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At least 145 records · Page 8Linked to original sources
PubMed2006

Use of whey protein beads as a new carrier system for recombinant yeasts in human digestive tract.

A new immobilizing protocol using whey protein isolates was developed to entrap recombinant Saccharomyces cerevisiae. The model yeast strain expresses the heterologous P45073A1 that converts trans-cinnamic acid into p-coumaric acid. Beads resulted from a cold-induced gelation of a whey protein solution (10%) containing yeasts (7.5 x 10(7)cells ml(-1)) into 0.1M CaCl(2). The viability and growth capability of yeasts were not altered by our entrapment process. The release and activity of immobilized yeasts were studied in simulated human gastric conditions. During the first 60 min of digestion, 2.2+/-0.9% (n=3) of initial entrapped yeasts were recovered in the gastric medium suggesting that beads should cross the gastric barrier in human. The P45073A1 activity of entrapped yeasts remained significantly higher (p<0.05) than that of free ones throughout digestion (trans-cinnamic acid conversion rate of 63.4+/-1.6% versus 51.5+/-1.8% (n=3) at 120 min). The protein matrix seemed to create a microenvironment favoring the activity of yeasts in the stringent gastric conditions. These results open up new opportunities for the development of drug delivery system using recombinant yeasts entrapped in whey protein beads. The main potential medical applications include biodetoxication or the correction of digestive enzyme deficiencies.

Artificial Organs↗
PubMed2006

Estimating the parameters of a model for protein-protein interaction graphs.

We find accurate approximations for the expected number of three-cycles and unchorded four-cycles under a stochastic distribution for graphs that has been proposed for modelling yeast two-hybrid protein-protein interaction networks. We show that unchorded four-cycles are characteristic motifs under this model and that the count of unchorded four-cycles in the graph is a reliable statistic on which to base parameter estimation. Finally, we test our model against a range of experimental data, obtain parameter estimates from these data and investigate possible improvements in the model. Characterization of this model lays the foundation for its use as a prior distribution in a Bayesian analysis of yeast two-hybrid networks that can potentially aid in identifying false-positive and false-negative results.

Algorithms↗
PubMed2004

Protein-only transmission of three yeast prion strains.

Key questions regarding the molecular nature of prions are how different prion strains can be propagated by the same protein and whether they are only protein. Here we demonstrate the protein-only nature of prion strains in a yeast model, the [PSI] genetic element that enhances the read-through of nonsense mutations in the yeast Saccharomyces cerevisiae. Infectious fibrous aggregates containing a Sup35 prion-determining amino-terminal fragment labelled with green fluorescent protein were purified from yeast harbouring distinctive prion strains. Using the infectious aggregates as 'seeds', elongated fibres were generated in vitro from the bacterially expressed labelled prion protein. De novo generation of strain-specific [PSI] infectivity was demonstrated by introducing sheared fibres into uninfected yeast hosts. The cross-sectional morphology of the elongated fibres generated in vitro was indistinguishable from that of the short yeast seeds, as visualized by electron microscopy. Electron diffraction of the long fibres showed the 4.7 A spacing characteristic of the cross-beta structure of amyloids. The fact that the amyloid fibres nucleated in vitro propagate the strain-specific infectivity of the yeast seeds implies that the heritable information of distinct prion strains must be encoded by different, self-propagating cross-beta folding patterns of the same prion protein.

Amyloid↗
PubMed2005

Triplicate genes for mitochondrial ADP/ATP carriers in the aerobic yeast Yarrowia lipolytica are regulated differentially in the absence of oxygen.

Yarrowia lipolytica is a strictly aerobic fungus, which differs from the extensively studied model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe with respect to its physiology, genetics and dimorphic growth habit. We isolated and sequenced cDNA and genomic clones (YlAAC1) from Y. lipolytica that encode a mitochondrial ADP/ATP carrier. The YlAAC1 gene can complement the S. cerevisiae Deltaaac2 deletion mutant. Southern hybridization, analysis of Yarrowia clones obtained in the course of the Genolevures project, and further sequencing revealed the existence of two paralogs of the YlAAC1 gene, which were named YlAAC2 and YlAAC3, respectively. Phylogenetic analysis showed that YlAAC1 and YlAAC2 were more closely related to each other than to YlAAC3, and are likely to represent the products of a recent gene duplication. All three Y. lipolytica YlAAC genes group together on the phylogenetic tree, suggesting that YlAAC3 is derived from a more ancient duplication within the Y. lipolytica lineage. A similar branching pattern for the three ScAAC paralogs in the facultative anaerobe S. cerevisiae demonstrates that two rounds of duplication of AAC genes occurred independently at least twice in the evolution of hemiascomycetous yeasts. Surprisingly, in both the aerobic Y. lipolytica and the facultative anaerobe S. cerevisiae, the three paralogs are differentially regulated in the absence of oxygen. Apparently, Y. lipolytica can sense hypoxia and down-regulate target genes in response.

Amino Acid Sequence↗
PubMed2004

Recombinogenic phenotype of human activation-induced cytosine deaminase.

Class switch recombination, gene conversion, and somatic hypermutation that diversify rearranged Ig genes to produce various classes of high affinity Abs are dependent on the enzyme activation-induced cytosine deaminase (AID). Evidence suggests that somatic hypermutation is due to error-prone DNA repair that is initiated by AID-mediated deamination of cytosine in DNA, whereas the mechanism by which AID controls recombination remains to be elucidated. In this study, using a yeast model system, we have observed AID-dependent recombination. Expression of human AID in wild-type yeast is mutagenic for G-C to A-T transitions, and as expected, this mutagenesis is increased upon inactivation of uracil-DNA glycosylase. AID expression also strongly induces intragenic mitotic recombination, but only in a strain possessing uracil-DNA glycosylase. Thus, the initial step of base excision repair is required for AID-dependent recombination and is a branch point for either hypermutagenesis or recombination.

Base Pair Mismatch↗
PubMed2002

Yeast protein kinase C.

The mammalian protein kinase C (PKC) superfamily plays regulatory roles in many different cellular processes. However, due to the many members that exist in cells, it is very complicated to present experimental evidence of the particular function of each member. In contrast, yeasts have only one or two PKC members and genetic tools have unveiled their role as main regulators of cell integrity. In this review, we will discuss the function of yeast protein kinase C homologues, their mechanism of activation and the signalling pathways that they regulate in two model yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Fungal Proteins↗
PubMed1996

Identification of an overexpressed yeast gene which prevents aminoglycoside toxicity.

Aminoglycoside antibiotics, used to treat bacterial infections by interfering with proofreading during protein synthesis, cause sensorineural hearing loss in genetically susceptible individuals. The only aminoglycoside-hypersensitivity mutations which have been described in humans are in the mitochondrial 125 rRNA gene, potentially allowing increased antibiotic binding to mitochondrial ribosomes. To identify additional predisposing mutations, a yeast model system was used to isolate genes which interact with or bypass the effects of aminoglycoside antibiotics. A novel yeast gene was isolated which, in high copy, confers neomycin resistance to yeast transformants. The neomycin-resistance 1 gene (NEO1) encodes a potential 1151 as integral membrane protein, most homologous to the yeast DRS2 gene product, a Ca(2+)-ATPase involved in cytoplasmic ribosome assembly. The N-terminus of Neo1p is partially homologous to abrin A-chain, another protein which interacts with cytoplasmic ribosomes. Mutagenesis experiments demonstrate that the NEO1 product is essential for vegetative growth and that the drug-resistance phenotype requires ATPase function.

Adenosine Triphosphatases↗
PubMed1996

Cell cycle control of S phase: a comparison of two yeasts.

The mechanisms responsible for correct timing of DNA synthesis within the cell cycle and for limiting replication to one round per cell cycle are basically similar in the two model yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, despite many differences in detail. In both cases, the timing of initiation and the prevention of additional rounds are controlled by the activity levels of B-type cyclins. These similarities are likely to extend to other eukaryotic organisms.

Cell Cycle↗
PubMed2002

Requirement of an intact microtubule cytoskeleton for aggregation and inclusion body formation by a mutant huntingtin fragment.

Huntington's disease is caused by the expansion of CAG repeats coding for a polyglutamine tract in the huntingtin protein. The major pathological feature found in Huntington's disease neurons is the presence of detergent-insoluble ubiquitinated inclusion bodies composed of the huntingtin protein. However, the mechanisms that underlie inclusion body formation, and the precise relationship between inclusion bodies and events that initiate toxicity, remain unclear. Here, we analyzed the effects of drugs or genetic mutations that disrupt the microtubule cytoskeleton in a Saccharomyces cerevisiae model of the aggregation of an amino-terminal polyglutamine-containing fragment of huntingtin exon 1 (HtEx1). Treatment of yeast with drugs that disrupt microtubules resulted in less than 2% of the detergent-insoluble HtEx1 observed in mock-treated cells and prevented the formation of large juxtanuclear inclusion bodies. Disruption of microtubules also unmasked a potent glutamine length-dependent toxicity of HtEx1 under conditions where HtEx1 exists in an entirely detergent-soluble nonaggregated form. Results from the yeast model paralleled those from neuronal pheochromocytoma cells, where disruption of microtubules eliminated the formation of juxtanuclear and intranuclear inclusion bodies by HtEx1. Our results suggest that active transport along microtubules may be required for inclusion body formation by HtEx1 and that inclusion body formation may have evolved as a cellular mechanism to promote the sequestration or clearance of soluble species of HtEx1 that are otherwise toxic to cells.

Cytoskeleton↗
PubMed2000

Unambiguous demonstration of triple-helix-directed gene modification.

Triple-helix-forming oligonucleotides (TFOs), which can potentially modify target genes irreversibly, represent promising tools for antiviral therapies. However, their effectiveness on endogenous genes has yet to be unambiguously demonstrated. To monitor endogenous gene modification by TFOs in a yeast model, we inactivated an auxotrophic marker gene by inserting target sequences of interest into its coding region. The genetically engineered yeast cells then were treated with psoralen-linked TFOs followed by UV irradiation, thus generating highly mutagenic covalent crosslinks at the target site whose repair could restore gene function; the number of revertants and spectrum of mutations generated were quantified. Results showed that a phosphoramidate TFO indeed reaches its target sequence, forms crosslinks, and generates mutations at the expected site via a triplex-mediated mechanism: (i) under identical conditions, no mutations were generated by the same TFO at two other loci in the target strain, nor in an isogenic control strain carrying a modified target sequence incapable of supporting triple-helix formation; (ii) for a given target sequence, whether the triplex was formed in vivo on an endogenous gene or in vitro on an exogenous plasmid, the nature of the mutations generated was identical, and consistent with the repair of a psoralen crosslink at the target site. Although the mutation efficiency was probably too low for therapeutic applications, our results confirm the validity of the triple-helix approach and provide a means of evaluating the effectiveness of new chemically modified TFOs and analogs.

Base Sequence↗
PubMed2004

The heat shock protein 70 cochaperone hip enhances functional maturation of glucocorticoid receptor.

Multiple molecular chaperones interact with steroid receptors to promote functional maturation and stability of receptor complexes. The heat shock protein (Hsp)70 cochaperone Hip has been identified in conjunction with Hsp70, Hsp90, and the Hsp70/Hsp90 cochaperone Hop/Sti1p in receptor complexes during an intermediate stage of receptor assembly, but a functional requirement for Hip in the receptor assembly process has not been established. Because the budding yeast Saccharomyces cerevisiae contains orthologs for most of the receptor-associated chaperones yet lacks an orthologous Hip gene, we exploited the well-established yeast model for steroid receptor function to ask whether Hip can alter steroid receptor function in vivo. Introducing human Hip into yeast enhances hormone-dependent activation of a reporter gene by glucocorticoid receptor (GR). Because Hip does not similarly enhance signaling by mineralocorticoid, progesterone, or estrogen receptors, a general effect on transcription can be excluded. Instead, Hip promotes functional maturation of GR without increasing steady-state levels of GR protein. Unexpectedly, Hip binding to Hsp70 is not critical for boosting GR responsiveness to hormone. In conclusion, Hip functions by a previously unrecognized mechanism to promote the efficiency of GR maturation in cells.

Animals↗
PubMed2004

A model for restriction point control of the mammalian cell cycle.

Inhibition of protein synthesis by cycloheximide blocks subsequent division of a mammalian cell, but only if the cell is exposed to the drug before the "restriction point" (i.e. within the first several hours after birth). If exposed to cycloheximide after the restriction point, a cell proceeds with DNA synthesis, mitosis and cell division and halts in the next cell cycle. If cycloheximide is later removed from the culture medium, treated cells will return to the division cycle, showing a complex pattern of division times post-treatment, as first measured by Zetterberg and colleagues. We simulate these physiological responses of mammalian cells to transient inhibition of growth, using a set of nonlinear differential equations based on a realistic model of the molecular events underlying progression through the cell cycle. The model relies on our earlier work on the regulation of cyclin-dependent protein kinases during the cell division cycle of yeast. The yeast model is supplemented with equations describing the effects of retinoblastoma protein on cell growth and the synthesis of cyclins A and E, and with a primitive representation of the signaling pathway that controls synthesis of cyclin D.

Animals↗
PubMed2002

Apoptosis in yeast: a new model system with applications in cell biology and medicine.

Apoptosis is a highly coordinated cellular suicide program crucial for metazoan health and diseases. Although its increasing importance in cancer, neurodegenerative disorders and AIDS led to intense research and a better understanding of apoptosis, many details of its regulation or the apoptotic phenotypes are poorly understood. The complex regulatory network and the often contradictory results obtained with human cell lines made application of an easier model system desirable. Apoptosis in yeast promises to provide a better understanding of the genetics of apoptosis. During the past 2 years, scientists were successful in identifying new cell-death regulators of humans, plants and fungi using Saccharomyces cerevisiae. The finding of apoptotic phenotypes, even in protists, suggests that apoptosis developed in unicellular organisms long before the evolutionary separation between fungi, plants and metazoan animals occurred.

Aging↗
PubMed2006

A network of protein interactions determines polyglutamine toxicity.

Several neurodegenerative diseases are associated with the toxicity of misfolded proteins. This toxicity must arise from a combination of the amino acid sequences of the misfolded proteins and their interactions with other factors in their environment. A particularly compelling example of how profoundly these intramolecular and intermolecular factors can modulate the toxicity of a misfolded protein is provided by the polyglutamine (polyQ) diseases. All of these disorders are caused by glutamine expansions in proteins that are broadly expressed, yet the nature of the proteins that harbor the glutamine expansions and the particular pathologies they produce are very different. We find, using a yeast model, that amino acid sequences that modulate polyQ toxicity in cis can also do so in trans. Furthermore, the prion conformation of the yeast protein Rnq1 and the level of expression of a suite of other glutamine-rich proteins profoundly affect polyQ toxicity. They can convert polyQ expansion proteins from toxic to benign and vice versa. Our work presents a paradigm for how a complex, dynamic interplay between intramolecular features of polyQ proteins and intermolecular factors in the cellular environment might determine the unique pathobiologies of polyQ expansion proteins.

DNA, Intergenic↗
PubMed2000

The influence of antisense gene location on target gene suppression in the fission yeast Schizosaccharomyces pombe.

A fission yeast model was employed to investigate the influence of antisense gene location on the efficacy of antisense RNA-mediated target gene suppression. Fission yeast transformants were generated that contained the target lacZ gene at a fixed position and a single copy antisense lacZ gene integrated into various genomic locations, including the same locus as the target gene. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus compared with other genomic locations, indicating that target and antisense gene colocalization is not a critical factor for efficient antisense RNA-mediated gene expression in vivo. Instead, increased lacZ downregulation correlated with an increase in antisense dose, with the steady-state levels of antisense RNA being dependent on genomic position effects and transgene copy number.

Down-Regulation↗
PubMed1994

Cdc25 is not the signal receiver for glucose induced cAMP response in S. cerevisiae.

The Ras/cAMP pathway in the yeast S. cerevisiae couples the cell cycle of this unicellular organism to the availability of nutrients. Glucose derepressed S. cerevisiae cells respond to glucose addition by an intracellular rise in cAMP. In the prevailing model, yeast Ras plays a similar role to that of heterotrimeric G-proteins coupled to cell surface receptors. A crucial element of this model is that the exchanger, Cdc25 is activated by glucose. Such activation would result in a glucose-dependent rise in GTP-bound Ras concentration. We here show, in contrast to this view, that Cdc25 cannot be the receiver of the glucose signal. We suggest that the Ras-GTP/cyclase complex is the molecular element directly receiving the signal while Cdc25-dependent exchange constitutes a prerequisite for complex formation.

Adenylyl Cyclases↗
PubMed2000

Destabilized green fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry.

Green fluorescent protein (GFP) has many advantages as a reporter molecule, but its stability makes it unsuitable for monitoring dynamic changes in gene expression, among other applications. Destabilized GFPs have been developed for bacterial and mammalian systems to counter this problem. Here, we extend such advances to the yeast model. We fused the PEST-rich 178 carboxyl-terminal residues of the G(1) cyclin Cln2 to the C terminus of yEGFP3 (a yeast- and FACS-optimized GFP variant), creating yEGFP3-Cln2(PEST). We tested the hybrid protein after integrating modules harbouring the yEGFP3 or yEGFP3-CLN2(PEST) ORFs into the Saccharomyces cerevisiae genome. yEGFP3- Cln2(PEST) had a markedly shorter half-life (t(1/2)) than yEGFP3; inhibition of protein synthesis with cycloheximide lead to a rapid decline in GFP content and fluorescence (t(1/2) approximately 30 min) in cells expressing yEGFP3-Cln2(PEST), whereas these parameters were quite stable in yEGFP3-expressing cells (t(1/2) approximately 7 h). We placed yEGFP3-CLN2(PEST) under the control of the CUP1 promoter, which is induced only transiently by copper. This transience was readily discernible with yEGFP3-Cln2(PEST), whereas yEGFP3 reported only on CUP1 switch-on, albeit more slowly than yEGFP3-Cln2(PEST). Cell cycle-regulated transcriptional activation/inactivation of the CLN2 promoter was also discernible with yEGFP3- Cln2(PEST), using cultures that were previously synchronized with nocodazole. In comparison to CLN2, expression from the ACT1 promoter was stable after release from nocodazole. We also applied a novel flow-cytometric technique for cell cycle analysis with asynchronous cultures. The marked periodicities of CLN2 and CLB2 (mitotic cyclin) transcription were readily evident from cellular yEGFP3-Cln2(PEST) levels with this non-perturbing approach. The results represent the first reported successful destabilization of a yeast-GFP. This new construct expands the range of GFP applications open to yeast workers.

Carrier Proteins↗
PubMed2005

Enteropathogenic Escherichia coli type III effectors alter cytoskeletal function and signalling in Saccharomyces cerevisiae.

Enteropathogenic Escherichia coli (EPEC) strains cause attaching/effacing lesions in enterocytes through the development of actin-supported pedestals at the site of bacterial adhesion. Pathogenesis requires a type III secretion system (TTSS), which injects into the host cell the intimin receptor, Tir, as well as other effectors called Esps (Escherichia secreted proteins). The genes encoding TTSS structural components and Esps are found within a pathogenicity island called the locus of enterocyte effacement (LEE). This paper describes the application of Saccharomyces cerevisiae as a model to probe the functions of LEE-encoded genes. In a systematic approach, the LEE-encoded translocator and effector proteins were endogenously expressed in yeast and their effects on cell growth, cytoskeletal function and signalling pathways were studied. EspD, EspG and Map inhibited growth by depolarizing the actin cortical cytoskeleton, whereas EspF expression altered the septin cytoskeleton. Specific yeast MAP kinase pathways were activated by EspF, EspG, EspH and Map. The yeast system was used to define functional domains in Map by expressing truncated versions; it was concluded that the C-terminal region of the protein is necessary for actin disruption and toxicity, but not for mitochondrial localization. The utility of the yeast model for functional analyses of EPEC pathogenesis is discussed.

Actins↗