPS from tobacco has a relatively high similarity to the TPS of Arabidopsis thaliana.

Fungal pathogens research: novel and improved molecular approaches for the discovery of antifungal drug targets.

With the rise of fungal infection incidence amongst the patient population, the importance of developing new antifungal drug targets is higher than ever. This review mainly focuses on the three most prevalent fungal pathogens, Candida, Aspergillus and Cryptococcus, and on the most recent progresses in molecular research that contribute to a better understanding of the pathogen itself, but also its host and the interaction with its host. We consider the progress made in comparative genomics following the huge effort of fungal genome sequence projects undertaken in the last few years. We focus not only on currently used mammalian animal models such as mice, but also on novel non-mammalian models, such as the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster, which offer useful tools in the area of the innate immune response to fungal infections. In addition we relate to the recent genomic and proteomic studies and focus on the use of these approaches in in vivo experiments in the pathogen itself as well as in the host. Finally, we describe the latest targeted mutagenesis strategy available in C. albicans and the use of RNA interference in both Cryptococcus neoformans and A. fumigatus. Our aim is not to give an exhaustive list of all new strategies but rather to give an overview of what will contribute most to the identification of new antifungal drug targets and the establishment of novel antifungal strategies.

Nutrient sensing systems for rapid activation of the protein kinase A pathway in yeast.

The cAMP-protein kinase A (PKA) pathway in the yeast Saccharomyces cerevisiae controls a variety of properties that depend on the nutrient composition of the medium. High activity of the pathway occurs in the presence of rapidly fermented sugars like glucose or sucrose, but only as long as growth is maintained. Growth arrest of fermenting cells or growth on a respiratory carbon source, like glycerol or ethanol, is associated with low activity of the PKA pathway. We have studied how different nutrients trigger rapid activation of the pathway. Glucose and sucrose activate cAMP synthesis through a G-protein-coupled receptor system, consisting of the GPCR Gpr1, the Galpha protein Gpa2 and its RGS protein Rgs2. Glucose is also sensed intracellularly through its phosphorylation. Specific mutations in Gpr1 abolish glucose but not sucrose signalling. Activation of the PKA pathway by addition of a nitrogen source or phosphate to nitrogen- or phosphate-starved cells, respectively, is not mediated by an increase in cAMP. Activation by amino acids is triggered by the general amino acid permease Gap1, which functions as a transporter/receptor. Short truncation of the C-terminus results in constitutively activating alleles. Activation by ammonium uses the ammonium permeases Mep1 and Mep2 as receptor. Specific point mutations in Mep2 uncouple signalling from transport. Activation by phosphate is triggered a.o. by the Pho84 phosphate permease. Several mutations in Pho84 separating transport and signalling or triggering constitutive activation have been obtained.

Carbon source induced yeast-to-hypha transition in Candida albicans is dependent on the presence of amino acids and on the G-protein-coupled receptor Gpr1.

Yeast-to-hypha transition in Candida albicans can be induced by a wide variety of factors, including specific nutrients. We have started to investigate the mechanism by which some of these nutrients may be sensed. The G-protein-coupled receptor Gpr1 is required for yeast-to-hypha transition on various solid hypha-inducing media. Recently we have shown induction of Gpr1 internalization by specific amino acids, e.g. methionine. This suggests a possible role for methionine as a ligand of CaGpr1. Here we show that there is a big variation in methionine-induced hypha formation depending on the type of carbon source present in the medium. In addition high glucose concentrations repress hypha formation whereas a concentration of 0.1%, which mimics the glucose concentration present in the bloodstream, results in maximal hypha formation. Hence, it remains unclear whether Gpr1 senses sugars, as in Saccharomyces cerevisiae, or specific amino acids like methionine.

Safety evaluation of phosphodiesterase derived from Leptographium procerum.

5'-Phosphodiesterase is produced by fermentation of the fungus Leptographium procerum and is used to hydrolyse yeast RNA to produce flavour enhancers. To establish the safety in use of this enzyme preparation a number of studies have been performed: analysis for the potential of the production strain to produce toxic secondary metabolites, 28-days oral toxicity study of the preparation in the rat, bacterial mutation assay and in vitro mammalian chromosome aberration test in human lymphocytes. The production strain did not produce any secondary metabolites that may be of significance in food. Administration of dosage levels of 1250, 2500 and 5000 mg/kg body weight/day to rats for 28 day did not result in any toxicological significant changes. The enzyme preparation showed no mutagenic activity in the bacterial mutation assay and no clastogenic potency in an in vitro test. These results together with existing knowledge of the production organism and the chemical and microbiological characterisation of the enzyme preparation lead to the conclusion that the enzyme preparation containing 5'-phosphodiesterase activity from Leptographium procerum can safely be used for the production of flavour enhancers from bakers yeast at the anticipated intake levels for these uses.

On the safety of Aspergillus niger--a review.

Aspergillus niger is one of the most important microorganisms used in biotechnology. It has been in use already for many decades to produce extracellular (food) enzymes and citric acid. In fact, citric acid and many A. niger enzymes are considered GRAS by the United States Food and Drug Administration. In addition, A. niger is used for biotransformations and waste treatment. In the last two decades, A. niger has been developed as an important transformation host to over-express food enzymes. Being pre-dated by older names, the name A. niger has been conserved for economical and information retrieval reasons and there is a taxonomical consensus based on molecular data that the only other common species closely related to A. niger in the Aspergillus series Nigri is A. tubingensis. A. niger, like other filamentous fungi, should be treated carefully to avoid the formation of spore dust. However, compared with other filamentous fungi, it does not stand out as a particular problem concerning allergy or mycopathology. A few medical cases, e.g. lung infections, have been reported, but always in severely immunocompromised patients. In tropical areas, ear infections (otomycosis) do occur due to A. niger invasion of the outer ear canal but this may be caused by mechanical damage of the skin barrier. A. niger strains produce a series of secondary metabolites, but it is only ochratoxin A that can be regarded as a mycotoxin in the strict sense of the word. Only 3-10% of the strains examined for ochratoxin A production have tested positive under favourable conditions. New and unknown isolates should be checked for ochratoxin A production before they are developed as production organisms. It is concluded, with these restrictions, that A. niger is a safe production organism.

Containment in industrial biotechnology within wastewater treatment plants.

Both physical and biological containment are considered to be essential parts in the risk analysis of industrial Good Industrial Large-Scale Practice (GILSP) processes using genetically modified organisms (GMOs). Biological containment of industrial microorganisms has become a more important issue since the introduction of recombinant DNA techniques. In the event of an accidental discharge in the production plant, a large amount of organisms could be released into the wastewater treatment (WWT) system. This WWT system should therefore be considered as a part of the containment. This study demonstrates both a hydrodynamic and a microbiological model for the containment aspects of industrial WWT plants. The models are verified by measurements using industrial hosts of GILSP GMOs at full scale. Both models describe the full-scale equipment accurately. The results are supplemented with microcosm studies on survival of GMOs in defined niches. It is shown that WWT plants can be considered as useful additional parts of the containment of microorganisms, in case of an accidental discharge. The effect of drainage of an enormous amount of microorganisms (several tons) through the WWT plant into the environment is shown to be comparable to the direct drainage of a small-scale fermenter. Microcosm experiments correlate well with the survival rates in the WWT and therefore can be of use to predict the behaviour of GMOs in this environment.

An unexpected plethora of trehalose biosynthesis genes in Arabidopsis thaliana.

Trehalose accumulation has been documented in many organisms, such as bacteria and fungi, where it serves a storage and stress-protection role. Although conspicuously absent in most plants, trehalose biosynthesis genes were discovered recently in higher plants. We have uncovered a family of 11 TPS genes in Arabidopsis thaliana, one of which encodes a trehalose-6-phosphate (Tre6P) synthase, and a subfamily of which might encode the still elusive Tre6P phosphatases. A regulatory role in carbon metabolism is likely but might not be restricted to the TPS control of hexokinase activity as documented for yeast. Incompatibility between high trehalose levels and chaperone-assisted protein folding might be a reason why plants have evolved to accumulate some alternative stress-protection compounds to trehalose.

A baker's yeast mutant (fil1) with a specific, partially inactivating mutation in adenylate cyclase maintains a high stress resistance during active fermentation and growth.

The initiation of fermentation in the yeast Saccharomyces cerevisiae is associated with a rapid drop in stress resistance. This is disadvantageous for several biotechnological applications, e.g. the preparation of freeze doughs. We have isolated mutants in a laboratory strain which are deficient in fermentation-induced loss of stress resistance ('fil' mutants) using a heat shock selection protocol. We show that the fil1 mutant contains a mutation in the CYR1 gene which encodes adenylate cyclase. It causes a change at position 1682 of glutamate into lysine and results in a tenfold drop in adenylate cyclase activity. The fil1 mutant displays a reduction in the glucose-induced cAMP increase, trehalase activation and loss of heat resistance. Interestingly, the fil1 mutant shows the same growth and fermentation rate as the wild type strain, as opposed to other mutants with reduced activity of the cAMP pathway. Introduction of the fil1 mutation in the vigorous Y55 strain and cultivation of the mutant under pilot scale conditions resulted in a yeast that displayed a higher freeze and drought resistance during active fermentation compared to the wild type Y55 strain. These results show that high stress resistance and high fermentation activity are compatible biological properties. Isolation of fil-type mutations appears a promising avenue for development of industrial yeast strains with improved stress resistance during active fermentation.

Expression of escherichia coli otsA in a Saccharomyces cerevisiae tps1 mutant restores trehalose 6-phosphate levels and partly restores growth and fermentation with glucose and control of glucose influx into glycolysis.

The TPS1 gene, encoding trehalose-6-phosphate synthase (TPS), exerts an essential control on the influx of glucose into glycolysis in the yeast Saccharomyces cerevisiae. The deletion of TPS1 causes an inability to grow on glucose because of a hyperaccumulation of sugar phosphates and depletion of ATP and phosphate. We show that expression of the Escherichia coli homologue, otsA, in a yeast tps1 mutant results in high TPS activity. Although the trehalose 6-phosphate (Tre6P) level during exponential growth on glucose was at least as high as in a wild-type yeast strain, growth on glucose was only partly restored and the lag phase was much longer. Measurement of the glycolytic metabolites immediately after the addition of glucose showed that in spite of a normal Tre6P accumulation there was still a partial hyperaccumulation of sugar phosphates. Strong elevation of the Tre6P level by the additional deletion of the TPS2 gene, which encodes Tre6P phosphatase, was not able to cause a strong decrease in the sugar phosphate levels in comparison with the wild-type strain. In addition, in chemostat experiments the short-term response to a glucose pulse was delayed, but normal metabolism was regained over a longer period. These results show that Tre6P synthesis from a heterologous TPS enzyme can to some extent restore the control of glucose influx into glycolysis and growth on glucose in yeast. However, they also indicate that the yeast TPS enzyme, as opposed to the E. coli otsA gene product, is able to increase the efficiency of the Tre6P control on glucose influx into yeast glycolysis.

Nutrient-induced signal transduction through the protein kinase A pathway and its role in the control of metabolism, stress resistance, and growth in yeast.

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."

A specific mutation in Saccharomyces cerevisiae adenylate cyclase, Cyr1K176M, eliminates glucose- and acidification-induced cAMP signalling and delays glucose-induced loss of stress resistance.

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.

Characterization of a new set of mutants deficient in fermentation-induced loss of stress resistance for use in frozen dough applications.

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.

Identification of genes responsible for improved cryoresistance in fermenting yeast cells.

Using repetitive freezing and thawing, different mutant industrial Saccharomyces cerevisiae strains with increased freeze resistance have been isolated. To get a better insight in the mechanisms responsible for this elevated resistance and to give us the opportunity to modify other strains so that they become more suitable for use in frozen dough preparations, we applied the microarray technology in order to identify genes that are differentially expressed in a freeze-resistant mutant when compared to a freeze-sensitive industrial yeast strain.

Deletion of SFI1, a novel suppressor of partial Ras-cAMP pathway deficiency in the yeast Saccharomyces cerevisiae, causes G(2) arrest.

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.