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PubMed2004

AtSGP1, AtSGP2 and MAP4K alpha are nucleolar plant proteins that can complement fission yeast mutants lacking a functional SIN pathway.

In the fission yeast Schizosaccharomyces pombe, the onset of septum formation is signalled via the septation initiation network (SIN) involving several protein kinases and a GTPase. Arabidopsis thaliana and Brassica napus proteins homologous to fission yeast spg1p (AtSGP1, AtSGP2), cdc7p (AtMAP3K epsilon 1, AtMAP3K epsilon 2, BnMAP3K epsilon 1) and sid1p (AtMAP4K alpha 1, AtMAP4K alpha 2, BnMAP4K alpha 2) exhibit a significant similarity. The plant proteins AtSGP1/2 and BnMAP4K alpha 2 are able to complement the S. pombe mutant proteins spg1-B8 and sid1-239, respectively and to induce mutisepta when overexpressed in wild-type yeast. Yeast two-hybrid assays demonstrated interactions both between plant proteins and between plant and yeast proteins of the SIN pathway. However, the primary structure of the proteins as well as the partial complementation of yeast mutants indicates that plant homologous proteins and their yeast counterparts have diverged during evolution. Real-time RT-PCR studies demonstrated plant SIN-related gene expression in all organs tested and a co-expression pattern during the cell cycle, with a higher accumulation at G(2)-M. During interphase, the plant SIN-related proteins were found to co-localise predominantly in the nucleolus of the plant cells, as shown by fusions to green fluorescent protein. These data suggest the existence of a plant SIN-related pathway.

Arabidopsis↗
PubMed2002

Sla1p couples the yeast endocytic machinery to proteins regulating actin dynamics.

Sla1p is a protein required for cortical actin patch structure and organisation in budding yeast. Here we use a combination of immunofluorescence microscopy and biochemical approaches to demonstrate interactions of Sla1p both with proteins regulating actin dynamics and with proteins required for endocytosis. Using Sla1p-binding studies we reveal association of Sla1p with two proteins known to be important for activation of the Arp2/3 complex in yeast, Abp1p and the yeast WASP homologue Las17p/Bee1p. A recent report of Sla1p association with Pan1p puts Sla1p in the currently unique position of being the only yeast protein known to interact with all three known Arp2/3-activating proteins in yeast. Localisation of Sla1p at the cell cortex is, however, dependent on the EH-domain-containing protein End3p, which is part of the yeast endocytic machinery. Using spectral variants of GFP on Sla1p (YFP) and on Abp1p (CFP) we show for the first time that these proteins can exist in discrete complexes at the cell cortex. However, the detection of a significant FRET signal means that these proteins also come close together in a single complex, and it is in this larger complex that we propose that Sla1p binding to Abp1p and Las17p/Bee1p is able to link actin dynamics to the endocytic machinery. Finally, we demonstrate marked defects in both fluid-phase and receptor-mediated endocytosis in cells that do not express SLA1, indicating that Sla1p is central to the requirement in yeast to couple endocytosis with the actin cytoskeleton.

Actins↗
PubMed1987

Preliminary characterization of maturation-promoting factor from yeast Saccharomyces cerevisiae.

It has been known for some time that maturation-promoting factor (MPF) appears in a wide variety of eukaryotic cells at M phase and exerts equal M-phase-promoting activity in both meiotic cells and mitotic cells in a non-specific manner. MPF was extracted from cdc20 mutant cells of the yeast Saccharomyces cerevisiae synchronized at M phase by incubation at the restrictive temperature. When injected into immature oocytes of Xenopus laevis, yeast MPF caused meiosis reinitiation in a dose-dependent manner and even in the presence of cycloheximide. Yeast MPF exerted its activity in starfish oocytes as well. MPF activity was obtained only from cells in M phase and not from G1, S or G2 phase cells, indicating cyclical changes during the yeast mitotic cell cycle. Preliminary characterization of yeast MPF revealed that its activity was associated with a heat-labile protein having a sedimentation coefficient value of 6 S. In contrast to the current assumption that MPF is a Ca-sensitive phosphoprotein stabilized by phosphorylated small molecules, such as ATP and Na-beta-glycerophosphate, the present study revealed that yeast MPF was still active even after treatment with either Ca2+ or alkaline phosphatase. Furthermore, it was found that yeast MPF and these phosphorylated small molecules were complementary in inducing reinitiation of meiosis, since the meiosis-reinitiating activity was detected only when both were present simultaneously and almost undetectable when either of them was present alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed2000

Overexpression of PRA2, a Rab/Ypt-family small GTPase from Pea Pisum sativum, aggravates the growth defect of yeast ypt mutants.

A large number of Rab/Ypt-family small GTPases have been identified from higher plants. While some of them can complement yeast ypt mutants, the expression of Arabidopsis Ara4 protein aggravated the growth defect of a subset of ypt mutants, probably because of the titration of common regulator(s) of yeast Ypt proteins [Ueda, T. et al. (1996) Plant Cell, 8: 2079-20911. PRA2 from pea Pisum sativum encodes an interesting Rab GTPase whose expression is regulated by light [Yoshida, K. et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 6636-6640]. We examined whether PRA2 complements any of the yeast ypt mutants and found again that PRA2 does not complement but rather confers the growth defect to some of the ypt mutants. No growth defect was observed when PRA2 was expressed in the wild-type yeast cells. Unlike the case of Ara4, neither Arabidopsis nor yeast GDI remedied the growth defect by Pra2, indicating that the mechanism of the exacerbation is different. Mutational analysis of PRA2 suggests that the growth inhibition can be ascribed to unidentified factor(s) which prefers the GTP-bound form of Pra2. This yeast system will be useful for identifying such putative regulatory factor(s) from yeast and plants and analyzing their interactions with Pra2.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Recognition of RNA by the p53 tumor suppressor protein in the yeast three-hybrid system.

The p53 tumor suppressor protein is a homotetrameric transcription factor whose gene is mutated in nearly half of all human cancers. In an unrelated screen of RNA/protein interactions using the yeast three-hybrid system, we inadvertently detected p53 interactions with several different RNAs. A literature review revealed previous reports of both sequence-specific and -non-specific interactions between p53 and RNA. Using yeast three-hybrid selections to identify preferred RNA partners for p53, we failed to identify primary RNA sequences or obvious secondary structures required for p53 binding. The cationic p53 C-terminus was shown to be required for RNA binding in yeast. We show that while p53 strongly discriminates between certain RNAs in the yeast three-hybrid assay, the same RNAs are bound equally by p53 in vitro. We further show that the p53 RNA-binding preferences in yeast are mirrored almost exactly by a recombinant tetrameric form of the HIV-1 nucleocapsid (NC) protein thought to be a sequence-nonspecific RNA-binding protein. However, the possibility of specific RNA binding by p53 could not be ruled out because p53 and HIV-1 NC displayed certain differences in RNA-binding preference. We conclude that (1) p53 binds RNA in vivo, (2) RNA binding by p53 is largely sequence-nonspecific in the yeast nucleus, (3) some structure-specific RNA binding by p53 cannot be ruled out, and (4) caution is required when interpreting results of RNA screens in the yeast three-hybrid system because sequence-dependent differences in RNA folding and display can masquerade as sequence-dependent differences in protein recognition.

Base Sequence↗
PubMed2001

Improving the freeze tolerance of bakers' yeast by loading with trehalose.

We examined the freeze tolerance of bakers' yeast loaded with exogenous trehalose. Freeze-tolerant and freeze-sensitive compressed bakers' yeast samples were soaked at several temperatures in 0.5 M and 1 M trehalose and analyzed. The intracellular trehalose contents in both types of bakers' yeast increased with increasing soaking period. The initial trehalose-accumulation rate increased with increasing exogenous trehalose concentration and soaking temperature. The maximum trehalose content was almost identical (200-250 mg/g of dry cells) irrespective of the soaking temperature and the type of bakers' yeast, but depended on the exogenous trehalose concentration. The leavening ability of both types of bakers' yeast loaded with trehalose was almost identical to that of the respective original cells, irrespective of the soaking conditions. The freeze-tolerant ratio (FTR) of both types of bakers' yeast increased with increasing intracellular trehalose content. However, FTR decreased during over-soaking after the maximum amount of trehalose had accumulated. FTR of the freeze-sensitive bakers' yeast was more efficiently improved than that of the freeze-tolerant type.

Freezing↗
PubMed2006

alpha-Synuclein fission yeast model: concentration-dependent aggregation without plasma membrane localization or toxicity.

Despite fission yeast's history of modeling salient cellular processes, it has not yet been used to model human neurodegeneration-linked protein misfolding. Because alpha-synuclein misfolding and aggregation are linked to Parkinson's disease (PD), here, we report a fission yeast (Schizosaccharomyces pombe) model that evaluates alpha-synuclein misfolding, aggregation, and toxicity and compare these properties with those recently characterized in budding yeast (Saccharomyces cerevisiae). Wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T) were expressed with thiamine-repressible promoters (using vectors of increasing promoter strength: pNMT81, pNMT41, and pNMT1) to test directly in living cells the nucleation polymerization hypothesis for alpha-synuclein misfolding and aggregation. In support of the hypothesis, wild-type and A53T alpha-synuclein formed prominent intracellular cytoplasmic inclusions within fission yeast cells in a concentration- and time-dependent manner, whereas A30P and A30P/A53T remained diffuse throughout the cytoplasm. A53T alpha-synuclein formed aggregates faster than wild-type alpha-synuclein and at a lower alpha-synuclein concentration. Unexpectedly, unlike in budding yeast, wild-type and A53T alpha-synuclein did not target to the plasma membrane in fission yeast, not even at low alpha-synuclein concentrations or as a precursor step to forming aggregates. Despite alpha-synuclein's extensive aggregation, it was surprisingly nontoxic to fission yeast. Future genetic dissection might yield molecular insight into this protection against toxicity. We speculate that alpha-synuclein toxicity might be linked to its membrane binding capacity. To conclude, S. pombe and S. cerevisiae model similar yet distinct aspects of alpha-synuclein biology, and both organisms shed insight into alpha-synuclein's role in PD pathogenesis.

Animals↗
PubMed1997

Human p53 restores DNA synthesis control in fission yeast.

The p53 gene is the most common target for genetic alterations in human cancers. As a transcriptional regulator p53 enhances the expression of proteins that control cellular proliferation. Although there is no evidence of a p53 homologous gene in yeast, the p53 protein was found to be functional in terms of growth repression and transactivation in yeast, suggesting that some features of p53 function are conserved. Here we report the construction and characterization of a p53 wild type expression strain of fission yeast. Upon induction of wild type p53 expression a dosage dependent growth arrest was observed rendering recipient yeast cells sensitive to UV irradiation in a p53 dosage dependent fashion. The observed growth arrest was efficiently suppressed by coexpression of human CDC25C phosphatase, which restored a normal resistance to UV irradiation in p53 and CDC25C coexpressing yeast cells. Furthermore, expression of CDC25C alone inactivated the DNA synthesis control whereas p53 and CDC25C coexpressing yeast cells showed an intact checkpoint control. Upon moderate expression of wild type p53 a restoration of the DNA synthesis control was also observed in a cdc2.3w mutant background, whereas a tumor mutant of p53 failed to restore this important checkpoint in fission yeast.

Cell Cycle Proteins↗
PubMedDate not supplied

Immobilization of yeast cells by radiation-induced polymerization.

Radiation-induced polymerization method was applied to the immobolization of yeast cells. The effects of irradiation, cooling and monomer, which are necessary for polymerization, were recovered completely bu subsequent aerobic incubation of yeast cells. The ethanol productive in immobilized yeast cells increased with the increase of aerobic incubation period. The growth of yeast cells in immobilized yeast cell was indicated. The maximum ethanol productivity in immobilized yeast cell system was around three times as much as that in free yeast cell system.

Dose-Response Relationship, Radiation↗
PubMed2003

Spectrophotometric assay of yeast vitality using 2,3,5,6-tetramethyl-1,4-benzoquinone and tetrazolium salts.

A method for the spectrophotometric assay of yeast vitality was developed using 2,3,5,6-tetramethyl-1,4-benzoquinone and tetrazolium salts. The metabolic efficiency of 2,3,5,6-tetramethyl-1,4-benzoquinone by yeast cells was used as an index of yeast vitality. 2,3,5,6-Tetramethyl-1,4-benzoquinone was reduced to 2,3,5,6-tetramethyl-1,4-hydroquinone by yeast cells. Then, the superoxide anion radicals generated from O2 by reduction with 2,3,5,6-tetramethyl-1,4-hydroquinone under alkaline conditions reduced tetrazolium salts to formazan, which exhibited absorbance maxima at 440 nm. A linear relationship between the absorbance and viable cell density was obtained in the range of 1.0 x 10(5)-2.0 x 10(7) cells/ml for a sample solution. During the cultivation of yeast cells, the absorbance showed almost an anti-parallel change with that of glucose in yeast growth and fermentation, suggesting that the absorbance change reflected the vitality of yeast cells.

Benzoquinones↗
PubMed1998

Protein expression both in mammalian cell lines and in yeast Pichia pastoris using a single expression plasmid.

We have designed and constructed a novel expression vector capable of producing recombinant proteins in both mammalian cell lines and the yeast strain Pichia pastoris. In this vector, a yeast promoter is placed inside an intron of the mammalian transcription unit. A yeast transcription termination sequence is placed immediately downstream of the mammalian polyadenylation site. In mammalian cells, transcription is driven by a mammalian promoter. The yeast promoter within the intron is removed by RNA processing. However, protein expression in yeast cells can be achieved utilizing the yeast promoter immediately upstream of the 3' splice site and the target genes. Our data indicate that this vector can express beta-galactosidase efficiently in both mammalian cell lines and the yeast strain P. pastoris.

Animals↗
PubMed1999

Budding yeast as a screening tool for discovery of nucleoside analogs for use in HSV-1 TK suicide-gene therapy.

We present a fast, convenient and inexpensive method that allows the automated, large-scale screening of chemical libraries for compounds that are converted by the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) into inhibitors of cell growth. The method is based on the use of budding yeast (Saccharomyces cerevisiae) transformed with the HSV-1 TK gene on a multicopy plasmid. Eight nucleoside analogs (acyclovir, ganciclovir, penciclovir, lobucavir, brivudin, sorivudine, IVDU and ara-T), for which the cytostatic action against mammalian cells expressing the HSV-1 TK gene has been well documented, were studied for their inhibitory effect on the growth of yeast expressing the viral TK. These nucleoside analogs had little or no inhibitory effect on the growth of yeasts transformed with the empty vector, but inhibited to a significant extent the growth of yeast expressing the viral TK. Use of HSV-1 TK-expressing yeast allows quick screening in multi-well plate format for compounds with potential use in HSV-1 TK suicide gene therapy. The method may also be used as a tool to selectively suppress or arrest the growth of one population of yeast out of mixed yeast cell cultures.

Acyclovir↗
PubMed1990

Effects of microbial supplements containing yeast and lactobacilli on roughage-fed ruminal microbial activities.

Effects of two microbial feed supplements on microbial activities in rumen-stimulating cultures and the rumens of steers fed a fescue hay-based roughage diet were evaluated. The yeast culture supplement contained Saccharomyces cerevisiae (1.4 to 4.2 x 10(9) colony-forming units [cfu]/g), whereas the mixed microbial supplement contained yeast, lactobacilli and enterococci (1.4 to 2.7 x 10(9) cfu/g, 1.2 to 2.3 x 10(9) cfu/g, and 1.5 to 2.6 x 10(10) cfu/g, respectively). Concentrations of viable yeast cells were increased consistently in continuous cultures and rumens of steers receiving either supplement (1 g/kg of feed). However, neither supplement consistently altered the relative concentrations of volatile fatty acids or ammonia in continuous cultures and rumens of steers. The pH tended to be greater (P = .13) in continuous cultures receiving yeast culture supplement than in cultures receiving the unsupplemented diet (6.50 vs 6.36), but pH in the rumens of steers was not affected by the supplements. Concentrations of cellulolytic microorganisms in cultures and the rumens of steers receiving supplements containing only yeast were from 5 to 40 times greater than those observed in cultures or steers receiving the unsupplemented diet. Supplements that had been treated with heat (121 degrees C for 15 min) to inactive yeast cells did not alter the concentrations of cellulolytic bacteria in rumen-stimulating cultures. These results suggest that live yeast culture supplements stimulate growth of cellulolytic microorganisms in the rumen.

Animals↗
PubMed1999

The influence of dietary selenium as selenium yeast or sodium selenite on the concentration of selenium in the milk of Suckler cows and on the selenium status of their calves.

The aim of this trial was to determine whether the selenium status of suckling calves could be improved by supplementing their dams' diet with organic Se instead of sodium selenite. A herd of 103 Hereford cows, which were on grass paddocks all year round, was divided into two groups. Both groups had free access to a mineral supplement that contained 30 mg of Se/kg; for one group the source of the Se was a Se yeast product, and for the other group the source was sodium selenite. The basal feed contained .02 mg of Se/kg DM. During the trial, the mean daily consumption of the mineral supplement was approximately 110 g/cow. The calving season started in the middle of March and ended in the middle of May. Blood samples were taken from 11 cows and their calves in the yeast group and from nine in the selenite group at the end of April and again at the beginning of June, and milk samples were taken at the same times. At both samplings, the concentration of Se in whole blood and the activity of glutathione peroxidase (GSH-Px) in the erythrocytes of the cows and calves in the yeast group were higher than in the samples from the animals in the selenite group. The same pattern was seen for plasma, except for the cows at the first sampling. The mean concentrations of Se in whole blood from calves in the yeast and selenite groups were 130 and 84 microg/L, respectively, and plasma concentrations were 48 and 34 microg/ L, respectively. Mean Se concentration in the milk from the yeast group (17.3 microg/L) was higher than that in milk from the selenite group (12.7 microg/L). There were significant correlations (r = .59 to .68) between the concentrations of Se in the cow's milk or cow's whole blood compared with Se concentrations in the calves whole blood and plasma or with the erythrocyte GSH-Px activity of the calves. The Se status of the calves in the selenite group was considered to be marginal, but the status of the calves in the yeast group was considered to be adequate. Supplementation of the suckler cows' diet with organic Se in the form of Se yeast rather than sodium selenite improved the Se status of their calves when the Se was mixed into a mineral supplement containing 30 mg of Se/kg. In practice, such supplementation would probably eliminate the risk of nutritional muscular degeneration in suckling calves.

Animal Feed↗
PubMed1995

Effects of varying forage types on milk production responses to whole cottonseed, tallow, and yeast.

Four forage treatments (45% corn silage, 33.75% corn silage plus 11.25% alfalfa hay, 11.25% bermudagrass hay, or 11.25% cottonseed hulls on a DM basis) were arranged factorially with no added fat, 12.5% whole cottonseed, or 2.5% tallow. Different diets were fed during three 28-d periods to 20 control Holstein cows and to 20 cows receiving yeast continuously in a split-plot design. Milk yield of cows fed cottonseed hulls with corn silage was 2.4 kg/d higher than with corn silage plus bermudagrass hay and .7 kg/d higher than with corn silage only or corn silage plus alfalfa hay. Whole cottonseed depressed milk yield by 1 kg/d. Cows fed yeast had increased DMI, and yeast interacted with forage so that more milk was produced by cows fed alfalfa diets. Yeast depressed milk protein percentage. Holstein cows in a commercial Florida dairy were fed no yeast or 10 g/d continuously for 60 d; milk fat percentage was greater (3.51 vs. 3.37%) with yeast. In summary, effects on milk and SCM were positive when cottonseed hulls were utilized with corn silage, negative with whole cottonseed, and neutral with supplemental tallow. Yeast effects on SCM, although not significant for either experiment, tended to be positive for both (mean +1.2 kg/d per cow).

Animal Feed↗
PubMed1997

Effect of yeast culture in the diets of early lactation dairy cows on ruminal fermentation and passage of nitrogen fractions and amino acids to the small intestine.

Eight early lactation, primiparous Holstein cows were fitted with ruminal and duodenal cannulas. The cows were used in a replicated 4 x 4 Latin square design to test the effects of yeast culture (0 vs. 10 g/ d) and dietary crude protein (CP) (16.1 vs. 18.8% of dry matter) in 44% forage diets. Dietary CP differed primarily in ruminally degradable CP (9.1 vs. 11.4% of dry matter). Dry matter intake tended to increase as amount of yeast culture increased. However, yeast culture had no effect on ruminal pH, concentrations of NH3 and volatile fatty acids in ruminal fluid, or ruminal digestibility. Yeast culture increased the ruminal concentration of isobutyrate in cows fed the low CP diet and decreased the concentration of isobutyrate in cows fed the high CP diet. The higher CP diets increased microbial N passage to the duodenum and had no effect on passage of nonmicrobial nonammonia N. Flows to the duodenum of nonmicrobial nonammonia N tended to be higher for cows fed yeast culture. Flows of essential amino acids to the duodenum and the essential amino acid profiles of duodenal digesta and of mixed ruminal bacteria were not altered by yeast culture. Yields of fat and 4% fat-corrected milk were increased by yeast culture supplementation of the low CP diet. Similar tendencies were noted for yields of milk and milk protein.

Amino Acids↗
PubMed2001

[Cloning and expression of Aspergillus niger glucose oxidase gene in methylotrophic yeast].

The DNA fragment encoding A. niger glucose oxidase was amplified by PCR using A. niger genomic DNA as template, and was cloned into vector of pPIC9 for expression in Pichia pastoris. When transformed into methylotrophic yeast Pichia pastoris GS115, The constructed plasmid pPICGOD1 directed the synthesis and secretion of functionally active GOD. After induction in MM medium for 4 days, the GOD activity in the medium reached 30-40 u/mL. SDS-PAGE revealed that recombinant yeast GOD was expressed up to 60%-70% of the total soluble protein, and the secreted GOD could be purified to electrophoretic homogeneity with one purification step using Q Sepharose Fast Flow ion exchange chromatography. The recombinant yeast GOD had very high catalytic activity, showed about 1.6-fold increase of specific activity over the commercial A. niger GOD. Kinetic analysis clearly demonstrated that recombinant yeast GOD showed similar substrate affinity for glucose to A. niger GOD, but the turnover number of the GOD from yeast was determined to be much higher than that of A. niger GOD. In addition, the linear range of glucose electrode made with recombinant yeast GOD was efficiently widened due to the high catalytic activity of yeast GOD.

Aspergillus niger↗
PubMed2002

Development of a new xenoestrogen screening system using fission yeast Schizosaccharomyces pombe.

Endocrine disrupters refer to environmental or chemical compounds, which interfere with the endocrine system of organisms. In this study, our aim was to develop a screening method to detect xenoestrogen (an endocrine disrupter that is commonly encountered in our daily life) by using fission yeast Schizosaccharomyces pombe. Although the yeast (the simplest eukaryotic cell) has no endocrine system, estrogen receptors that are created to express in the yeast cell can be activated by estrogen in a similar manner to mammalian cells. First, in order to express the human estrogen receptor (hER) in the yeast cell, we constructed a yeast expression vector that contained hER (pREP42MHN-hER). In the yeast cells that are transformed with the pREP42MHN-hER vector, estrogen receptors could recognize xenoestrogen, which allowed the determination of the presence of xenoestrogen in any given sample. Furthermore, we constructed a yeast strain that contained an estrogen responsive element (ERE) that fused with the Escherichia coli LacZ gene (pERE-LacZ) as a reporter for binding of xenoestrogen with the estrogen receptor. Since this vector system allows determination of the presence and level of xenoestrogen with simple procedures, it is expected that they can serve as an efficient assay system to detect xenoestrogen.

Artificial Gene Fusion↗