Enhanced viability of probiotic Saccharomyces boulardii encapsulated by layer-by-layer approach in pH responsive chitosan-dextran sulfate polyelectrolytes

Saccharomyces boulardii was encapsulated by layer-by-layer technique (LbL) using oppositely charged polyelectrolytes, chitosan and dextran sulfate to protect from degradation during its gastrointestinal transit. The protective effect of the coating was evaluated by checking viability after subjecting the coated cells to lyophilisation and simulated gastrointestinal conditions. During lyophilization, coated S. boulardii was found to have an enhanced viability of 7.74 +/- 2.00 log CFU/100 mg (5.62 x 10(6) +/- 2.12 CFU/100 mg) and 5.53 +/- 1.85 log CFU/100 mg (3.46 x 10(5) 1.73 CFU/100 mg) for uncoated cells. On sequential treatment with simulated gastric and intestinal juice, the coated cells had a viability of 4.59 +/- 1.52 log CFU/100 mg (3.8 x 104 +/- 1.52 CFU/100 mg) while only 1.90 +/- 0.80 log CFU/100 mg (0.79 x 102 +/- 0.81 CFU/100 mg) of uncoated cells survived. Confocal studies displayed the selective permeability of the coated cells which plays a significant role in maintaining the integrity and viability of the yeast cells. This clearly indicates that LbL is an efficient protective encapsulation technique and it could be potentially used for improving therapeutic applications of yeast. (C) 2014 Elsevier Ltd. All rights reserved.

Positional Preferences of Polypurine/Polypyrimidine Tracts in Saccharomyces cerevisiae Genome: Implications for cis Regulation of Gene Expression

The complete genome of the baker's yeast S. cerevisiae was analyzed for the presence of polypurine/polypyrimidine (poly[pu/py]) repeats and their occurrences were classified on the basis of their location within and outside open reading frames (ORFs). The analysis reveals that such sequence motifs are present abundantly both in coding as well as noncoding regions. Clear positional preferences are seen when these tracts occur in noncoding regions. These motifs appear to occur predominantly at a unit nucleosomal length both upstream and downstream of ORFs. Moreover, there is a biased distribution of polypurines in the coding strands when these motifs occur within open reading frames. The significance of the biased distribution is discussed with reference to the occurrence of these motifs in other known mRNA sequences and expressed sequence tags. A model for cis regulation of gene expression is proposed based on the ability of these motifs to form an intermolecular triple helix structure when present within the coding region and/or to modulate nucleosome positioning via enhanced histone affinity when present outside coding regions.

Genetic studies of the PRP17 gene of Saccharomyces cerevisiae: a domain essential for function maps to a nonconserved region of the protein

The PRP17 gene product is required for the second step of pre-mRNA splicing reactions. The C-terminal half of this protein bears four repeat units with homology to the beta transducin repeat. Missense mutations in three temperature-sensitive prp17 mutants map to a region in the N-terminal half of the protein. We have generated, in vitro, 11 missense alleles at the beta transducin repeat units and find that only one affects function in vivo. A phenotypically silent missense allele at the fourth repeat unit enhances the slow-growing phenotype conferred by an allele at the third repeat, suggesting an interaction between these domains. Although many missense mutations in highly conserved amino acids lack phenotypic effects, deletion analysis suggests an essential role for these units. Only mutations in the N-terminal nonconserved domain of PRP17 are synthetically lethal in combination with mutations in PRP16 and PRP18, two other gene products required for the second splicing reaction. A mutually allele-specific interaction between Prp17 and snr7, with mutations in U5 snRNA, was observed. We therefore suggest that the functional region of Prp17p that interacts with Prp18p, Prp16p, and U5 snRNA is the N terminal region of the protein.

An extragenic suppressor of prp24-1 defines genetic interaction between PRP24 and PRP21 gene products of Saccharomyces cerevisiae

The temperature-sensitive prp24-1 mutation defines a gene product required for the first step in pre-mRNA splicing. PRP24 is probably a component of the U6 snRNP particle. We have applied genetic reversion analysis to identify proteins that interact with PRP24. Spontaneous revertants of the temperature-sensitive (ts) prp24-1 phenotype were analyzed for those that are due to extragenic suppression. We then extended our analysis to screen for suppressors that confer a distinct conditional phenotype. We have identified a temperature-sensitive extragenic suppressor, which was shown by genetic complementation analysis to be allelic to prp21-1. This suppressor, prp21-2, accumulates pre-mRNA at the non-permissive temperature, a phenotype similar to that of prp21-1. prp21-2 completely suppresses the splicing defect and restores in vivo levels of the U6 snRNA in the prp24-1 strain. Genetic analysis of the suppressor showed that prp21-2 is not a bypass suppressor of prp24-1. The suppression of prp24-1 by prp21-2 is gene specific and also allele specific with respect to both the loci. Genetic interactions with other components of the pre-spliceosome have also been studied. Our results indicate an interaction between PRP21, a component of the U2 snRNP, and PRP24, a component of the U6 snRNP. These results substantiate other data showing U2-U6 snRNA interactions.

Limiting the Extent of the RDN1 Heterochromatin Domain by a Silencing Barrier and Sir2 Protein Levels in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, transcriptional silencing occurs at the cryptic mating-type loci (HML and HMR), telomeres, and ribosomal DNA ( rDNA; RDN1). Silencing in the rDNA is unusual in that polymerase II (Pol II) promoters within RDN1 are repressed by Sir2 but not Sir3 or Sir4. rDNA silencing unidirectionally spreads leftward, but the mechanism of limiting its spreading is unclear. We searched for silencing barriers flanking the left end of RDN1 by using an established assay for detecting barriers to HMR silencing. Unexpectedly, the unique sequence immediately adjacent to RDN1, which overlaps a prominent cohesin binding site (CARL2), did not have appreciable barrier activity. Instead, a fragment located 2.4 kb to the left, containing a tRNA(Gln) gene and the Ty1 long terminal repeat, had robust barrier activity. The barrier activity was dependent on Pol III transcription of tRNA(Gln), the cohesin protein Smc1, and the SAS1 and Gcn5 histone acetyltransferases. The location of the barrier correlates with the detectable limit of rDNA silencing when SIR2 is overexpressed, where it blocks the spreading of rDNA heterochromatin. We propose a model in which normal Sir2 activity results in termination of silencing near the physical rDNA boundary, while tRNA(Gln) blocks silencing from spreading too far when nucleolar Sir2 pools become elevated.

Unstructured N Terminus of the RNA Polymerase II Subunit Rpb4 Contributes to the Interaction of Rpb4·Rpb7 Subcomplex with the Core RNA Polymerase II of Saccharomyces cerevisiae

Two subunits of eukaryotic RNA polymerase II, Rpb7 and Rpb4, form a subcomplex that has counterparts in RNA polymerases I and III. Although a medium resolution structure has been solved for the 12-subunit RNA polymerase II, the relative contributions of the contact regions between the subcomplex and the core polymerase and the consequences of disrupting them have not been studied in detail. We have identified mutations in the N-terminal ribonucleoprotein-like domain of Saccharomyces cerevisiae Rpb7 that affect its role in certain stress responses, such as growth at high temperature and sporulation. These mutations increase the dependence of Rpb7 on Rpb4 for interaction with the rest of the polymerase. Complementation analysis and RNA polymerase pulldown assays reveal that the Rpb4 center dot Rbp7 subcomplex associates with the rest of the core RNA polymerase II through two crucial interaction points: one at the N-terminal ribonucleoprotein-like domain of Rpb7 and the other at the partially ordered N-terminal region of Rpb4. These findings are in agreement with the crystal structure of the 12-subunit polymerase. We show here that the weak interaction predicted for the N-terminal region of Rpb4 with Rpb2 in the crystal structure actually plays a significant role in interaction of the subcomplex with the core in vivo. Our mutant analysis also suggests that Rpb7 plays an essential role in the cell through its ability to interact with the rest of the polymerase.

Cloning,overexpression and purification of functionally active Saccharomyces cerevisiae Hop1 protein from scherichia coli

One of the major limitations to the application of high-resolution biophysical techniques such as X-crystallography and spectroscopic analyses to structure-function studies of Saccharomyces cerevisiae Hop1 protein has been the non-availability of sufficient quantities of functionally active pure protein. This has, indeed, been the case of many proteins, including yeast synaptonemal complex proteins. In this study, we have performed expression screening in Escherichia coli host strains, capable of high-level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed for expression and purification of S. cerevisiae Hop1 protein, based on the presence of hexa-histidine tag and double-stranded DNA-Cellulose chromatography. Recombinant S. cerevisiae Hop1 protein was >98% pure and exhibited DNA-binding activity with high-affinity to the Holliday junction. The availability of the recombinant HOP1 expression vector and active Hop1 protein would facilitate structure-function investigations as well as the generation of appropriate truncated and site-directed mutant proteins, respectively. (C) 2010 Elsevier Inc. All rights reserved.

The nucleus of Saccharomyces bayanus

The extra-vacuolar nucleus is visible in a small percentage of living cells from 72–96 hour wort cultures. The vacuoles show a luminous boundary under dark ground illumination. The details observed in living nuclei could be stained with haematoxylin after fixation in iodine-formaldehyde-acetic acid mixture. The Feulgen-negative nature of the vacuole and the limitation of the Feulgen-positive material to the area bounded by the nuclear membrane would imply that the ‘centrosome’ described by Lindegren and Rafalko (1950) is the real nucleus. The nucleus ofS. bayanus conforms in its structure to those of higher organisms.

The reaction of the nuclei of the vegetative cells and zygotes of Saccharomyces carlsbergensis to some staining techniques

Vegetative cells and zygotes of Saccharomyces carlsbergensis fixed in iodine formaldehyde acetic acid solution and stained after acid hydrolysis in hæmatoxylin, Feulgen and Giemsa show a remarkable similarity in the size and orientation of the structures in the nuclear matrix with reference to the nuclear membrane. The nucleolus described by Guilliermond may either be the chromocenter or the nucleolar equivalent.

Triacylglycerol lipolysis is linked to sphingolipid and phospholipid metabolism of the yeast Saccharomyces cerevisiae

Previous work from our laboratory had demonstrated that deletion of TGL3 encoding the major yeast triacylglycerol (TAG) lipase resulted in decreased mobilization of TAG, a sporulation defect and a changed pattern of fatty acids, especially increased amounts of C22:0 and C26:0 very long chain fatty acids in the TAG fraction K. Athenstaedt and G. Daum, J. Biol. Chem. 278 (2003) 23317-23323]. To study a possible link between TAG lipolysis and membrane lipid biosynthesis, we carried out metabolic labeling experiments with wild type and deletion strains bearing defects in the three major yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. Using H-3]inositol. P-32]orthophosphate, 3H]palmitate and C-14]acetate as precursors for complex lipids we demonstrated that tgl mutants had a lower level of sphingolipids and glycerophospholipids than wild type. ESI-MS/MS analyses confirmed that TAG accumulation in these mutant cells resulted in reduced amounts of phospholipids and sphingolipids. In vitro and in vivo experiments revealed that TAG lipolysis markedly affected the metabolic flux of long chain fatty acids and very long chain fatty acids required for sphingolipid and glycerophospholipid synthesis. Activity and expression level of fatty acid elongases, Elo1p and Elo2p were enhanced as a consequence of reduced TAG lipolysis. Finally, the pattern of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine molecular species was altered in tgl deletion strain underlining the important role of TAG turnover in maintaining the pool size of these compounds and the remodeling of complex membrane lipids. (C) 2010 Elsevier B.V. All rights reserved.

Small Ubiquitin-related Modifier Ligase Activity of Mms21 Is Required for Maintenance of Chromosome Integrity during the Unperturbed Mitotic Cell Division Cycle in Saccharomyces cerevisiae

The SUMO ligase activity of Mms21/Nse2, a conserved member of the Smc5/6 complex, is required for resisting extrinsically induced genotoxic stress. We report that the Mms21 SUMO ligase activity is also required during the unchallenged mitotic cell cycle in Saccharomyces cerevisiae. SUMO ligase-defective cells were slow growing and spontaneously incurred DNA damage. These cells required caffeine-sensitive Mec1 kinase-dependent checkpoint signaling for survival even in the absence of extrinsically induced genotoxic stress. SUMO ligase-defective cells were sensitive to replication stress and displayed synthetic growth defects with DNA damage checkpoint-defective mutants such as mec1, rad9, and rad24. MMS21 SUMO ligase and mediator of replication checkpoint 1 gene (MRC1) were epistatic with respect to hydroxyurea-induced replication stress or methyl methanesulfonate-induced DNA damage sensitivity. Subjecting Mms21 SUMO ligase-deficient cells to transient replication stress resulted in enhancement of cell cycle progression defects such as mitotic delay and accumulation of hyperploid cells. Consistent with the spontaneous activation of the DNA damage checkpoint pathway observed in the Mms21-mediated sumoylation-deficient cells, enhanced frequency of chromosome breakage and loss was detected in these mutant cells. A mutation in the conserved cysteine 221 that is engaged in coordination of the zinc ion in Loop 2 of the Mms21 SPL-RING E3 ligase catalytic domain resulted in strong replication stress sensitivity and also conferred slow growth and Mec1 dependence to unchallenged mitotically dividing cells. Our findings establish Mms21-mediated sumoylation as a determinant of cell cycle progression and maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in budding yeast.

Glutathione peroxidase3 of Saccharomyces cerevisiae protects phospholipids during cadmium-induced oxidative stress

The present study was undertaken to determine the role of glutathione peroxidase3 (gpx3) in phospholipid protection in cells. Wild-type (WT) cells showed an overall increase in phospholipids upon 50 mu M cadmium (Cd)-treatment, whereas an untreated gpx3 Delta strain showed a drastic reduction in overall phospholipids which was further reduced with 50 mu M Cd. In WT cells, Cd-exposure increased the short chain fatty acids and decreased the unsaturated fatty acids and the magnitude was high in Cd-treated gpx3 Delta cells. Purified recombinant gpx3p showed higher activity with phospholipid hydroperoxides than shorter hydroperoxides. An increase in gpx activity was observed in Cd-treated WT cells and no such alteration was observed in gpx3 Delta. WT cells treated with Cd showed an increase in MDA over untreated, while untreated gpx3 Delta cells themselves showed a higher level of MDA which was further enhanced with Cd-treatment. Iron, zinc and calcium levels were significantly altered in WT and gpx3 Delta cells during Cd-treatment.

Primary Sequence That Determines the Functional Overlap between Mitochondrial Heat Shock Protein 70 Ssc1 and Ssc3 of Saccharomyces cerevisiae

The evolutionary diversity of the HSP70 gene family at the genetic level has generated complex structural variations leading to altered functional specificity and mode of regulation in different cellular compartments. By utilizing Saccharomyces cerevisiae as a model system for better understanding the global functional cooperativity between Hsp70 paralogs, we have dissected the differences in functional properties at the biochemical level between mitochondrial heat shock protein 70 (mtHsp70) Ssc1 and an uncharacterized Ssc3 paralog. Based on the evolutionary origin of Ssc3 and a high degree of sequence homology with Ssc1, it has been proposed that both have a close functional overlap in the mitochondrial matrix. Surprisingly, our results demonstrate that there is no functional cross-talk between Ssc1 and Ssc3 paralogs. The lack of in vivo functional overlap is due to altered conformation and significant lower stability associated with Ssc3. The substrate-binding domain of Ssc3 showed poor affinity toward mitochondrial client proteins and Tim44 due to the open conformation in ADP-bound state. In addition to that, the nucleotide-binding domain of Ssc3 showed an altered regulation by the Mge1 co-chaperone due to a high degree of conformational plasticity, which strongly promotes aggregation. Besides, Ssc3 possesses a dysfunctional inter-domain interface thus rendering it unable to perform functions similar to generic Hsp70s. Moreover, we have identified the critical amino acid sequence of Ssc1 and Ssc3 that can ``make or break'' mtHsp70 chaperone function. Together, our analysis provides the first evidence to show that the nucleotide-binding domain of mtHsp70s plays a critical role in determining the functional specificity among paralogs and orthologs across kingdoms.

Microbially induced separation of quartz from calcite using Saccharomyces cerevisiae

Cells of Saccharomyces cerevisiae and their metabolites were successfully utilized to achieve selective separation of quartz and calcite through microbially induced flotation and flocculation. S. cerevisiae was adapted to calcite and quartz minerals. Adsorption studies and electrokinetic investigations were carried out to understand the changes in the surface chemistry of yeast cells and the minerals after mutual interaction. Possible mechanisms in microbially induced flotation and flocculation are outlined. (C) 2011 Elsevier B.V. All rights reserved.

Saccharomyces cerevisiae NineTeen Complex (NTC)-associated Factor Bud31/Ycr063w Assembles on Precatalytic Spliceosomes and Improves First and Second Step Pre-mRNA Splicing Efficiency

Pre-mRNA splicing occurs in spliceosomes whose assembly and activation are critical for splice site selection and catalysis. The highly conserved NineTeen complex protein complex stabilizes various snRNA and protein interactions early in the spliceosome assembly pathway. Among several NineTeen complex-associated proteins is the nonessential protein Bud31/Ycr063w, which is also a component of the Cef1p subcomplex. A role for Bud31 in pre-mRNA splicing is implicated by virtue of its association with splicing factors, but its specific functions and spliceosome interactions are uncharacterized. Here, using in vitro splicing assays with extracts from a strain lacking Bud31, we illustrate its role in efficient progression to the first catalytic step and its requirement for the second catalytic step in reactions at higher temperatures. Immunoprecipitation of functional epitope-tagged Bud31 from in vitro reactions showed that its earliest association is with precatalytic B complex and that the interaction continues in catalytically active complexes with stably bound U2, U5, and U6 small nuclear ribonucleoproteins. In complementary experiments, wherein precatalytic spliceosomes are selected from splicing reactions, we detect the occurrence of Bud31. Cross-linking of proteins to pre-mRNAs with a site-specific 4-thio uridine residue at the -3 position of exon 1 was tested in reactions with WT and bud31 null extracts. The data suggest an altered interaction between a similar to 25-kDa protein and this exonic residue of pre-mRNAs in the arrested bud31 null spliceosomes. These results demonstrate the early spliceosomal association of Bud31 and provide plausible functions for this factor in stabilizing protein interactions with the pre-mRNA.