Showing papers on "Yeast published in 1995"
TL;DR: The results showed that T‐DNA circles were formed in yeast with precise fusions between the left and right borders, suggesting that the process of T‐ DNA integration is predominantly determined by host factors.
Abstract: Agrobacterium tumefaciens transfers part of its tumour-inducing (Ti) plasmid, the transferred or T-DNA, to plants during tumourigenesis This represents the only example of naturally occurring trans-kingdom transfer of genetic material Here we report that Atumefaciens can transfer its T-DNA not only to plant cells, but also to another eukaryote, namely the yeast Saccharomyces cerevisiae The Ti plasmid virulence (vir) genes that mediate T-DNA transfer to plants were found to be essential for transfer to yeast as well Transgenic Scerevisiae strains were analysed for their T-DNA content Results showed that T-DNA circles were formed in yeast with precise fusions between the left and right borders Such T-DNA circles were stably maintained by the yeast if the replicator from the yeast 2 mu plasmid was present in the T-DNA Integration of T-DNA in the Scerevisiae genome was found to occur via homologous recombination This contrasts with integration in the plant genome, where T-DNA integrates preferentially via illegitimate recombination Our results thus suggest that the process of T-DNA integration is predominantly determined by host factors
634 citations
TL;DR: It was concluded that ETR1 acts as an ethylene receptor in Arabidopsis and that the amino-terminal hydrophobic domain of the protein is the site of ethylene binding.
Abstract: Mutations in the ETR1 gene of Arabidopsis thaliana confer insensitivity to ethylene, which indicates a role for the gene product in ethylene signal transduction. Saturable binding sites for [14C]ethylene were detected in transgenic yeast expressing the ETR1 protein, whereas control yeast lacking ETR1 showed no detectable ethylene binding. Yeast expressing a mutant form of ETR1 (etr1-1) also showed no detectable ethylene binding, which provides an explanation for the ethylene-insensitive phenotype observed in plants carrying this mutation. Expression of truncated forms of ETR1 in yeast provided evidence that the amino-terminal hydrophobic domain of the protein is the site of ethylene binding. It was concluded from these results that ETR1 acts as an ethylene receptor in Arabidopsis.
593 citations
TL;DR: An overall view of the current understanding of cell wall dynamics and of the complex network that controls polarized growth at particular stages of the budding yeast cell cycle and life cycle is offered.
456 citations
TL;DR: Work in yeast and filamentous fungi has revealed a mechanism for glucose repression in eukaryotes that is different from that found in bacteria, and zinc finger proteins play a key role in mediating this response.
441 citations
TL;DR: A dual function of the trehalose-6-phosphate synthase subunit has been found in several yeast species, suggesting that this control system might be widespread in fungi and possibly also in other organisms.
438 citations
TL;DR: Biosorption of uranium by S. cerevisiae was a rapid process reaching 60% of the final uptake value within the first 15 min of contact, and uranium was deposited as fine-needle-like crystals both on the inside and outside of the S. Cerevisiae cells.
Abstract: Abundant and common yeast biomass has been examined for its capacity to sequester heavy metals from dilute aqueous solutions. Live and non-living biomass of Saccharomyces cerevisiae differs in the uptake of uranium, zinc and copper at the optimum pH 4-5. Culture growth conditions can influence the biosorbent metal uptake capacity which normally was: living and non-living brewer's yeast: U > Zn > Cd > Cu; non-living baker's yeast: Zn > (Cd) > U > Cu; living baker's yeast: Zn > Cu approximately (Cd) > U. Non-living brewer's yeast biomass accumulated 0.58 mmol U/g. The best biosorbent of zinc was non-living baker's yeast (approximately 0.56 mmol Zn/g). Dead cells of S. cerevisiae removed approximately 40% more uranium or zinc than the corresponding live cultures. Biosorption of uranium by S. cerevisiae was a rapid process reaching 60% of the final uptake value within the first 15 min of contact. Its deposition differing from that of other heavy metals more associated with the cell wall, uranium was deposited as fine needle-like crystals both on the inside and outside of the S. cerevisiae cells.
376 citations
TL;DR: Oligonucleotide-directed mutagenesis is used to alter the COOH- terminal portion of Sec8 with a 6-histidine tag, a 9E10 c-myc epitope, or both to allow the isolation of the Sec8/15 complex from yeast lysates either by immobilized metal affinity chromatography or by immunoprecipitation.
Abstract: In the yeast Saccharomyces cerevisiae, the products of at least 14 genes are involved specifically in vesicular transport from the Golgi apparatus to the plasma membrane. Two of these genes, SEC8 and SEC15, encode components of a 1-2-million D multi-subunit complex that is found in the cytoplasm and associated with the plasma membrane. In this study, oligonucleotide-directed mutagenesis is used to alter the COOH-terminal portion of Sec8 with a 6-histidine tag, a 9E10 c-myc epitope, or both, to allow the isolation of the Sec8/15 complex from yeast lysates either by immobilized metal affinity chromatography or by immunoprecipitation. Sec6 cofractionates with Sec8/15 by immobilized metal affinity chromatography, gel filtration chromatography, and by sucrose velocity centrifugation. Sec6 and Sec15 coimmunoprecipitate from lysates with c-myc-tagged Sec8. These data indicate that the Sec8/15 complex contains Sec6 as a stable component. Additional proteins associated with Sec6/8/15 were identified by immunoprecipitations from radiolabeled lysates. The entire Sec6/8/15 complex contains at least eight polypeptides which range in molecular mass from 70 to 144 kD. Yeast strains containing temperature sensitive mutations in the SEC genes were also transformed with the SEC8-c-myc-6-histidine construct and analyzed by immunoprecipitation. The composition of the Sec6/8/15 complex is disrupted specifically in the sec3-2, sec5-24, and sec10-2 strain backgrounds. The c-myc-Sec8 protein is localized by immunofluorescence to small bud tips indicating that the Sec6/8/15 complex may function at sites of exocytosis.
308 citations
TL;DR: A second methionine aminopeptidase (Met-AP2) is described in Saccharomyces cerevisiae, encoded by MAP2, which was cloned as a suppressor of the slow-growth phenotype of the map1 null strain and was purified by immunoaffinity chromatography and shown to contain Met-AP activity.
Abstract: We previously characterized a methionine aminopeptidase (EC 3.4.11.18; Met-AP1; also called peptidase M) in Saccharomyces cerevisiae, which differs from its prokaryotic homologues in that it (i) contains an N-terminal zinc-finger domain and (ii) does not produce lethality when disrupted, although it does slow growth dramatically; it is encoded by a gene called MAP1. Here we describe a second methionine aminopeptidase (Met-AP2) in S. cerevisiae, encoded by MAP2, which was cloned as a suppressor of the slow-growth phenotype of the map1 null strain. The DNA sequence of MAP2 encodes a protein of 421 amino acids that shows 22% identity with the sequence of yeast Met-AP1. Surprisingly, comparison with sequences in the GenBank data base showed that the product of MAP2 has even greater homology (55% identity) with rat p67, which was characterized as an initiation factor 2-associated protein but not yet shown to have Met-AP activity. Transformants of map1 null cells expressing MAP2 in a high-copy-number plasmid contained 3- to 12-fold increases in Met-AP activity on different peptide substrates. The epitope-tagged suppressor gene product was purified by immunoaffinity chromatography and shown to contain Met-AP activity. To evaluate the physiological significance of Met-AP2, the MAP2 gene was deleted from wild-type and map1 null yeast strains. The map2 null strain, like the map1 null strain, is viable but with a slower growth rate. The map1, map2 double-null strains are nonviable. Thus, removal of N-terminal methionine is an essential function in yeast, as in prokaryotes, but yeast require two methionine aminopeptidases to provide the essential function which can only be partially provided by Met-AP1 or Met-AP2 alone.
276 citations
TL;DR: The data suggest that a plant metal transporter, which is most likely involved in the transport of copper, has been identified and is associated with an increased sensitivity to copper toxicity.
271 citations
TL;DR: The isolation and characterization of another gene in the yeast Saccharomyces cerevisiae that plays a critical role in detoxification of reactive oxygen species is described, named ATX1, which protects cells against the toxicity of both superoxide anion and hydrogen peroxide.
Abstract: In aerobic organisms, protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes, such as superoxide dismutase (SOD) and catalase. Here we describe the isolation and characterization of another gene in the yeast Saccharomyces cerevisiae that plays a critical role in detoxification of reactive oxygen species. This gene, named ATX1, was originally isolated by its ability to suppress oxygen toxicity in yeast lacking SOD. ATX1 encodes a 8.2-kDa polypeptide exhibiting significant similarity and identity to various bacterial metal transporters. Potential ATX1 homologues were also identified in multicellular eukaryotes, including the plants Arabidopsis thaliana and Oryza sativa and the nematode Caenorhabditis elegans. In yeast cells, ATX1 evidently acts in the transport and/or partitioning of copper, and this role in copper homeostasis appears to be directly relevant to the ATX1 suppression of oxygen toxicity: ATX1 was incapable of compensating for SOD when cells were depleted of exogenous copper. Strains containing a deletion in the chromosomal ATX1 locus were generated. Loss of ATX1 function rendered both mutant and wild-type SOD strains hypersensitive toward paraquat (a generator of superoxide anion) and was also associated with an increased sensitivity toward hydrogen peroxide. Hence, ATX1 protects cells against the toxicity of both superoxide anion and hydrogen peroxide.
267 citations
TL;DR: It is proposed that fab1p is a PI(4)P 5-kinase and that the product of the Fab1p reaction, PIP2, functions as an important regulator of vacuole homeostasis perhaps by controlling membrane flux to and/or from the vacuoles.
Abstract: The FAB1 gene of budding yeast is predicted to encode a protein of 257 kDa that exhibits significant sequence homology to a human type II PI(4)P 5-kinase (PIP5K-II). The recently cloned human PIP5K...
TL;DR: Two interacting heat shock cognate proteins in the lumen of the yeast endoplasmic reticulum, Sec63p and Kar2p, are required for posttranslational translocation of yeast alpha-factor precursor in vitro and the import of invertase into wild-type, sec63, and kar2 mutant yeast membranes is examined.
Abstract: Two interacting heat shock cognate proteins in the lumen of the yeast endoplasmic reticulum (ER), Sec63p and BiP (Kar2p), are required for posttranslational translocation of yeast alpha-factor precursor in vitro. To investigate the role of these proteins in cotranslational translocation, we examined the import of invertase into wild-type, sec63, and kar2 mutant yeast membranes. We found that Sec63p and Kar2p are necessary for both co- and posttranslational translocation in yeast. Several kar2 mutants, one of which had normal ATPase activity, were defective in cotranslational translocation of invertase. We conclude that the requirement for BiP/Kar2p, which is not seen in a reaction reconstituted with pure mammalian membrane proteins [Gorlich, D. & Rapoport, T.A. (1993) Cell 75, 615-630], is not due to a distinction between cotranslational translocation in mammalian cells and posttranslational translocation in yeast cells.
TL;DR: The rate of spontaneous reversion was shown to increase when the mutant gene was highly transcribed and transcriptionally active DNA and enhanced spontaneous mutation rates are associated in yeast.
Abstract: Complex processes such as transcription, replication, repair, and recombination require changes in chromatin structure and the interactions of numerous trans-acting factors with DNA sequences, raising the possibility that these processes may be interrelated. Here the effect of transcription on the rate of spontaneous mutation in the yeast Saccharomyces cerevisiae was examined. With the use of a lys2 frameshift allele under the control of a highly inducible promoter, the rate of spontaneous reversion was shown to increase when the mutant gene was highly transcribed. Thus, transcriptionally active DNA and enhanced spontaneous mutation rates are associated in yeast.
TL;DR: It is shown that in animal cells the “independent” activity of AF-1 is embodied in a rather hydrophobic proline-rich 99-amino acid activating domain (amino acids 51-149), whereas in yeast, three discrete activating domains are almost as active on their own as the whole A/B region, indicating that multiple activating domains can operate independently in yeast.
TL;DR: The results suggest that Rev directly promotes the cytoplasmic transport of suitable transcripts by targeting them to the nuclear pore.
TL;DR: This work has noted an additional conserved motif (UUXGHXH, where U specifies a hydrophobic residue) located 84-153 amino acids from the GNHD/E sequence, and in preparation for this paper discovered that this fourth motif had also been independently identified.
Abstract: The destruction of long palindromic DNA sequences in Escherichia coli is mediated by the products of sbcC and sbcD (Leach, 1994, BioEssays 16: 893-900). Mutations in these genes also function as co-suppressors with sbcB of recombination and repair defective recB or recC strains. The sbcB gene product is a 3' ~ 5' single-stranded DNA exonuclease. SbcC and SbcD proteins have ATPdependent double-stranded DNA exonuclease and ATPindependent single-stranded DNA endonuclease activities (J. C. Connelly and D. R. F. Leach, unpublished). Inactivation of these nucleases is proposed to achieve suppression of recBC by stabilizing DNA ends, allowing other recombination enzymes access in order to initiate recombination and DNA repair. Previous studies have shown structural similarities between E. coil SbcD and proteins from bacteriophages T4 (gp47), T5 (gpD12) and Bacillus subti/is (ORF3) (Leach et al., 1992, Genetica 87: 95-100; Sharpies and Lloyd, 1993, Nuc/Acids Res 21:2010). Bacteriophages T4 and T5 also have homologous equivalents of SbcC (T4 gp46 and T5 gpD13). In keeping with their relatedness to E. coli SbcCD, they are responsible for the exonuclease activities which normally degrade chromosomal DNA and are also important for phage replication, recombination and DNA repair. Recently it has been noted that a large number of diverse proteins that cleave phosphoester bonds share a common sequence structure. These enzymes include Ser/Thr protein phosphatases, diadenosine tetraphosphatases, 5' nucleotidases, 2',3'-cyclic phosphodiesterases, sphingomyelinases, an RNA lariat 2 ' -5 ' phosphodiesterase, and exonucleases; SbcD, gp47 and gpD12 belong to the latter group. The phosphoesterase signature sequence is DXH(~X25)GDXXD(~X25)GNHD/E (Zhuo et al., 1994, J Biol Chem 269: 26234-26238). We have noted an additional conserved motif (UUXGHXH, where U specifies a hydrophobic residue) located 84-153 amino acids from the GNHD/E sequence (Fig. 1A and data not shown). While this paper was in preparation we discovered that this fourth motif had also been independently identified (Yeast Update 3.6). Our particular interest focused on members of the phosphoesterase
TL;DR: Sequence comparison shows that this beta-1,3-glucanase, first described for filamentous fungi, belongs to a family different from that of its previously described bacterial, yeast, and plant counterparts.
Abstract: The mycoparasitic fungus Trichoderma harzianum CECT 2413 produces at least three extracellular beta-1,3-glucanases. The most basic of these extracellular enzymes, named BGN13.1, was expressed when either fungal cell wall polymers or autoclaved mycelia from different fungi were used as the carbon source. BGN13.1 was purified to electrophoretic homogeneity and was biochemically characterized. The enzyme was specific for beta-1,3 linkages and has an endolytic mode of action. A synthetic oligonucleotide primer based on the sequence of an internal peptide was designed to clone the cDNA corresponding to BGN13.1. The deduced amino acid sequence predicted a molecular mass of 78 kDa for the mature protein. Analysis of the amino acid sequence indicates that the enzyme contains three regions, one N-terminal leader sequence; another, nondefined sequence; and one cysteine-rich C-terminal sequence. Sequence comparison shows that this beta-1,3-glucanase, first described for filamentous fungi, belongs to a family different from that of its previously described bacterial, yeast, and plant counterparts. Enzymatic-activity, protein, and mRNA data indicated that bgn13.1 is repressed by glucose and induced by either fungal cell wall polymers or autoclaved yeast cells and mycelia. Finally, experimental evidence showed that the enzyme hydrolyzes yeast and fungal cell walls.
TL;DR: It is suggested that Nup170p and Nup157p may be part of a morphologically symmetrical but functionally distinct substructure of the yeast NPC, e.g., the nucleoplasmic and cytoplasmic rings.
Abstract: We have taken a combined genetic and biochemical approach to identify major constituents of the yeast nuclear pore complex (NPC). A synthetic lethal screen was used to identify proteins which interact genetically with the major pore-membrane protein Pom152p. In parallel, polypeptides present in similar amounts to Pom152p in a highly enriched preparation of yeast NPCs have been characterized by direct microsequencing. These approaches have led to the identification of two novel and major nucleoporins, Nup170p and Nup157p. Both Nup170p and Nup157p are similar to each other and to an abundant mammalian nucleoporin, Nup155p (Radu, A., G. Blobel, and R. W. Wozniak. 1993. J. Cell Biol. 121: 1-9) and interestingly, nup170 mutants can be complemented with mammalian NUP155. In addition, the synthetic lethal screen identified genetic interactions between Pom152p and two other major nucleoporins, Nup188p (Nehrbass, U., S. Maguire, M. Rout, G. Blobel, and R. W. Wozniak, manuscript submitted for publication), and Nic96p (Grandi, P., V. Doye, and E. C. Hurt. 1993. EMBO J. 12: 3061-71). We have determined that together, Nup170p, Nup157p, Pom152p, Nup188p, and Nic96p comprise greater than one-fifth of the mass of the isolated yeast NPC. Examination of the genetic interactions between these proteins indicate that while deletion of either POM152, NUP170, or NUP188 alone is not lethal, pairwise combinations are. Deletion of NUP157 is also not lethal. However, nup157 null mutants, while lethal in combination with nup170 and nup188 null alleles, are not synthetically lethal with pom152 null alleles. We suggest that Nup170p and Nup157p may be part of a morphologically symmetrical but functionally distinct substructure of the yeast NPC, e.g., the nucleoplasmic and cytoplasmic rings. Finally, we observed morphological abnormalities in the nuclear envelope as a function of alterations in the expression levels of NUP170 suggesting a specific stoichiometric relationship between NPC components is required for the maintenance of normal nuclear structure.
TL;DR: Yeast is particularly amenable for analyzing transcriptional regulatory mechanisms in vivo under true physiological conditions, and classical and molecular yeast genetics has permitted the discovery and functional characterization of transcriptional Regulatory proteins that were not identified in biochemical studies.
Abstract: Transcriptional regulation directly influences many biological phenomena such as cell growth, response to environmental change, development of multicellular organisms, and disease. Transcriptional regulatory mechanisms are fundamentally similar in eukaryotic organisms (93). Components of the basic RNA polymerase II (Pol II) machinery are highly conserved and, in some cases, functionally interchangeable. Transcription factors with similar structures and DNA-binding specificities are found throughout the eukaryotic kingdom, and acidic activation domains stimulate transcription across a wide range of species. Complex promoters with multiple protein binding sites are typical in all eukaryotic organisms, and efficient transcription generally requires the combinatorial and synergistic action of activator proteins that function at long and variable distances from the mRNA initiation site. Molecular mechanisms of eukaryotic transcriptional regulation have been elucidated from the studies that involve a wide variety of genes, promoters, proteins, organisms, and experimental approaches. This review focuses on transcriptional regulatory mechanisms in the baker's yeast Saccharomyces cerevisiae. Studies in yeast have emphasized powerful genetic approaches that are not available in other eukaryotic organisms. As a consequence, yeast is particularly amenable for analyzing transcriptional regulatory mechanisms in vivo under true physiological conditions. Furthermore, classical and molecular yeast genetics has permitted the discovery and functional characterization of transcriptional regulatory proteins that were not identified in biochemical studies. Thus, genetic analysis in yeast has often generated information complementary to that obtained from biochemical studies of transcription in vitro, and it has provided unique insights into mechanisms of eukaryotic transcriptional regulation.
TL;DR: It is demonstrated that both phenomena can be dissociated clearly once the cells initiate fermentation and that the initiation of fermentation causes the disappearance of any other factor required for the maintenance of stress resistance, even in the presence of a high trehalose content.
Abstract: The trehalose content in laboratory and industrial baker's yeast is widely believed to be a major determinant of stress resistance. Fresh and dried baker's yeast is cultured to obtain a trehalose content of more than 10% of the dry weight. Initiation of fermentation, e.g., during dough preparation, is associated with a rapid loss of stress resistance and a rapid mobilization of trehalose. Using specific Saccharomyces cerevisiae mutants affected in trehalose metabolism, we confirm the correlation between trehalose content and stress resistance but only in the absence of fermentation. We demonstrate that both phenomena can be dissociated clearly once the cells initiate fermentation. This was accomplished both for cells with moderate trehalose levels grown under laboratory conditions and for cells with trehalose contents higher than 10% obtained under pilot-scale conditions. Retention of a high trehalose level during fermentation also does not prevent the loss of fermentation capacity during preparation of frozen doughs. Although higher trehalose levels are always correlated with higher stress resistance before the addition of fermentable sugar, our results show that the initiation of fermentation causes the disappearance of any other factor(s) required for the maintenance of stress resistance, even in the presence of a high trehalose content.
TL;DR: Studies of cell-type determination in the yeast Saccharomyces cerevisiae have revealed a regulatory network of proteins that are highly conserved in evolutionary terms.
TL;DR: The genetic interactions suggest that budding-yeast 14-3-3 proteins are multifunctional and may play a role in both vesicular transport and Ras signaling pathways.
Abstract: Deletion of the clathrin heavy-chain gene, CHC1, in the budding yeast Saccharomyces cerevisiae results in growth, morphological, and membrane trafficking defects, and in some strains chc1-delta is lethal. A previous study identified five genes which, in multicopy, rescue inviable strains of Chc- yeast. Now we report that one of the suppressor loci, BMH2/SCD3, encodes a protein of the 14-3-3 family. The 14-3-3 proteins are abundant acidic proteins of approximately 30 kDa with numerous isoforms and a diverse array of reported functions. The Bmh2 protein is > 70% identical to the mammalian epsilon-isoform and > 90% identical to a previously reported yeast 14-3-3 protein encoded by BMH1. Single deletions of BMH1 or BMH2 have no discernable phenotypes, but deletion of both BMH1 and BMH2 is lethal. High-copy BMH1 also rescues inviable strains of Chc- yeast, although not as well as BMH2. In addition, the slow growth of viable strains of Chc- yeast is further impaired when combined with single bmh mutations, often resulting in lethality. Overexpression of BMH genes also partially suppresses the temperature sensitivity of the cdc25-1 mutant, and high-copy TPK1, encoding a cAMP-dependent protein kinase, restores Bmh- yeast to viability. High-copy TPK1 did not rescue Chc- yeast. These genetic interactions suggest that budding-yeast 14-3-3 proteins are multifunctional and may play a role in both vesicular transport and Ras signaling pathways.
TL;DR: An essential role of the 14-3-3 proteins is suggested, possibly at a later stage of the yeast cell cycle, possibly in the presence of calcium/calmodulin-dependent protein kinase II and stimulation of exocytosis.
Abstract: The 14-3-3 proteins comprise a family of highly conserved acidic proteins. Several activities have been ascribed to these proteins, including activation of tyrosine and tryptophan hydroxylases in the presence of calcium/calmodulin-dependent protein kinase II, regulation of protein kinase C, phospholipase A2 activity, stimulation of exocytosis and activation of bacterial exoenzyme S (ExoS) during ADP-ribosy-lation of host proteins. In addition, a plant 14-3-3 protein is present in a G-box DNA/protein-binding complex. Previously, we isolated the BMH1 gene from Saccharomyces cerevisiae encoding a putative 14-3-3 protein. Using the polymerase chain reaction method, we have isolated a second yeast gene encoding a 14-3-3 protein (BMH2). While disruption of either BMH1 or BMH2 alone had little effect, it was impossible to obtain viable cells with both genes disrupted. The cDNA encoding a plant 14-3-3 protein under the control of the inducible GAL1 promoter complemented the double disruption. Transfer of the complemented double disruptant to a medium with glucose resulted in the appearance of a high percentage of large budded cells. After prolonged incubation, these cells became enlarged with irregular buds and chains of cells defective in cell-cell separation became visible. These results suggest an essential role of the 14-3-3 proteins, possibly at a later stage of the yeast cell cycle.
TL;DR: Viability was shown to be dependent on temperature and dissolved CO2 concentration, and can be described by a sigmoidal (S-shaped) curve, and cell death was mainly due to an 'anaesthesia effect' rather than cell rupture.
TL;DR: The identification of the yeast CTP gene, and the expression and purification of large quantities of its protein product, pave the way for investigations into the roles of specific amino acids in the CTP translocation mechanism, as well as for the initiation of crystallization trials.
TL;DR: The results suggest that hsk1+ is a strong candidate for the functional fission yeast homologue of budding yeast CDC7 and that a mechanism through which initiation of chromosomal replication is regulated may be conserved between the two yeast species.
Abstract: Degenerate oligonucleotide-directed polymerase chain reaction was conducted to clone a possible Schizosaccharomyces pombe homologue [hsk1 for a putative homologue of CDC7 (seven) kinase 1] of Saccharomyces cerevisiae Cdc7 kinase. The cloned cDNA for hsk1+ contains an open reading frame consisting of 507 amino acids with predicted mol. wt of 58,370 that possesses overall amino acid identity of 46% (65% including similar residues) to CDC7. In addition to conserved domains for serine-threonine kinases, the predicted primary structure of Hsk1 contains three 'kinase insert' sequences characteristic to Cdc7 at the positions identical to those of Cdc7. Whereas the length and sequences of the kinase inserts are diverged between the two yeast species, 58% identity (76% including similar residues) is detected within the kinase conserved domains. The hsk1+ gene, which is present as a single copy on the S.pombe chromosome, contains two introns within the coding frame. Disruption of the hsk1+ gene by insertion of the ura4+ gene is lethal to growth. Analysis of the DNA content of germinating spores that contain hsk1 null alleles indicates that DNA replication is inhibited in the mutant. The morphology of these mutant spores after germination indicates abnormal nuclear division in some population of germinating spores, suggesting either that Hsk1 may be required for inhibition of mitosis until completion of S phase or that it may also be involved in proper execution of mitosis. Our results suggest that hsk1+ is a strong candidate for the functional fission yeast homologue of budding yeast CDC7 and that a mechanism through which initiation of chromosomal replication is regulated may be conserved between the two yeast species.
TL;DR: A mutation in the SST2 gene, which confers supersensitivity to pheromone, was found to significantly enhance the growth response to S-14, and these yeast strains should serve as a useful in vivo reconstitution system for examination of molecular interactions of the G-protein-coupled receptors and G proteins.
Abstract: A detailed analysis of structural and functional aspects of G-protein-coupled receptors, as well as discovery of novel pharmacophores that exert their effects on members of this class of receptors, will be facilitated by development of a yeast-based bioassay. To that end, yeast strains that functionally express the rat somatostatin receptor subtype 2 (SSTR2) were constructed. High-affinity binding sites for somatostatin ([125I-Tyr-11]S-14) comparable to those in native tissues were detected in yeast membrane extracts at levels equivalent to the alpha-mating pheromone receptor (Ste2p). Somatostatin-dependent growth of strains modified by deletion of genes encoding components of the pheromone response pathway was detected through induction of a pheromone-responsive HIS3 reporter gene, enabling cells to grow on medium lacking histidine. Dose-dependent growth responses to S-14 and related SSTR2 subtype-selective agonists that were proportional to the affinity of the ligands for SSTR2 were observed. The growth response required SSTR2, G alpha proteins, and an intact signal transduction pathway. The sensitivity of the bioassay was affected by intracellular levels of the G alpha protein. A mutation in the SST2 gene, which confers supersensitivity to pheromone, was found to significantly enhance the growth response to S-14. In sst2 delta cells, SSTR2 functionally interacted with both a chimeric yeast/mammalian G alpha protein and the yeast G alpha protein, Gpa1p; to promote growth. These yeast strains should serve as a useful in vivo reconstitution system for examination of molecular interactions of the G-protein-coupled receptors and G proteins.
TL;DR: Subjects with a healthy metabolism do not need to be advised against cultivating Kombucha, however, those suffering from immunosuppression should preferably consume controlled commercial Kombsucha beverages.
Abstract: The tea fungus 'Kombucha' is a symbiosis of Acetobacter, including Acetobacter xylinum as a characteristic species, and various yeasts. A characteristic yeast species or genus has not yet been identified. Kombucha is mainly cultivated in sugared black tea to produce a slightly acidulous effervescent beverage that is said to have several curative effects. In addition to sugar, the beverage contains small amounts of alcohol and various acids, including acetic acid, gluconic acid and lactic acid, as well as some antibiotic substances. To characterize the yeast spectrum with special consideration given to facultatively pathogenic yeasts, two commercially available specimens of tea fungus and 32 from private households in Germany were analysed by micromorphological and biochemical methods. Yeasts of the genera Brettanomyces, Zygosaccharomyces and Saccharomyces were identified in 56%, 29% and 26% respectively. The species Saccharomycodes ludwigii and Candida kefyr were only demonstrated in isolated cases. Furthermore, the tests revealed pellicle-forming yeasts such as Candida krusei or Issatchenkia orientalis/occidentalis as well as species of the apiculatus yeasts (Kloeckera, Hanseniaspora). Thus, the genus Brettanomyces may be a typical group of yeasts that are especially adapted to the environment of the tea fungus. However, to investigate further the beneficial effects of tea fungus, a spectrum of the other typical genera must be defined. Only three specimens showed definite contaminations. In one case, no yeasts could be isolated because of massive contamination with Penicillium spp. In the remaining two samples (from one household), Candida albicans was demonstrated. The low rate of contamination might be explained by protective mechanisms, such as formation of organic acids and antibiotic substances. Thus, subjects with a healthy metabolism do not need to be advised against cultivating Kombucha. However, those suffering from immunosuppression should preferably consume controlled commercial Kombucha beverages.
TL;DR: Results indicate that presumptive killer yeast strains, together with their killer toxins, may have potential as novel antimycotic biocontrol agents.
Abstract: A total of 17 presumptive killer yeast strains were tested in vitro for growth inhibitory and killing activity against a range of fungal pathogens of agronomic, environmental and clinical significance. Several yeasts were identified which displayed significant activity against important pathogenic fungi. For example, isolates of the opportunistic human pathogen, Candida albicans, were generally very sensitive to Williopsis mrakii killer yeast activity, whilst killer strains of Saccharomyces cerevisiae and Pichia anomala markedly inhibited the growth of certain wood decay basidiomycetes and plant pathogenic fungi. Results indicate that such yeasts, together with their killer toxins, may have potential as novel antimycotic biocontrol agents.
TL;DR: Results suggest that RbAp48 and Rb Ap46 may have shared as well as unique functions in the regulation of cell proliferation and differentiation.