The germ tubes of Candida albicans hyphae and pseudohyphae show different patterns of septin ring localization.

The location of the septin ring in the germ tubes of Candida albicans hyphae and pseudohyphae was studied using an antibody to Saccharomyces cerevisiae Cdc11p. In pseudohyphae induced by growth at 35 degrees C in YEPD or Lee's medium, a septin ring formed at or near (mean 1.8 microm) the neck between the mother cell and the germ tube. This became double later in the cycle, and the first mitosis took place across the plane of this double ring. A septin ring also formed at the germ tube neck of developing hyphae induced by serum or growth on Lee's medium at 37 degrees C. However, at later times, this ring became disorganized and disappeared. A second double ring then appeared 10-15 microm (mean 12.5 microm) along the length of the germ tube. The nucleus subsequently migrated out of the mother cell into the germ tube, and the first mitosis took place across the plane of this second septin ring. The relocation of the septin ring in developing hyphae provides a clear-cut molecular distinction between hyphae and pseudohyphae. Commitment to one type of septin localization and mitosis was shown to occur early in the first mitotic cycle, well before evagination. Germ tubes of hyphae and pseudohyphae also have different widths. A point of commitment to germ tube width was also demonstrated, but occurred later in the cycle, approximately coincident with the time of evagination.

Rad6p represses yeast-hypha morphogenesis in the human fungal pathogen Candida albicans.

Rad6p plays important roles in post-replication DNA repair, chromatin organization, gene silencing and meiosis. In this study, we show that Rad6p also regulates yeast-hypha morphogenesis in the human pathogen Candida albicans. CaRAD6 gene and cDNAs were isolated and characterized revealing that the gene carries two 5'-proximal introns. CaRad6p shows a high degree of sequence similarity to Rad6 proteins from fungi to man (60-83% identity), and it suppresses the UV sensitivity and lack of induced mutagenesis displayed by a Saccharomyces cerevisiae rad6 mutant. In C. albicans, CaRAD6 expression is induced in response to UV, and CaRad6p depletion confers UV sensitivity, confirming that Rad6p serves a role in protecting this fungus against UV damage. CaRAD6 overexpression inhibits hyphal development, whereas CaRad6p depletion enhances hyphal growth. Also, CaRAD6 mRNA levels decrease during the yeast-hypha transition. These effects are dependent on Efg1p, but not Cph1p, indicating that CaRad6p acts specifically through the Efg1p morphogenetic signalling pathway to repress yeast-hypha morphogenesis.

The MET3 promoter: a new tool for Candida albicans molecular genetics.

A central technique used to investigate the role of a Candida albicans gene is to study the phenotype of a cell in which both copies of the gene have been deleted. To date, such investigations can only be undertaken if the gene is not essential. We describe the use of the Candida albicans MET3 promoter to express conditionally an essential gene, so that the consequences of depletion of the gene product may be investigated. The effects of environmental conditions on its expression were investigated, using GFP as a reporter gene. The promoter showed an approximately 85-fold range of expression, according to the presence or absence of either methionine or cysteine in concentrations in excess of 1 mM. In the presence of either amino acid, expression was reduced to levels that were close to background. We used URA3 as a model to demonstrate that the MET3 promoter could control the expression of an essential gene, provided that a mixture of both methionine and cysteine was used to repress the promoter. We describe an expression vector that may be used to express any gene under the control of the MET3 promoter and a vector that may be used to disrupt a gene and simultaneously place an intact copy under the control of the MET3 promoter. During the course of these experiments, we discovered that directed integration into the RP10 locus gives a high frequency of transformation, providing a means to solve a long-standing problem in this field.

The yeast PRS3 gene is required for cell integrity, cell cycle arrest upon nutrient deprivation, ion homeostasis and the proper organization of the actin cytoskeleton.

PRS3 is one of a family of five genes encoding phosphoribosylpyrophosphate synthetase, an enzyme which catalyses the first step in a variety of biosynthetic pathways, including purine and pyrimidine biosynthesis. We report here that prs3Delta mutants have a number of phenotypes that suggest an unexpected role for PRS3 in linking nutrient availability to cell cycle progression, cell integrity and the actin cytoskeleton. Upon nutrient limitation, prs3Delta mutants fail to arrest in G(1)-cells remain budded and a significant fraction have a G(2) DNA content. Furthermore, in such conditions, prs3Delta mutants have a disorganized actin cytoskeleton: actin accumulates in one or two intensely staining clumps per cell. Prs3Delta mutants also show defects in ion homeostasis and cell integrity. They fail to grow on medium containing 1.0 M NaCl, 5 mM caffeine or when incubated at 37 degrees C. The caffeine and temperature sensitivity are rescued by supplementing the growth medium with 1.0 M sorbitol. These phenotypes resemble those of whi2Delta mutations and indeed, a prs3 allele was recovered in a colony-sectoring screen for mutations that are co-lethal with whi2Delta. However, further investigation showed that the prs3Delta whi2Delta double mutant was viable, with no obvious growth defect compared to either single mutant. In the same colony-sectoring assay, an mpk1 allele was also recovered. Multicopy PRS3 rescued the caffeine sensitivity of this mpk1 allele.

Filamentous growth of the budding yeast Saccharomyces cerevisiae induced by overexpression of the WHi2 gene.

The WHI2 gene of the budding yeast Saccharomyces cerevisiae is required for the arrest of cell proliferation upon nutrient exhaustion: whi2 mutants carry on dividing and in the absence of growth become abnormally small. It is reported here that overexpression of Whi2 from the GAL1 promoter results in filamentous growth - cells fail to complete cytokinesis, the budding pattern changes from axial to polar, cells become elongated and cell size increases threefold. In many ways, these filaments resemble the pseudohyphae which result from nitrogen-limited growth and the filaments seen during the invasive growth of haploids. However, Whi2-induced filament formation is reduced, but not blocked, by mutations in STE7, STE12 or STE20 which do block pseudohypha formation. Furthermore, pseudohypha formation can still occur in a diploid in which both copies of the WHI2 gene have been deleted. Thus Whi2-induced filament formation and pseudohypha formation must come about through the action of different pathways. Despite this, a mutation in the STE11 gene, which is required for pseudohypha formation, does block Whi2-induced filament formation. Concanavalin A pulse-chase experiments show that new cell wall material is incorporated only into the tips of the apical cells. An extragenic suppressor of the Whi2 allele also results in filamentous growth. These results suggest that Whi2 negatively regulates a function required for the budding mode of cell proliferation including cytokinesis. This function is defined wholly or in part by the fsw1 allele.

The expression of recombinant proteins in yeasts.

The methylotrophic yeasts Hansenula polymorpha and Pichia pastoris are rapidly becoming the systems of choice for the expression of recombinant proteins in yeast. However, the powerful genetic techniques available in Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are still exploited to establish models to study medically important cell processes and screen for pharmacologically active compounds.

Expression of the major bean proteins from Theobroma cacao (cocoa) in the yeasts Hansenula polymorpha and Saccharomyces cerevisiae.

The production in two yeast expression systems of recombinant forms of the major proteins from the cocoa bean is described. Three major protein species are found in the cocoa bean: an albumin of molecular mass 21 kDa (p21) and two insoluble vicilin-like proteins of molecular mass 31 kDa and 47 kDa (p31 and p47, respectively). The p31 and p47 species are known to be derived from a common 67-kDa precursor (p67) by post-translational processing that includes the deletion of a hydrophilic domain located immediately after an N-terminal signal sequence. All three proteins appear to be targeted to membrane-bound storage organelles by N-terminal signal sequences. The p21 and p67 coding sequences were expressed in Hansenula polymorpha using the powerful methanol oxidase (MOX) promoter and in Saccharomyces cerevisiae using the promoter of the pyruvate kinase (PYK) gene. The expression constructs contained the native plant signal sequence, or various yeast signals. The p21 protein was successfully expressed and secreted from both yeasts. The insoluble p67 protein proved more difficult. Species of the correct molecular mass were recovered internally and small amounts of a p47 species were secreted using a yeast leader sequence. However, proteolytic cleavage, probably due to Kex2p-like processing, led to the appearance of other protein species.

Development of a strain of Hansenula polymorpha for the efficient expression of guar alpha-galactosidase.

A strain of the methylotrophic yeast Hansenula polymorpha, A16, has been developed that expresses the guar alpha-galactosidase gene to 22.4 mg/g dry cell weight in chemostat cultures at a dilution rate of 0.1 h(-1). This corresponds to more than 13.1% of soluble cell protein, of which 56-62% is secreted into the medium. The alpha-galactosidase gene was flanked by the promoter and terminator sequences of the H.polymorpha mox gene, which can direct expression of the mox gene itself more than 30% of total cell protein under methanol growth. The expression cassette (pUR3510) based on the Saccharomyces cerevisiae plasmid, YEp13, was integrated into the genome. Such transformants were stable in chemostat cultures and exhibited 100% stability for both alpha-galactosidase+ and leu+ phenotypes. Chemostat cultures produced higher levels of alpha-galactosidase with higher specific productivities expressed as mg alpha-galactosidase g(-1) h(-1) compared to batch cultures.

Expression of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) by Hansenula polymorpha.

The methylotrophic yeast Hansenula polymorpha, a host organism for the production of heterologous proteins, has been applied to produce the alpha-galactosidase from the plant Cyamopsis tetragonoloba (guar). The yeast/Escherichia coli shuttle expression vector used is based on the origin of replication of the endogenous 2 microns plasmid of Saccharomyces cerevisiae and the LEU2 gene of S. cerevisiae for selection in H. polymorpha. In the expression vector, the alpha-galactosidase is controlled by the methanol-regulated promoter from the methanol oxidase gene, MOX, of H. polymorpha. The signal sequence of SUC2 (invertase) from the yeast S. cerevisiae, was used to ensure secretion of the alpha-galactosidase enzyme. After transformation and stabilization, the expression vector was stably integrated in the genome. The active alpha-galactosidase enzyme was efficiently secreted (greater than 85%) and after methanol induction, the expression level was 42 mg/l. Amino-terminal sequencing of the purified alpha-galactosidase enzyme synthesized by H. polymorpha showed that the S. cerevisiae invertase signal sequence was correctly processed by H. polymorpha. The secreted alpha-galactosidase was glycosylated and had a sugar content of 9.5%. The specific activity of the alpha-galactosidase produced by H. polymorpha was 38 U mg-1 compared to 100 U mg-1 for the guar alpha-galactosidase. Deglycosylation of the H. polymorpha alpha-galactosidase restored the specific activity completely.

Regulation of the Saccharomyces cerevisiae WHI2 gene.

WHI2 mRNA levels were followed through the growth cycle in WHI2 mutant and wild-type cells of Saccharomyces cerevisiae. Levels were high during the first (glucose) phase of growth, and were reduced sharply during the second (ethanol) phase of growth. Transcript levels of the glycolytic genes PDC1 and PYK1 were also measured; they each showed a pattern similar to that of WHI2, whereas transcript levels of the CDC7 gene remained constant throughout the cycle, showing that a decrease in transcription is not a general feature of genes. These results make it unlikely that the WHI2 product acts as an inhibitor of cell proliferation which is activated upon carbon starvation. No difference was observed between the pattern of expression of mutant and wild-type strains, showing that the mutant phenotype was not the result of a change in regulation at the transcriptional level.

The relationship of growth rate and catabolite repression with WHI2 expression and cell size in Saccharomyces cerevisiae.

Mixtures of D-glucosamine and glucose were used to slow the growth of wild-type and whi2 mutant strains of Saccharomyces cerevisiae without affecting the level of catabolite repression. The following observations were made. Firstly, mutant cells were found to be partially resistant to the inhibitory effect of glucosamine. Secondly, slow growth induced by glucosamine resulted in cells becoming larger, in direct contrast to the effect of slowing growth by glucose limitation in a chemostat or by carbon source substitution. It is concluded that the level of repression/derepression, rather than absolute growth rate, is responsible for controlling cell size. Thirdly, when WHI2 transcript levels were measured it was found that expression was correlated with growth rate rather than the level of repression. These results are interpreted in terms of a model which envisages that the WHI2 product acts as a negative regulator of catabolite repression. A test of this model is reported: it is shown that mutant cells respired more actively in the presence of glucose and grew more rapidly on glycerol, whereas overexpression of WHI2 from multicopy plasmids prevented growth on glycerol and depressed respiration.

Hansenula polymorpha as a novel yeast system for the expression of heterologous genes.

The advantages of Hansenula polymorpha as a new yeast expression system are discussed in terms of the powerful and regulatable methanol oxidase promoter and the organism's ability to grow on cheap carbon sources. The development of techniques for conventional genetic analysis is described. A total of 218 mutants have been assigned to 62 complementation groups, three genes have been found to be linked forming the first linkage group in this organism. Methods for molecular transformation have been developed allowing the expression of heterologous genes. The disruptive integration and expression of the neomycin phosphotransferase is described.

The effect of dissolved oxygen concentration on the growth physiology of Saccharomyces cerevisiae whi2 mutants.

Isogenic whi2 and WHI2+ strains of Saccharomyces cerevisiae were grown in a 2-litre bioreactor as batch cultures on a medium containing yeast extract and peptone with either glucose or ethanol as carbon and energy source. The concentration of dissolved oxygen within the medium was varied over the range of 0 to 100% saturation. Expression of the whi2 phenotype only occurred above 40% oxygen saturation with either glucose or ethanol as carbon and energy source. Under these conditions the whi2 cells could be distinguished from WHI2+ cells in that they were phase dark, highly budded and very small during the stationary growth phase, and reached final cell densities four to six times higher than WHI2+ cells. The results clearly show that the WHI2 gene of S. cerevisiae plays an important role in cell proliferation and that the availability of oxygen, or some product of oxidative metabolism, is involved in regulating the phenotypic expression of mutations within this gene.

Transcript characterisation, gene disruption and nucleotide sequence of the Saccharomyces cerevisiae WH12 gene.

WH12 is a gene which plays a prominent role in regulating growth and proliferation in Saccharomyces cerevisiae. It is expressed as a 2.0-kb mRNA transcript. Disruption of this transcript in a WH12+ cell results in the mutant phenotype being displayed. The nucleotide sequence of the cloned gene has been determined and found to include a 487-codon long open reading frame coding for a 55.3-kDa protein. The protein showed no extensive homologies to any previously identified protein. The 5' and 3' noncoding regions contained many of the features found in other yeast genes.

Molecular cloning of WHI2, a gene involved in the regulation of cell proliferation in Saccharomyces cerevisiae.

The WHI2 gene of Saccharomyces cerevisiae plays a key role in coordinating cell proliferation and nutrient availability. A 2.6 kb yeast DNA sequence has been cloned which fully suppresses the whi2 mutation. Integration of this sequence to demonstrate that the structural gene itself had been cloned proved difficult. Integration occurred only rarely and only at the LEU2 locus which was also present on the integrating plasmid. To circumvent these difficulties an adjacent sequence, present on the original isolate from the gene library, was subcloned onto the integrating vector YIp5, after which directed integration proved straightforward. The integrated sequence was closely linked to WHI2, confirming that the structural gene had been cloned. A chromosomal restriction map of the WHI2 region is presented; no gross changes were observed in the region as cells entered stationary phase.

Small-sized mutants of Saccharomyces cerevisiae.

The isolation of mutants of Saccharomyces cerevisiae that divide at approximately half the size of the wild type is described. Three mutants have been isolated in which the small size at bud initiation is due to a mutation in a single nuclear gene.

Genes which control cell proliferation in the yeast Saccharomyces cerevisiae.

In many eukaryotes it is thought that cell proliferation is regulated at a point in G1 close to the initiation of DNA synthesis. Hartwell and his colleagues have shown such a point in G1 phase in the budding yeast, Saccharomyces cerevisiae, defined by the cdc 28 mutation. He has termed this point 'start' and showed that for cells to proceed beyond start, initiate DNA synthesis and produce a bud, various conditions must be met. Two of these conditions are the presence of adequate nutrients in the medium and attainment of a critical size. We identify here some of the genes controlling start by isolating mutants which are altered with respect to the conditions in which start occurs. Two types of mutant have been isolated. One results in bud initiation when the parent cell is only half the size at which bud initiation occurs in wild-type cells. Such mutants define a single gene, whi-1, and they are apparently analogous to the size mutants isolated by Nurse and his colleagues in Schizosaccharomyces pombe. A second type of mutation affects a second gene, whi-2, which is involved in the mechanism whereby cells arrest in G1 in stationary phase. whi-2- cells growing exponentially initiate buds at the same size as wild-type cells. In stationary phase, however, whi-2- cells growing exponentially initiate buds at the same size as wild-type cells. In stationary phase, however, whi-2- cells, unlike wild-type cells, are predominantly budded and are smaller than wild-type cells.

The control of mitosis in Physarum polycephalum: the effect of delaying mitosis and evidence for the operation of the control mechanism in the absence of growth.

Possible mechanisms coordinating the control of mitosis and DNA synthesis with growth were experimentally tested in Physarum polycephalum by the response of plasmodia to 2 different kinds of perturbation of the DNA:mass ratio. Mitosis and DNA synthesis were delayed without stopping growth either by the use of fluorodeoxyuridine (FUdR) or puromycin, in both cases the delayed mitosis was followed by a single shortened intermitotic period, as predicted by all the mechanisms considered and substantiating a basic assumption made that the time of mitosis is homeostatically controlled to maintain a constant DNA:mass ratio. When more than 50% of the DNA was destroyed by ultraviolet irradiation 2 consecutive mitoses could occur even when growth was completely stopped by starvation. This result can only be accounted for by 2 of the 5 models considered, a finding which agrees with the results of previous experiments which were also consistent with only these 2 mechanisms.