Addition Of Galactose Research Articles

Functional characterization of the polyketide synthase gene required for ochratoxin A biosynthesis in Penicillium verrucosum

The ochratoxin A (OTA) polyketide synthase otapks gene has been cloned from Penicillium verrucosum. A P. verrucosum mutant in which the otapksPV gene has been interrupted cannot synthesize ochratoxin A. The protein is most similar to the citrinin polyketide synthase CtnpksMa from Monascus anka (83% identity at the amino acid level). Different nutritional conditions influence OTA production in P. verrucosum, with the addition of glycerol and galactose to MCB resulting in approximately 19 and 32 fold increases in OTA production respectively. These effects are mirrored in increased levels of otapksPV gene transcription. In contrast, the addition of glucose to MCB containing galactose results in an approximate 10 fold repression in OTA production, with this repression again being mirrored in decreased levels of otapksPV gene transcription. Thus the effects of different carbon sources on OTA production in P. verucosum appear to be regulated at the level of gene transcription. Two additional open reading frames, otaE and otaT, were identified in the 5′ and 3′ flanking regions of otapksPV, respectively. The otaT and otaE genes are co-expressed with P. verrucosum otapksPv, indicating a possible role for these genes in OTA biosynthesis. Furthermore, otaT and otaE were identified as putative homologues of the M. anka citrinin transporter ctnC (72% amino acid identity) and M. anka citrinin oxidoreductase ctnB (83% amino acid identity); suggesting that the genes involved in OTA production in P. verrucosum may be very similar to those involved in citrinin production in M. anka.

Saccharomyces cerevisiae Dmc1 and Rad51 Proteins Preferentially Function with Tid1 and Rad54 Proteins, Respectively, to Promote DNA Strand Invasion during Genetic Recombination

The Saccharomyces cerevisiae Dmc1 and Tid1 proteins are required for the pairing of homologous chromosomes during meiotic recombination. This pairing is the precursor to the formation of crossovers between homologs, an event that is necessary for the accurate segregation of chromosomes. Failure to form crossovers can have serious consequences and may lead to chromosomal imbalance. Dmc1, a meiosis-specific paralog of Rad51, mediates the pairing of homologous chromosomes. Tid1, a Rad54 paralog, although not meiosis-specific, interacts with Dmc1 and promotes crossover formation between homologs. In this study, we show that purified Dmc1 and Tid1 interact physically and functionally. Dmc1 forms stable nucleoprotein filaments that can mediate DNA strand invasion. Tid1 stimulates Dmc1-mediated formation of joint molecules. Under conditions optimal for Dmc1 reactions, Rad51 is specifically stimulated by Rad54, establishing that Dmc1-Tid1 and Rad51-Rad54 function as specific pairs. Physical interaction studies show that specificity in function is not dictated by direct interactions between the proteins. Our data are consistent with the hypothesis that Rad51-Rad54 function together to promote intersister DNA strand exchange, whereas Dmc1-Tid1 tilt the bias toward interhomolog DNA strand exchange.

Lysine post-translational modifications of collagen

Type I collagen is the most abundant structural protein in vertebrates. It is a heterotrimeric molecule composed of two α1 chains and one α2 chain, forming a long uninterrupted triple helical structure with short non-triple helical telopeptides at both the N- and C-termini. During biosynthesis, collagen acquires a number of post-translational modifications, including lysine modifications, that are critical to the structure and biological functions of this protein. Lysine modifications of collagen are highly complicated sequential processes catalysed by several groups of enzymes leading to the final step of biosynthesis, covalent intermolecular cross-linking. In the cell, specific lysine residues are hydroxylated to form hydroxylysine. Then specific hydroxylysine residues located in the helical domain of the molecule are glycosylated by the addition of galactose or glucose-galactose. Outside the cell, lysine and hydroxylysine residues in the N- and C-telopeptides can be oxidatively deaminated to produce reactive aldehydes that undergo a series of non-enzymatic condensation reactions to form covalent intra- and inter-molecular cross-links. Owing to the recent advances in molecular and cellular biology, and analytical technologies, the biological significance and molecular mechanisms of these modifications have been gradually elucidated. This chapter provides an overview on these enzymatic lysine modifications and subsequent cross-linking.

Flavobacterium johnsoniae RemA Is a Mobile Cell Surface Lectin Involved in Gliding

Cells of Flavobacterium johnsoniae move rapidly over surfaces by a process known as gliding motility. Gld proteins are thought to comprise the motor that propels the cell surface adhesin SprB. Cells with mutations in sprB are partially defective in motility and are also resistant to some bacteriophages. Transposon mutagenesis of a strain carrying a deletion spanning sprB identified eight mutants that were resistant to additional phages and exhibited reduced motility. Four of the mutants had transposon insertions in remA, which encodes a cell surface protein that has a lectin domain and appears to interact with polysaccharides. Three other genes identified in this screen (remC, wza, and wzc) encode proteins predicted to be involved in polysaccharide synthesis and secretion. Myc-tagged versions of RemA localized to the cell surface and were propelled rapidly along the cell at speeds of 1 to 2 μm/s. Deletion of gldN and gldO, which encode components of a bacteroidete protein secretion system, blocked the transport of RemA to the cell surface. Overexpression of RemA resulted in the formation of cell aggregates that were dispersed by the addition of galactose or rhamnose. Cells lacking RemC, Wza, and Wzc failed to aggregate. Cells of a remC mutant and cells of a remA mutant, neither of which formed aggregates in isolation, aggregated when they were mixed together, suggesting that polysaccharides secreted by one cell may interact with RemA on another cell. Fluorescently labeled lectin Ricinus communis agglutinin I detected polysaccharides secreted by F. johnsoniae. The polysaccharides bound to cells expressing RemA and were rapidly propelled on the cell surface. RemA appears to be a mobile cell surface adhesin, and secreted polysaccharides may interact with the lectin domain of RemA and enhance motility.

Interplay of a Ligand Sensor and an Enzyme in Controlling Expression of the Saccharomyces cerevisiae GAL Genes

The regulation of the Saccharomyces cerevisiae GAL genes in response to galactose as a source of carbon has served as a paradigm for eukaryotic transcriptional control over the last 50 years. Three proteins--a transcriptional activator (Gal4p), an inhibitor (Gal80p), and a ligand sensor (Gal3p)--control the switch between inert and active gene expression. The molecular mechanism by which the recognition of galactose within the cell is converted into a transcriptional response has been the subject of considerable debate. In this study, using a novel and powerful method of localizing active transcription factors within the nuclei of cells, we show that a short-lived complex between Gal4p, Gal80p, and Gal3p occurs soon after the addition of galactose to cells to activate GAL gene expression. Gal3p is subsequently replaced in this complex by Gal1p, and a Gal4p-Gal80p-Gal1p complex is responsible for the continued expression of the GAL genes. The transient role of the ligand sensor indicates that current models for the induction and continued expression of the yeast GAL genes need to be reevaluated.

Identification of a galactose‐specific flocculin essential for non‐sexual flocculation and filamentous growth in Schizosaccharomyces pombe

Although various mutant strains of the fission yeast Schizosaccharomyces pombe exhibit non-sexual flocculation, little is known about the mechanistic basis for this phenomenon, nor have genes encoding the implicated flocculin been identified. In the budding yeast Saccharomyces cerevisiae, the transcription factor Flo8 controls expression of some of the genes involved in non-sexual flocculation. We have found that overexpression of S. cerevisiae FLO8 induced non-sexual flocculation in S. pombe. This non-sexual flocculation was Ca(2+) -dependent, and was inhibited by addition of galactose, but not by mannose, glucose or sucrose. In the FLO8-overexpressing strain, a gene designated gsf2(+) (galactose-specific flocculation) was specifically induced. The gsf2(+) gene was also highly expressed in lkh1Δ, tup12Δ and gsf1 mutants, all of which exhibited non-sexual flocculation dependent on gsf2(+) . We show that the N-terminal region of Gsf2 recognizes galactose in mediating cell-cell interaction. Disruption of gsf2(+) also abolished the adhesion phenotype and invasive growth of the wild-type strain cultured in low ammonium medium. The newly identified flocculin Gsf2 in fission yeast was not only required for non-sexual flocculation but was also required for adhesion and filamentous growth through recognition of galactose residues on cell surface glycoconjugates.

Rapid GAL Gene Switch of Saccharomyces cerevisiae Depends on Nuclear Gal3, Not Nucleocytoplasmic Trafficking of Gal3 and Gal80

The yeast transcriptional activator Gal4 localizes to UAS(GAL) sites even in the absence of galactose but cannot activate transcription due to an association with the Gal80 protein. By 4 min after galactose addition, Gal4-activated gene transcription ensues. It is well established that this rapid induction arises through a galactose-triggered association between the Gal80 and Gal3 proteins that decreases the association of Gal80 and Gal4. How this happens mechanistically remains unclear. Strikingly different hypotheses prevail concerning the possible roles of nucleocytoplasmic distribution and trafficking of Gal3 and Gal80 and where in the cell the initial Gal3-Gal80 association occurs. Here we tested two conflicting hypotheses by evaluating the subcellular distribution and dynamics of Gal3 and Gal80 with reference to induction kinetics. We determined that the rates of nucleocytoplasmic trafficking for both Gal80 and Gal3 are slow relative to the rate of induction. We find that depletion of the nuclear pool of Gal3 slows the induction kinetics. Thus, nuclear Gal3 is critical for rapid induction. Fluorescence-recovery-after-photobleaching experiments provided data suggesting that the Gal80-Gal4 complex exhibits kinetic stability in the absence of galactose. Finally, we detect Gal3 at the UAS(GAL) only if Gal80 is covalently linked to the DNA-binding domain. Taken altogether, these new findings lead us to propose that a transient interaction of Gal3 with Gal4-associated Gal80 could explain the rapid response of this system. This notion could also explain earlier observations.

Serum butyrylcholinesterase activity and phenotype associations with lipids, apolipoproteins, sex hormones and nonenzymatic and enzymatic antioxidants systems during various phases of menstrual cycle

We, herein, investigated the in vitro effects of galactose on thiobarbituric acid-reactive substances (TBA-RS), total sulfhydryl content, and on the activities of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and butyrylcholinesterase (BuChE) in the blood of 30- and 60-day-old rats. We also determined the influence of the antioxidants, trolox, ascorbic acid and glutathione, on the effects elicited by galactose on the parameters tested. Galactose was added to the assay at final concentrations of 0.1, 3.0, 5.0 and 10.0 mM. Control experiments were performed without the addition of galactose. Rats were sacrificed by decapitation without anesthesia and a blood sample was removed for analysis. Galactose, at 3.0 mM, 5.0 mM and 10.0 mM, enhanced TBA-RS in the plasma of 60-day-old rats, while 10.0 mM galactose reduced total sulfhydryl content in the plasma of 30-day-old rats; 5.0 mM and 10.0 mM galactose enhanced CAT activity in the erythrocytes of 30- and 60-day-old rats and 10.0 mM galactose reduced SOD activity in the erythrocytes of 60-day-old rats. Galactose did not alter BuChE activity. Data showed that at the pathologically high concentration (greater than 5.0 mM), galactose induces lipid peroxidation, reduces total sulfhydryl content and alters antioxidant defenses in the blood of rats. Trolox, ascorbic acid and glutathione addition prevented most alterations in oxidative stress parameters that were caused by galactose. Our findings lend support to a potential therapeutic strategy for this disease, which may include the use of antioxidants for ameliorating the damage caused by galactose.

Peracetylated 4-Fluoro-glucosamine Reduces the Content and Repertoire of N- and O-Glycans without Direct Incorporation

Prior studies have shown that treatment with the peracetylated 4-fluorinated analog of glucosamine (4-F-GlcNAc) elicits anti-skin inflammatory activity by ablating N-acetyllactosamine (LacNAc), sialyl Lewis X (sLeX), and related lectin ligands on effector leukocytes. Based on anti-sLeX antibody and lectin probing experiments on 4-F-GlcNAc-treated leukocytes, it was hypothesized that 4-F-GlcNAc inhibited sLeX formation by incorporating into LacNAc and blocking the addition of galactose or fucose at the carbon 4-position of 4-F-GlcNAc. To test this hypothesis, we determined whether 4-F-GlcNAc is directly incorporated into N- and O-glycans released from 4-F-GlcNAc-treated human sLeX (+) T cells and leukemic KG1a cells. At concentrations that abrogated galectin-1 (Gal-1) ligand and E-selectin ligand expression and related LacNAc and sLeX structures, MALDI-TOF and MALDI-TOF/TOF mass spectrometry analyses showed that 4-F-GlcNAc 1) reduced content and structural diversity of tri- and tetra-antennary N-glycans and of O-glycans, 2) increased biantennary N-glycans, and 3) reduced LacNAc and sLeX on N-glycans and on core 2 O-glycans. Moreover, MALDI-TOF MS did not reveal any m/z ratios relating to the presence of fluorine atoms, indicating that 4-F-GlcNAc did not incorporate into glycans. Further analysis showed that 4-F-GlcNAc treatment had minimal effect on expression of 1200 glycome-related genes and did not alter the activity of LacNAc-synthesizing enzymes. However, 4-F-GlcNAc dramatically reduced intracellular levels of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), a key precursor of LacNAc synthesis. These data show that Gal-1 and E-selectin ligand reduction by 4-F-GlcNAc is not caused by direct 4-F-GlcNAc glycan incorporation and consequent chain termination but rather by interference with UDP-GlcNAc synthesis.

Lectin 주형을 이용한 탄수화물 기능화된 금 나노입자의 자기조립에 관한 연구

The unique structure produced by biological systems provides inspiration for the fabrication of functional materials. Proteins themselves are nanoscopic entities as well as can be used as useful nanomaterial components possessing novel properties. In addition to their exquisite features, they are obtained easily from the natural sources. There are a number of examples which employ viruses and protein assemblies for the design of new functional materials. Freeman and co-workers at Tel Aviv University have shown the formation of the predesigned diamondlike protein crystals using the self assembly of a tetrahedral letin. This strategy can be applied for the controlled fabrication of new three-dimensional protein lattices and scaffolds. Rotello and co-workers at the University of Massachusetts demonstrated the controlled assembly of protein-nanoparticle composites via protein surface recognition. These protein-mediated assemblies are also important tools for the detection of biomolecules as well as for the manufacturing of functional nanomaterials with their unique properties. Herein, we report the use of the lectin protein for the spontaneous self-assembly of carbohydratefunctionalized gold nanoparticles. We envisage the precise control over the spatial arrangements of carbohydrate-functionalized gold nanoparticles using the specific carbohydrateprotein interaction. This interaction plays a crucial role in a wide variety of intercellular processes in our nature. Gold nanoparticles were suggested as excellent fluorescence quenchers. In this report, we have observed the recognition process via quantitative fluorescence quenching experiments by monitoring of the fluorescence of TritcConcanavalin A (Con A) as the addition of the nanoparticles. Self-assembly process was observed using qualitative TEM, dynamic light scattering technique as well. We observed the aggregation process can be reversed by the addition of free mannose sugar which was not observed by the addition of galactose.

Yeast Nej1 Is a Key Participant in the Initial End Binding and Final Ligation Steps of Nonhomologous End Joining

In Saccharomyces cerevisiae, the key components of the nonhomologous end joining (NHEJ) pathway that repairs DNA double-strand breaks (DSBs) are yeast Ku (yKu), Mre11-Rad50-Xrs2, Dnl4-Lif1, and Nej1. Here, we examined the role of Nej1 in NHEJ by a combination of molecular genetic and biochemical approaches. As expected, the recruitment of Nej1 to in vivo DSBs is dependent upon yKu. Surprisingly, Nej1 is required for the stable binding of yKu to in vivo DSBs, in addition to Dnl4-Lif1. Thus, Nej1 and Dnl4-Lif1 are independently recruited by yKu to in vivo DSBs, forming a stable ternary complex that channels DSBs into the NHEJ pathway. In accord with these results, purified Nej1 interacts with yKu and preferentially binds to DNA ends bound by yKu. Furthermore, the binding of a mixture of Nej1 and Dnl4-Lif1 to DNA ends bound by yKu is greater than the sum of the binding of the individual proteins, indicating that pairwise interactions among yKu, Nej1, and Dnl4-Lif1 contribute to complex assembly at DNA ends. Nej1 stimulates intermolecular ligation by Dnl4-Lif1, but, more interestingly, the addition of Nej1 results in more than one intermolecular ligation per Dnl4 molecule. Thus, Nej1 not only plays an important role in determining repair pathway choice by participating in the initial NHEJ complex formed at DSBs but also contributes to the reactivation of Dnl4-Lif1 after repair is complete, thereby increasing the capacity of the NHEJ repair pathway.

Inhibition of raffinose family oligosaccharides and galactosyl pinitols breakdown delays germination of winter vetch (Vicia villosa Roth.) seeds

Beside RFOs, which are commonly present in legume seeds, seeds of some species contain galactosyl pinitols (GPs). These carbohydrates, like RFOs, have been hypothesized to constitute an important energy and carbon skeletal source during germination. To test this hypothesis we have applied a specific α-galactosidase inhibitor (1-deoxygalactonojirimycin, DGJ) to germinating winter vetch (<em>Vicia villosa</em> Roth.) seeds, containing more galactosyl pinitols than RFOs. The breakdown of RFOs but not that of GPs was completely blocked in both embryonic axes and cotyledons tissues, during the first 18 h of imbibition in DGJ. The inhibitor only decreased the rate of GPs degradation. The inhibitory effect of DGJ on GPs degradation was partially alleviated by addition of sucrose or galactose to DGJ solutions. After three days of germination in water, RFOs and GPs disappeared in axial tissues of seeds imbibed in water, galactose or sucrose. Eighteen-hour imbibition of seeds in DGJ drastically reduced germination, by ca 50%, during the first three days. The inhibitory effect of DGJ decreased during the next seven days of germination. The presence of galactose or sucrose in imbibition solution initially stimulated seed germination, but later this effect was not statistically significant. Our study provides clear evidence that galactosyl pinitols play an important role in early winter vetch seeds germination. Additionally, we suggest that galactosyl pinitols can replace RFOs as reserve material necessary for early germination.

Production of heterologous polygalacturonase I from Aspergillus kawachii in Saccharomyces cerevisiae in batch and fed-batch cultures

The pg1 gene from the filamentous fungus Aspergillus kawachii, which codifies for an acid polygalacturonase, was cloned into the pYES2 expression vector, giving rise to the pYES2:pg1ΔI construct. Engineered Saccharomyces cerevisiae, transformed with pYES2:pg1ΔI construct, both expressed and exported an active polygalacturonase with a MW of ~60 kDa and an isoelectric point of 3.7, similar to those reported for the wild-type enzyme. The recombinant enzyme has the ability to hydrolyze polygalacturonic acid at pH 2.5. Heterologous PG1 production was studied under controlled conditions in batch and fed-batch systems. A simultaneous addition of glucose and galactose was found to be the most suitable feeding strategy assayed, resulting in a final PG1 production of 50 U/ml. The production process proposed in this study could be applied for the industrial production of a novel and useful polygalacturonase.

Subcompartment localization of the side chain xyloglucan-synthesizing enzymes within Golgi stacks of tobacco suspension-cultured cells

Xyloglucan is the dominant hemicellulosic polysaccharide of the primary cell wall of dicotyledonous plants that plays a key role in plant development. It is well established that xyloglucan is assembled within Golgi stacks and transported in Golgi-derived vesicles to the cell wall. It is also known that the biosynthesis of xyloglucan requires the action of glycosyltransferases including α-1,6-xylosyltransferase, β-1,2-galactosyltransferase and α-1,2-fucosyltransferase activities responsible for the addition of xylose, galactose and fucose residues to the side chains. There is, however, a lack of knowledge on how these enzymes are distributed within subcompartments of Golgi stacks. We have undertaken a study aiming at mapping these glycosyltransferases within Golgi stacks using immunogold-electron microscopy. To this end, we generated transgenic lines of tobacco (Nicotiana tabacum) BY-2 suspension-cultured cells expressing either the α-1,6-xylosyltransferase, AtXT1, the β-1,2-galactosyltransferase, AtMUR3, or the α-1,2-fucosyltransferase AtFUT1 of Arabidopsis thaliana fused to green-fluorescent protein (GFP). Localization of the fusion proteins within the endomembrane system was assessed using confocal microscopy. Additionally, tobacco cells were high pressure-frozen/freeze-substituted and subjected to quantitative immunogold labelling using anti-GFP antibodies to determine the localization patterns of the enzymes within subtypes of Golgi cisternae. The data demonstrate that: (i) all fusion proteins, AtXT1-GFP, AtMUR3-GFP and AtFUT1-GFP are specifically targeted to the Golgi apparatus; and (ii) AtXT1-GFP is mainly located in the cis and medial cisternae, AtMUR3-GFP is predominantly associated with medial cisternae and AtFUT1-GFP mostly detected over trans cisternae suggesting that initiation of xyloglucan side chains occurs in early Golgi compartments in tobacco cells.

New insights into galactose metabolism by Schizosaccharomyces pombe: Isolation and characterization of a galactose-assimilating mutant

The fission yeast Schizosaccharomyces pombe cannot use galactose as a carbon or energy source, and little is known about galactose metabolism in this species. Here we report isolation of a galactose-assimilating mutant that grows on a medium containing galactose as a sole carbon source through use of a proofreading-deficient DNA polymerase δ variant encoded by cdc6-1. Based on comparative analysis of gene expression profiles in the wild-type and the mutant (FG2-8), we found that SPBPB2B2.10c (gal7+), SPBPB2B2.12c (gal10+) and SPBPB2B2.13 (gal1+), homologous to Saccharomyces cerevisiae GAL7, GAL10 and GAL1, respectively, and SPBPB2B2.08, SPBPB2B2.09c, and SPBPB2B2.11 that localize close to the gal genes, were highly expressed and dramatically induced by addition of galactose. The gal7Δ strain, carrying an integrated ura4+ marker at the gal7+ locus, grew on 5-fluoroorotic acid (5-FOA)-containing medium. In contrast, the FG2-8 gal7Δ strain could not grow on 5-FOA medium. In addition, expression of gal7+, SPBPB2B2.13, gal10+ and gal1+ genes increased in the wild-type strain when carried on a vector, and these transformants grew on galactose medium. We suggest that gal7+, gal10+, and gal1+ are localized close to a chromosomal terminal repressed by gene silencing in S. pombe. In contrast, gene silencing was defective in the FG2-8 strain making galactose assimilation possible.

Reduction of galactose inhibition via the mutation of β-galactosidase from Caldicellulosiruptor saccharolyticus for lactose hydrolysis

For the removal of galactose inhibition, the predicted galactose binding residues, which were determined by sequence alignment, were replaced separately with Ala. The activities of the Ala-substituted mutant enzymes were assessed with the addition of galactose. As a consequence, amino acid at position 349 was correlated with the reduction in galactose inhibition. The F349S mutant exhibited the highest activity in the presence of galactose relative to the activity measured in the absence of galactose among the tested mutant enzymes at position 349. The K (i) of the F349S mutant (160 mM), which was 13-fold that of the wild-type enzyme, was the highest among the reported values of β-galactosidase. The wild-type enzyme hydrolyzed 62% of 100 g lactose/l with the addition of 30 g galactose/l, whereas the F349S mutant hydrolyzed more than 99%.

Biodistribution and Excretion of Monosaccharide−Albumin Conjugates Measured with in Vivo Near-Infrared Fluorescence Imaging

Target specific small molecules as modulators of drug delivery may play a significant role in the future development of therapeutics. Small molecules can alter the in vivo pharmacokinetics of therapeutic macromolecules leading to more efficient drug delivery with less systemic toxicity. The potential of creating a more effective drug delivery system through glycosylation has led, for instance, to the addition of galactose to increase drug delivery to the liver. However, there are many other monosaccharides with potentially useful targeting properties that require further characterization. Here, we investigate the potential of glycosylation to guide molecular therapies using five different monosaccharides conjugated to human serum albumin (HSA). Additionally, we investigate how the amount of glycosylation may alter the pharmacokinetic profile of HSA. We introduce the use of in vivo near-infrared optical imaging to characterize the effect of differential glycosylation on the pharmacokinetics of macromolecules.

Identification of Pep4p as the Protease Responsible for Formation of the SAGA-related SLIK Protein Complex

The Saccharomyces cerevisiae Spt-Ada-Gcn5 acetyltransferase (SAGA) protein complex is a coactivator for transcription by RNA polymerase II and has various activities, including acetylation and deubuiqitination of histones and recruitment of TATA-binding protein to promoters. The Spt7p subunit is subject to proteolytic cleavage at its C terminus resulting in removal of the Spt8p-binding domain and generation of the SAGA-related SALSA/SAGA-like (SLIK) protein complex. Here, we report identification of the protease responsible for this cleavage. Screening of a protease knock-out collection revealed PEP4 to be required for cleavage of Spt7p within SAGA in vitro. Endogenous formation of truncated Spt7p was abolished in cells lacking PEP4. Purified Pep4p but not catalytic dead mutant Pep4p or unrelated Prc1p protease specifically cleaved Spt7p within SAGA into SLIK-related Spt7p. Interestingly, SAGA lacking Spt8p was more sensitive to Pep4p-mediated truncation of Spt7p, suggesting that Spt8p counteracted its own release from SAGA. Strains mimicking constitutive SLIK formation showed increased resistance to rapamycin treatment, suggesting a role for SLIK in regulating cellular responses to nutrient stress.

Derivation, identification and validation of a computational model of a novel synthetic regulatory network in yeast

Systems biology aims at building computational models of biological pathways in order to study in silico their behaviour and to verify biological hypotheses. Modelling can become a new powerful method in molecular biology, if correctly used. Here we present step-by-step the derivation and identification of the dynamical model of a biological pathway using a novel synthetic network recently constructed in the yeast Saccharomyces cerevisiae for In-vivo Reverse-Engineering and Modelling Assessment. This network consists of five genes regulating each other transcription. Moreover, it includes one protein-protein interaction, and its genes can be switched on by addition of galactose to the medium. In order to describe the network dynamics, we adopted a deterministic modelling approach based on non-linear differential equations. We show how, through iteration between experiments and modelling, it is possible to derive a semi-quantitative prediction of network behaviour and to better understand the biology of the pathway of interest.

A food-grade industrial arming yeast expressing β-1,3-1,4-glucanase with enhanced thermal stability

The aim of this work was to construct a novel food-grade industrial arming yeast displaying beta-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase (GAL1) and phosphoglycerate kinase 1 (PGK1) promoters in different orientations was constructed. The beta-1,3-1,4-glucanase gene from Bacillus subtilis was fused to alpha-agglutinin and expressed under the control of the GAL1 promoter. alpha-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the alpha-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of beta-1,3-1,4-glucanase. The analysis of beta-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that beta-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed beta-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of beta-1,3-1,4-glucanase displayed in the recombinant yeast cells was enhanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer.