| 1. |
Kurtzman CP,
Robnett CJ,
( 2003 ) Phylogenetic relationships among yeasts of the 'Saccharomyces complex' determined from multigene sequence analyses. PMID : 12748053 : DOI : 10.1016/S1567-1356(03)00012-6 Abstract >>
Species of Saccharomyces, Arxiozyma, Eremothecium, Hanseniaspora (anamorph Kloeckera), Kazachstania, Kluyveromyces, Pachytichospora, Saccharomycodes, Tetrapisispora, Torulaspora, and Zygosaccharomyces, as well as three related anamorphic species assigned to Candida (C. castellii, C. glabrata, C. humilis), were phylogenetically analyzed from divergence in genes of the rDNA repeat (18S, 26S, ITS), single copy nuclear genes (translation elongation factor 1alpha, actin-1, RNA polymerase II) and mitochondrially encoded genes (small-subunit rDNA, cytochrome oxidase II). Single-gene phylogenies were congruent for well-supported terminal lineages but deeper branches were not well resolved. Analysis of combined gene sequences resolved the 75 species compared into 14 clades, many of which differ from currently circumscribed genera.
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2. |
Hansen J,
Bruun SV,
Bech LM,
Gjermansen C,
( 2002 ) The level of MXR1 gene expression in brewing yeast during beer fermentation is a major determinant for the concentration of dimethyl sulfide in beer. PMID : 12702301 : DOI : 10.1111/j.1567-1364.2002.tb00078.x Abstract >>
DMS (dimethyl sulfide) is an important beer flavor compound which is derived either from the beer wort production process or via the brewing yeast metabolism. We investigated the contribution of yeast MXR1 gene activity to the final beer DMS content. The MXR1-CA gene from Saccharomyces carlsbergensis (synonym of Saccharomyces pastorianus) lager brewing yeast was isolated and sequenced, and found to be 88% identical with Saccharomyces cerevisiae MXR1. Inactive deletion alleles of both genes were substituted for their functional counterparts in S. carlsbergensis. Such yeasts fermented well and did not form DMS from dimethyl sulfoxide. Overexpression in brewing yeast of MXR1 from non-native promoters with various strengths and transcription profiles resulted in an enhanced and correlated DMS production. The promoters of MXR1 and MXR1-CA contain conserved Met31p/Met32p binding sites, and in accordance with this were found to be co-regulated with the genes of the sulfur assimilation pathway. In addition, conserved YRE-like DNA sequences are present in these promoters, indicating that Yap1p may also take part in the control of these genes.
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3. |
Okuda Y,
Sasaki D,
Nogami S,
Kaneko Y,
Ohya Y,
Anraku Y,
( 2003 ) Occurrence, horizontal transfer and degeneration of VDE intein family in Saccharomycete yeasts. PMID : 12734795 : DOI : 10.1002/yea.984 Abstract >>
VDE is a homing endonuclease gene originally discovered as an intervening element in VMA1s of Saccharomyces cerevisiae. There have been two independent subfamilies of VDE, one from S. cerevisiae strain X2180-1A and the other from Saccharomyces sp. DH1-1A in the host VMA1 gene, and they share the identity of 96.3%. In order to search the occurrence, intra/interspecies transfer and molecular degeneration of VDE, complete sequences of VMA1 in 10 strains of S. cerevisiae, eight species of saccharomycete yeasts, Candida glabrata and Kluyveromyces lactis were determined. We found that six of 10 S. cerevisiae strains contain VDEs 99.7-100% identical to that of the strain X2180-1A, one has no VDE, whereas the other three harbour VDEs 100% identical to that of the strain DH1-1A. S. carlsbergensis has two VMA1s, one being 99.8% identical to that of the strain X2180-1A with VDE 100% identical to that of the strain DH1-1A and the other containing the same VMA1 in S. pastorianus with no VDE. This and other evidence indicates that intra/interspecies transmissions of VDEs have occurred among saccharomycete yeasts. Phylogenetic analyses of VMA1 and VDE suggest that the S. cerevisiae VDEs had branched earlier than other VDEs from an ancestral VDE and had invaded into the host loci as relatively late events. The two VDEs seemed to degenerate in individual host loci, retaining their splicing capacity intact. The degeneration of the endonuclease domains was distinct and, if compared, its apparent rate was much faster than that of the protein-splicing domains.
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4. |
Verstrepen KJ,
Van Laere SD,
Vanderhaegen BM,
Derdelinckx G,
Dufour JP,
Pretorius IS,
Winderickx J,
Thevelein JM,
Delvaux FR,
( 2003 ) Expression levels of the yeast alcohol acetyltransferase genes ATF1, Lg-ATF1, and ATF2 control the formation of a broad range of volatile esters. PMID : 12957907 : DOI : 10.1128/aem.69.9.5228-5237.2003 PMC : PMC194970 Abstract >>
Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction. In order to investigate and compare the roles of the known Saccharomyces cerevisiae alcohol acetyltransferases, Atf1p, Atf2p and Lg-Atf1p, in volatile ester production, the respective genes were either deleted or overexpressed in a laboratory strain and a commercial brewing strain. Subsequently, the ester formation of the transformants was monitored by headspace gas chromatography and gas chromatography combined with mass spectroscopy (GC-MS). Analysis of the fermentation products confirmed that the expression levels of ATF1 and ATF2 greatly affect the production of ethyl acetate and isoamyl acetate. GC-MS analysis revealed that Atf1p and Atf2p are also responsible for the formation of a broad range of less volatile esters, such as propyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate, heptyl acetate, octyl acetate, and phenyl ethyl acetate. With respect to the esters analyzed in this study, Atf2p seemed to play only a minor role compared to Atf1p. The atf1Delta atf2Delta double deletion strain did not form any isoamyl acetate, showing that together, Atf1p and Atf2p are responsible for the total cellular isoamyl alcohol acetyltransferase activity. However, the double deletion strain still produced considerable amounts of certain other esters, such as ethyl acetate (50% of the wild-type strain), propyl acetate (50%), and isobutyl acetate (40%), which provides evidence for the existence of additional, as-yet-unknown ester synthases in the yeast proteome. Interestingly, overexpression of different alleles of ATF1 and ATF2 led to different ester production rates, indicating that differences in the aroma profiles of yeast strains may be partially due to mutations in their ATF genes.
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5. |
Johannesen PF,
Hansen J,
( 2002 ) Differential transcriptional regulation of sulfur assimilation gene homologues in the Saccharomyces carlsbergensis yeast species hybrid. PMID : 12702335 : DOI : 10.1111/j.1567-1364.2002.tb00050.x Abstract >>
The allopolyploid yeast Saccharomyces carlsbergensis appears to be a relatively newly formed species hybrid, and therefore constitutes a good model for studying early steps in hybrid speciation. Using reverse transcription-coupled polymerase chain reaction to monitor derepression of the S. carlsbergensis homologues of the sulfur assimilation genes MET14 and MET2, we found that both homologues of these genes are regulated in the same pathway-specific manner, but surprisingly, with different kinetics, as the genes derived from one of the parent species (the non-Saccharomyces cerevisiae-like) are alleviated from repression much faster than the genes from the other parent (the S. cerevisiae-like). This probably reflects differing physiological adaptation of the parent species, and the finding may contribute to the general understanding of hybrid speciation.
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6. |
Daniel HM,
Meyer W,
( 2003 ) Evaluation of ribosomal RNA and actin gene sequences for the identification of ascomycetous yeasts. PMID : 12892922 : Abstract >>
Highly similar gene sequences of the 5' region of the large subunit (LSU) are commonly interpreted to predict the organism's identity. However, it was recognised that closely related taxa do not always show sufficiently diverged D1/D2 LSU sequences to differentiate between them. The effectiveness of species separation using D1/D2 LSU sequences, small subunit (SSU) sequences and actin gene sequences was determined by pair-wise comparisons. The LSU data showed coinciding similarities among and within species. The actin data resolved all investigated species. Examples strengthened the value of almost complete SSU sequences for species separation. The larger number of differences in the highly conserved actin gene, compared to the overall more variable LSU gene, is due to the tolerance of protein coding genes to synonymous nucleotide changes. In contrast, the pairing in secondary structures of the rRNA, ensuring the functionality of the molecule, relies on longer and uninterrupted sequence sections. In conclusion, D1/D2 LSU sequences are not specific enough to identify closely related taxa. The actin gene is a better marker in these cases. However, because of the availability of a large database of fungal D1/D2 LSU sequences, this gene region is currently still the preferred target for sequence-based identification.
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7. |
Maxfield Boumil R,
Kemp B,
Angelichio M,
Nilsson-Tillgren T,
Dawson DS,
( 2003 ) Meiotic segregation of a homeologous chromosome pair. PMID : 12655401 : DOI : 10.1007/s00438-002-0796-9 Abstract >>
During meiosis, the alignment of homologous chromosomes facilitates their subsequent migration away from one another to opposite spindle poles at anaphase I. Recombination is part of the mechanism by which chromosomes identify their homologous partners, and serves to link the homologs in a way that, in some organisms, has been shown to promote proper attachment to the meiotic spindle. We have built a diploid strain that contains a pair of homeologous chromosomes V': one is derived from Saccharomyces cerevisiae and one originates from S. carlsbergensis. Sequence analysis reveals that these chromosomes share 71% sequence identity. The homeologs experience high levels of meiotic double-stranded breaks. Despite their relatedness and their competence to initiate recombination, the meiotic segregation behavior of the homeologous chromosomes suggests that, in most meioses, they are partitioned by a meiotic segregation system that has been shown previously to partition non-exchange chromosomes and pairs with no homology. Though the homeologous chromosomes show a degree of meiotic segregation fidelity similar to that of other non-exchange pairs, our data provide evidence that their limited sequence homology may provide some bias in meiotic partner choice.
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8. |
Evans SK,
Lundblad V,
( 2002 ) The Est1 subunit of Saccharomyces cerevisiae telomerase makes multiple contributions to telomere length maintenance. PMID : 12454059 : PMC : PMC1462332 Abstract >>
The telomerase-associated Est1 protein of Saccharomyces cerevisiae mediates enzyme access by bridging the interaction between the catalytic core of telomerase and the telomere-binding protein Cdc13. In addition to recruiting telomerase, Est1 may act as a positive regulator of telomerase once the enzyme has been brought to the telomere, as previously suggested by the inability of a Cdc13-Est2 fusion protein to promote extensive telomere elongation in an est1-Delta strain. We report here three classes of mutant Est1 proteins that retain association with the telomerase enzyme but confer different in vivo consequences. Class 1 mutants display a telomere replication defect but are capable of promoting extensive telomere elongation in the presence of a Cdc13-Est2 fusion protein, consistent with a defect in telomerase recruitment. Class 2 mutants fail to elongate telomeres even in the presence of the Cdc13-Est2 fusion, which is the phenotype predicted for a defect in the proposed second regulatory function of EST1. A third class of mutants impairs an activity of Est1 that is potentially required for the Ku-mediated pathway of telomere length maintenance. The isolation of mutations that perturb separate functions of Est1 demonstrates that a telomerase holoenzyme subunit can contribute multiple regulatory roles to telomere length maintenance.
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9. |
Brown BJ,
Hyun JW,
Duvvuri S,
Karplus PA,
Massey V,
( 2002 ) The role of glutamine 114 in old yellow enzyme. PMID : 11668181 : DOI : 10.1074/jbc.M108453200 Abstract >>
Glutamine 114 of OYE1 is a well conserved residue in the active site of the Old Yellow Enzyme family. It forms hydrogen bonds to the O2 and N3 of the flavoprotein prosthetic group, FMN. Glutamine 114 was mutated to asparagine, introducing an R-group that is one methylene group shorter. The resultant enzyme was characterized to determine the effect of the mutation on the mechanistic behavior of the enzyme, and the crystal structure was solved to determine the effect of the mutation on the structure of the protein. The Q114N mutation results in little change in the protein structure, moving the amide group of residue 114 out of H-bonding distance, allowing repositioning of the FMN prosthetic group to form new interactions that replace the lost H-bonds. The mutation decreases the ability to bind ligands, as all dissociation constants for substituted phenols are larger than for the wild type enzyme. The rate constant for the reductive half-reaction with beta-NADPH is slightly greater, whereas that for the oxidative half-reaction with 2-cyclohexenone is smaller than for the wild type enzyme. Oxidation with molecular oxygen is biphasic and involves formation and reaction with O(2), a phenomenon that is more pronounced with this mutation than with wild type enzyme. When superoxide dismutase is added to the reaction, we observe a single-phase reaction typical of the wild type enzyme. Turnover reactions using beta-NADPH with 2-cyclohexenone and molecular oxygen were studied to further characterize the mutant enzyme.
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10. |
Kodama Y,
Omura F,
Ashikari T,
( 2001 ) Isolation and characterization of a gene specific to lager brewing yeast that encodes a branched-chain amino acid permease. PMID : 11472919 : DOI : 10.1128/AEM.67.8.3455-3462.2001 PMC : PMC93043 Abstract >>
We found two types of branched-chain amino acid permease gene (BAP2) in the lager brewing yeast Saccharomyces pastorianus BH-225 and cloned one type of BAP2 gene (Lg-BAP2), which is identical to that of Saccharomyces bayanus (by-BAP2-1). The other BAP2 gene of the lager brewing yeast (cer-BAP2) is very similar to the Saccharomyces cerevisiae BAP2 gene. This result substantiates the notion that lager brewing yeast is a hybrid of S. cerevisiae and S. bayanus. The amino acid sequence homology between S. cerevisiae Bap2p and Lg-Bap2p was 88%. The transcription of Lg-BAP2 was not induced by the addition of leucine to the growth medium, while that of cer-BAP2 was induced. The transcription of Lg-BAP2 was repressed by the presence of ethanol and weak organic acid, while that of cer-BAP2 was not affected by these compounds. Furthermore, Northern analysis during beer fermentation revealed that the transcription of Lg-BAP2 was repressed at the beginning of the fermentation, while cer-BAP2 was highly expressed throughout the fermentation. These results suggest that the transcription of Lg-BAP2 is regulated differently from that of cer-BAP2 in lager brewing yeasts.
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11. |
Casaregola S,
Nguyen HV,
Lapathitis G,
Kotyk A,
Gaillardin C,
( 2001 ) Analysis of the constitution of the beer yeast genome by PCR, sequencing and subtelomeric sequence hybridization. PMID : 11491364 : DOI : 10.1099/00207713-51-4-1607 Abstract >>
The lager brewing yeasts, Saccharomyces pastorianus (synonym Saccharomyces carlsbergensis), are allopolyploid, containing parts of two divergent genomes. Saccharomyces cerevisiae contributed to the formation of these hybrids, although the identity of the other species is still unclear. The presence of alleles specific to S. cerevisiae and S. pastorianus was tested for by PCR/RFLP in brewing yeasts of various origins and in members of the Saccharomyces sensu stricto complex. S. cerevisiae-type alleles of two genes, HIS4 and YCL008c, were identified in another brewing yeast, S. pastorianus CBS 1503 (Saccharomyces monacensis), thought to be the source of the other contributor to the lager hybrid. This is consistent with the hybridization of S. cerevisiae subtelomeric sequences X and Y' to the electrophoretic karyotype of this strain. S. pastorianus CBS 1503 (S. monacensis) is therefore probably not an ancestor of S. pastorianus, but a related hybrid. Saccharomyces bayanus, also thought to be one of the contributors to the lager yeast hybrid, is a heterogeneous taxon containing at least two subgroups, one close to the type strain, CBS 380T, the other close to CBS 395 (Saccharomyces uvarum). The partial sequences of several genes (HIS4, MET10, URA3) were shown to be identical or very similar (over 99%) in S. pastorianus CBS 1513 (S. carlsbergensis), S. bayanus CBS 380T and its close derivatives, showing that S. pastorianus and S. bayanus have a common ancestor. A distinction between two subgroups within S. bayanus was made on the basis of sequence analysis: the subgroup represented by S. bayanus CBS 395 (S. uvarum) has 6-8% sequence divergence within the genes HIS4, MET10 and MET2 from S. bayanus CBS 380T, indicating that the two S. bayanus subgroups diverged recently. The detection of specific alleles by PCR/RFLP and hybridization with S. cerevisiae subtelomeric sequences X and Y' to electrophoretic karyotypes of brewing yeasts and related species confirmed our findings and revealed substantial heterogeneity in the genome constitution of Czech brewing yeasts used in production.
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12. |
Tamai Y,
Tanaka K,
Umemoto N,
Tomizuka K,
Kaneko Y,
( 2000 ) Diversity of the HO gene encoding an endonuclease for mating-type conversion in the bottom fermenting yeast Saccharomyces pastorianus. PMID : 11015730 : DOI : 10.1002/1097-0061(200010)16:14<1335::AID-YEA623>3.0.CO;2-P Abstract >>
Two types of HO gene were cloned, sequenced and characterized from the bottom fermenting yeast Saccharomyces pastorianus. The HO gene present on the 1500 kb chromosome was designated Sc-HO (S. cerevisiae-type HO), because the nucleotide sequence of its promoter region and the open reading frame (ORF) was almost identical to that of the S. cerevisiae laboratory strain HO gene (Lab-HO). The other HO gene, designated Lg-HO (Lager-fermenting-yeast specific HO), showed 64% and 83% homology with the promoter and ORF of the Lab-HO at the nucleotide sequence level, respectively, and was located on the 1100 kb chromosome. Analysis of the 4 kb DNA fragment amplified from S. bayanus type strain indicated that the nucleotide sequence of S. bayanus-HO is almost identical to that of the Lg-HO. The SSB1 gene located downstream of the HO gene in S. cerevisiae was also found in the 3' distal region of the Sc-HO, Lg-HO and S. bayanus HO genes. These results showed that the genetic arrangement around the HO loci both of S. pastorianus and S. bayanus is identical to S. cerevisiae. Southern analysis has revealed that Saccharomyces sensu stricto contain four types of HO genes; S. paradoxus-type HO, the Sc-HO, the Lg-HO and S. uvarum-type HO genes. This HO gene diversity provides useful information for the classification of strains belonging to Saccharomyces sensu stricto. The S. pastorianus Sc-HO, Lg-HO and S. bayanus-HO Accession Nos in the DDBJ Nucleotide Sequence Database are AB027449, AB027450 and AB027451, respectively.
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13. |
Hansen J,
Groth C,
( 1999 ) A natural chimeric yeast containing genetic material from three species. PMID : 10555378 : DOI : 10.1099/00207713-49-4-1933 Abstract >>
The Saccharomyces sp. CID1 isolate (CBS 8614) and several other Saccharomyces sensu stricto yeasts were analysed for their mitochondrial and nuclear genes. The data show that Saccharomyces sp. CID1, found so far only in one location in Europe, is a natural hybrid between three different Saccharomyces yeast species. Two of them, Saccharomyces cerevisiae-like and Saccharomyces bayanus-like, are ubiquitous and contributed parts of the nuclear genome; the third, Saccharomyces sp. IFO 1802-like, which has been found only in Japan, contributed the mitochondrial DNA molecule. These data suggest that the yeast cell is able to accommodate, express and propagate genetic material that originates from different species, and the very existence of the resulting natural hybrids indicates that such hybrids are well adapted to their habitats.
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14. |
Yang C,
Schaefer CB,
( 1999 ) DNA sequence and functional analysis of homologous ARS elements of Saccharomyces cerevisiae and S. carlsbergensis. PMID : 10388814 : PMC : PMC1460646 Abstract >>
ARS elements of Saccharomyces cerevisiae are the cis-acting sequences required for the initiation of chromosomal DNA replication. Comparisons of the DNA sequences of unrelated ARS elements from different regions of the genome have revealed no significant DNA sequence conservation. We have compared the sequences of seven pairs of homologous ARS elements from two Saccharomyces species, S. cerevisiae and S. carlsbergensis. In all but one case, the ARS308-ARS308(carl) pair, significant blocks of homology were detected. In the cases of ARS305, ARS307, and ARS309, previously identified functional elements were found to be conserved in their S. carlsbergensis homologs. Mutation of the conserved sequences in the S. carlsbergensis ARS elements revealed that the homologous sequences are required for function. These observations suggested that the sequences important for ARS function would be conserved in other ARS elements. Sequence comparisons aided in the identification of the essential matches to the ARS consensus sequence (ACS) of ARS304, ARS306, and ARS310(carl), though not of ARS310.
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15. |
Nagasawa N,
Sone H,
Momma T,
Yoshimoto H,
( 1999 ) Isolation and characterization of the ATF2 gene encoding alcohol acetyltransferase II in the bottom fermenting yeast Saccharomyces pastorianus. PMID : 10219999 : DOI : 10.1002/(SICI)1097-0061(19990330)15:5<409::AID-YEA366>3.0.CO;2-Q Abstract >>
The ATF2 gene encodes alcohol acetyltransferase II, which catalyses the synthesis of isoamyl acetate from acetyl coenzyme A and isoamyl alcohol. To characterize the ATF2 gene from the bottom fermenting yeast Saccharomyces pastorianus, the S. pastorianus ATF2 gene was cloned by colony hybridization using the S. cerevisiae ATF2 gene as a probe. When an atf1 null mutant strain was transformed with a multi-copy plasmid carrying the S. pastorianus ATF2 gene, the AATase activity of this strain was increased by 2.5-fold compared to the control. The S. pastorianus ATF2 gene has 99% nucleic acid homology in the coding region and 100% amino acid homology with the S. cerevisiae ATF2 gene. Southern blot analysis of chromosomes separated by pulse-field gel electrophoresis indicated that the ATF2 gene probe hybridized to chromosome VII in S. cerevisiae and to the 1100 kb chromosome in S. pastorianus. As S. pastorianus is thought to be a hybrid of S. cerevisiae and S. bayanus, the S. bayanus-type gene, which has a relatively low level of homology with the S. cerevisiae-type gene, is also usually detected. Interestingly, an S. bayanus-type ATF2 gene could not be detected. These results suggested that the cloned ATF2 gene was derived from S. cerevisiae. Analysis using an ATF2-lacZ fusion gene in S. pastorianus showed that expression of the ATF2 gene was relatively lower than that of the ATF1 gene and that it is repressed by aeration but activated by the addition of unsaturated fatty acids. The S. pastorianus ATF1, Lg-ATF1 and ATF2 Accession Numbers in the DDBJ Nucleotide Sequence Database are D63449, D63450 and D86480, respectively.
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16. |
Omura F,
Hatanaka H,
Nakao Y,
( 2007 ) Characterization of a novel tyrosine permease of lager brewing yeast shared by Saccharomyces cerevisiae strain RM11-1a. PMID : 17825063 : DOI : 10.1111/j.1567-1364.2007.00310.x Abstract >>
In Saccharomyces cerevisiae yeast, the uptake of aromatic amino acids is mediated by the relatively specific permeases Tat1p, Tat2p, Bap2p, and Bap3p, as well as by two other permeases with broader specificities: Gap1p and Agp1p. Here, a novel permease gene TAT3 (Tyrosine Amino acid Transporter) identified in the S. cerevisiae-type subset genome of the lager brewing yeast strain Weihenstephan Nr.34 (34/70) is reported. The TAT3 sequence was also found in the genome of S. cerevisiae strain RM11-1a, but not in S. cerevisiae strain S288C. Tat3p showed a significant similarity to Penicillium chrysogenum ArlP permease, which has transport activity for aromatic amino acids and leucine. When overexpressed in ssy1Delta gap1Delta mutant cells, Tat3p exhibited a tyrosine transport activity with an apparent K(m) of 160 microM. TAT3 transcription in lager brewing yeast was subjected to nitrogen catabolite repression in a manner similar to that of GAP1. Furthermore, the subcellular localization of Tat3p-green fluorescent protein (GFP) fusion protein was dependent on the quality of the nitrogen source, indicating a post-translational control of Tat3p function.
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17. |
Sawyer SL,
Malik HS,
( 2006 ) Positive selection of yeast nonhomologous end-joining genes and a retrotransposon conflict hypothesis. PMID : 17101967 : DOI : 10.1073/pnas.0605468103 PMC : PMC1693795 Abstract >>
Transposable elements have clearly played a major role in shaping both the size and organization of eukaryotic genomes. However, the evolution of essential genes in core biological processes may also have been shaped by coevolution with these elements. This would be predicted to occur in instances where host proteins are either hijacked for use by mobile elements or recruited to defend against them. To detect such cases, we have used the Saccharomyces cerevisiae-Saccharomyces paradoxus sibling species pair to identify genes that have evolved under positive selection. We identify 72 such genes, which participate in a variety of biological processes but are enriched for genes involved in meiosis and DNA repair by nonhomologous end-joining (NHEJ). We confirm the signature of positive selection acting on NHEJ genes using orthologous sequences from all seven Saccharomyces sensu stricto species. Previous studies have found altered rates of Ty retrotransposition when these NHEJ genes are disrupted. We propose that the evolution of these repair proteins is likely to have been shaped by their interactions with Ty elements. Antagonistic pleiotropy, where critical genes like those involved in DNA repair are also subject to selective pressures imposed by mobile elements, could favor alleles that might be otherwise deleterious for their normal roles related to genome stability.
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18. |
Rainieri S,
Kodama Y,
Kaneko Y,
Mikata K,
Nakao Y,
Ashikari T,
( 2006 ) Pure and mixed genetic lines of Saccharomyces bayanus and Saccharomyces pastorianus and their contribution to the lager brewing strain genome. PMID : 16751504 : DOI : 10.1128/AEM.02769-05 PMC : PMC1489639 Abstract >>
The yeast species Saccharomyces bayanus and Saccharomyces pastorianus are of industrial importance since they are involved in the production process of common beverages such as wine and lager beer; however, they contain strains whose variability has been neither fully investigated nor exploited in genetic improvement programs. We evaluated this variability by using PCR-restriction fragment length polymorphism analysis of 48 genes and partial sequences of 16. Within these two species, we identified "pure" strains containing a single type of genome and "hybrid" strains that contained portions of the genomes from the "pure" lines, as well as alleles termed "Lager" that represent a third genome commonly associated with lager brewing strains. The two pure lines represent S. uvarum and S. bayanus, the latter a novel group of strains that may be of use in strain improvement programs. Hybrid lines identified include (i) S. cerevisiae/S. bayanus/Lager, (ii) S. bayanus/S. uvarum/Lager, and (iii) S. cerevisiae/S. bayanus/S. uvarum/Lager. The genome of the lager strains may have resulted from chromosomal loss, replacement, or rearrangement within the hybrid genetic lines. This study identifies brewing strains that could be used as novel genetic sources in strain improvement programs and provides data that can be used to generate a model of how naturally occurring and industrial hybrid strains may have evolved.
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19. |
Thomson JM,
Gaucher EA,
Burgan MF,
De Kee DW,
Li T,
Aris JP,
Benner SA,
( 2005 ) Resurrecting ancestral alcohol dehydrogenases from yeast. PMID : 15864308 : DOI : 10.1038/ng1553 PMC : PMC3618678 Abstract >>
Modern yeast living in fleshy fruits rapidly convert sugars into bulk ethanol through pyruvate. Pyruvate loses carbon dioxide to produce acetaldehyde, which is reduced by alcohol dehydrogenase 1 (Adh1) to ethanol, which accumulates. Yeast later consumes the accumulated ethanol, exploiting Adh2, an Adh1 homolog differing by 24 (of 348) amino acids. As many microorganisms cannot grow in ethanol, accumulated ethanol may help yeast defend resources in the fruit. We report here the resurrection of the last common ancestor of Adh1 and Adh2, called Adh(A). The kinetic behavior of Adh(A) suggests that the ancestor was optimized to make (not consume) ethanol. This is consistent with the hypothesis that before the Adh1-Adh2 duplication, yeast did not accumulate ethanol for later consumption but rather used Adh(A) to recycle NADH generated in the glycolytic pathway. Silent nucleotide dating suggests that the Adh1-Adh2 duplication occurred near the time of duplication of several other proteins involved in the accumulation of ethanol, possibly in the Cretaceous age when fleshy fruits arose. These results help to connect the chemical behavior of these enzymes through systems analysis to a time of global ecosystem change, a small but useful step towards a planetary systems biology.
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20. |
Salema-Oom M,
Valadão Pinto V,
Gonçalves P,
Spencer-Martins I,
( 2005 ) Maltotriose utilization by industrial Saccharomyces strains: characterization of a new member of the alpha-glucoside transporter family. PMID : 16151085 : DOI : 10.1128/AEM.71.9.5044-5049.2005 PMC : PMC1214619 Abstract >>
Maltotriose utilization by Saccharomyces cerevisiae and closely related yeasts is important to industrial processes based on starch hydrolysates, where the trisaccharide is present in significant concentrations and often is not completely consumed. We undertook an integrated study to better understand maltotriose metabolism in a mixture with glucose and maltose. Physiological data obtained for a particularly fast-growing distiller's strain (PYCC 5297) showed that, in contrast to what has been previously reported for other strains, maltotriose is essentially fermented. The respiratory quotient was, however, considerably higher for maltotriose (0.36) than for maltose (0.16) or glucose (0.11). To assess the role of transport in the sequential utilization of maltose and maltotriose, we investigated the presence of genes involved in maltotriose uptake in the type strain of Saccharomyces carlsbergensis (PYCC 4457). To this end, a previously constructed genomic library was used to identify maltotriose transporter genes by functional complementation of a strain devoid of known maltose transporters. One gene, clearly belonging to the MAL transporter family, was repeatedly isolated from the library. Sequence comparison showed that the novel gene (designated MTY1) shares 90% and 54% identity with MAL31 and AGT1, respectively. However, expression of Mty1p restores growth of the S. cerevisiae receptor strain on both maltose and maltotriose, whereas the closely related Mal31p supports growth on maltose only and Agt1p supports growth on a wider range of substrates, including maltose and maltotriose. Interestingly, Mty1p displays higher affinity for maltotriose than for maltose, a new feature among all the alpha-glucoside transporters described so far.
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21. |
Nguyen HV,
Gaillardin C,
( 2005 ) Evolutionary relationships between the former species Saccharomyces uvarum and the hybrids Saccharomyces bayanus and Saccharomyces pastorianus; reinstatement of Saccharomyces uvarum (Beijerinck) as a distinct species. PMID : 15691752 : DOI : 10.1016/j.femsyr.2004.12.004 Abstract >>
Analysis of the nucleotide sequence of the GDH1 homologues from Saccharomyces bayanus strain CBS 380T and S. pastorianus strains showed that they share an almost identical sequence, SuGDH1*, which is a diverged form of the SuGDH1 from the type strain of the former species S. uvarum, considered as synonym of S. bayanus. SuGDH1* is close to but differs from SuGDH1 by the accumulation of a high number of neutral substitutions designated as Multiple Neutral Mutations Accumulation (MNMA). Further analysis carried out with three other markers, BAP2, HO and MET2 showed that they have also diverged from their S. uvarum counterparts by MNMA. S. bayanus CBS 380T is placed between S. uvarum and S. pastorianus sharing MET2, CDC91 sequences with the former and BAP2, GDH1, HO sequences with the latter. S. bayanus CBS 380T has been proposed to be a S. uvarum/S. cerevisiae hybrid and this proposal is confirmed by the presence in its genome a S. cerevisiae SUC4 gene. Strain S. bayanus CBS 380T, with a composite genome, is genetically isolated from strains of the former S. uvarum species, thus justifying the reinstatement of S. uvarum as a distinct species.
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22. |
Marinangeli P,
Clementi F,
Ciani M,
Mannazzu I,
( 2004 ) SED1 polymorphism within the genus Saccharomyces. PMID : 15381124 : DOI : 10.1016/j.femsyr.2004.07.001 Abstract >>
The SED1 gene is characterised by abundant length and sequence polymorphisms within the species Saccharomyces cerevisiae, due to the expansion and contraction of minisatellite-like sequences located within the ORF. A survey of the SED1 ORFs of 26 yeasts ascribed to the species S. cerevisiae, S. bayanus, S. pastorianus, S. paradoxus, S. cariocanus, S. kudriavzevii and S. mikatae revealed SED1 gene length and sequence variations between the species of the genus. Moreover, results obtained by Neighbour-Joining analysis of a dataset comprising the partial predicted amino acid sequences of SED1 ORFs agreed with the phylogenetic relationships of the seven species. Thus, the SED1 gene may represent a further molecular target for the identification of Saccharomyces isolates.
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23. |
Johnson LJ,
Koufopanou V,
Goddard MR,
Hetherington R,
Schäfer SM,
Burt A,
( 2004 ) Population genetics of the wild yeast Saccharomyces paradoxus. PMID : 15020405 : PMC : PMC1470673 Abstract >>
Saccharomyces paradoxus is the closest known relative of the well-known S. cerevisiae and an attractive model organism for population genetic and genomic studies. Here we characterize a set of 28 wild isolates from a 10-km(2) sampling area in southern England. All 28 isolates are homothallic (capable of mating-type switching) and wild type with respect to nutrient requirements. Nine wild isolates and two lab strains of S. paradoxus were surveyed for sequence variation at six loci totaling 7 kb, and all 28 wild isolates were then genotyped at seven polymorphic loci. These data were used to calculate nucleotide diversity and number of segregating sites in S. paradoxus and to investigate geographic differentiation, population structure, and linkage disequilibrium. Synonymous site diversity is approximately 0.3%. Extensive incompatibilities between gene genealogies indicate frequent recombination between unlinked loci, but there is no evidence of recombination within genes. Some localized clonal growth is apparent. The frequency of outcrossing relative to inbreeding is estimated at 1.1% on the basis of heterozygosity. Thus, all three modes of reproduction known in the lab (clonal replication, inbreeding, and outcrossing) have been important in molding genetic variation in this species.
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24. |
Poothong J,
Sopha P,
Kaufman RJ,
Tirasophon W,
( 2010 ) Domain compatibility in Ire1 kinase is critical for the unfolded protein response. PMID : 20541549 : DOI : 10.1016/j.febslet.2010.06.003 PMC : PMC3762510 Abstract >>
The unfolded protein response is a mechanism to cope with endoplasmic reticulum stress. In Saccharomyces cerevisiae, Ire1 senses the stress and mediates a signaling cascade to upregulate responsive genes through an unusual HAC1 mRNA splicing. The splicing requires interconnected activity (kinase and endoribonuclease (RNase)) of Ire1 to cleave HAC1 mRNA at the non-canonical splice sites before translation into Hac1 transcription factor. Analysis of the truncated kinase domain from Ire1 homologs revealed that this domain is highly conserved. Characterization by domain swapping indicated that a functional ATP/ADP binding domain is minimally required. However the overall domain compatibility is critical for eliciting its full RNase function.
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25. |
Yamagishi H,
Ohnuki S,
Nogami S,
Ogata T,
Ohya Y,
( 2010 ) Role of bottom-fermenting brewer's yeast KEX2 in high temperature resistance and poor proliferation at low temperatures. PMID : 20953094 : Abstract >>
Variants of bottom-fermenting brewer's yeast that grew at high temperatures and showed poor proliferation and fermentation at low temperatures were isolated. Similar variants of laboratory yeast were also isolated and found to be incapable of mating. The KEX2 gene was cloned by complementation. It was shown to be responsible for these traits, because a KEX2 disruptant of Saccharomyces cerevisiae (S. cerevisiae) laboratory yeast grew poorly at low temperatures and was resistant to high temperatures. In addition, a Saccharomyces bayanus (S. bayanus)-type KEX2 (Sb-KEX2) disruptant of bottom-fermenting brewer's yeast grew poorly at low temperatures and was resistant to high temperatures. The KEX2 gene product plays an important role in proliferation of yeast at low temperatures, which is an important trait of bottom-fermenting brewer's yeast. These findings advance our understanding of the proliferation of yeast at low temperatures, especially that of bottom-fermenting brewer's yeast.
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26. |
Dietvorst J,
Walsh MC,
van Heusden GP,
Steensma HY,
( 2010 ) Comparison of the MTT1- and MAL31-like maltose transporter genes in lager yeast strains. PMID : 20698888 : DOI : 10.1111/j.1574-6968.2010.02056.x Abstract >>
Maltose transporter genes were isolated from four lager yeast strains and sequenced. All four strains contain at least two different types of maltose transporter genes, MTT1 and MAL31. In addition, 'long' 2.7 kb, and 'short' 2.4 kb, versions of each type exist. The size difference is caused by the insertion of two repeats of 147 bp into the promoter regions of the long versions of the genes. As a consequence of the insertion, two Mal63-binding sites move 294 bp away from the transcription initiation site. The 2.4- and 2.7-kb versions are further highly similar. Only the 2.4-kb versions and not the 2.7-kb versions of MTT1 could restore the rapid growth of lager yeast strain A15 on maltotriose in the presence of antimycin A. These results suggest that insertion of the two repeats into the promoter region of the 'long versions' of MTT1 genes led to a diminished expression of these genes. None of the tested long and short versions of the MAL31 genes were able to restore this growth. As the promoter regions of the MTT1 and MAL31 genes are identical, small differences in the protein sequence may be responsible for the different properties of these genes.
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27. |
Ogata T,
Izumikawa M,
Tadami H,
( 2009 ) Chimeric types of chromosome X in bottom-fermenting yeasts. PMID : 19486390 : DOI : 10.1111/j.1365-2672.2009.04289.x Abstract >>
To determine the structure of the chimeric chromosome X of bottom-fermenting yeasts. Eight cosmid clones carrying DNA from chromosome X of bottom-fermenting yeasts were selected by end-sequencing. Four of the cosmid clones had Saccharomyces cerevisiae (SC)-type and Saccharomyces bayanus (SB)-type chimeric ends, two had SC-type ends and two had SB-type ends. Sequencing revealed that the bottom-fermenting yeast strains in this study had four types of chromosome X: SC-SC, SC-SB, SB-SC and SB-SB. The translocation site in the chimeric chromosome is conserved among bottom-fermenting yeast strains, and was created by homologous recombination within a region of high sequence identity between the SC-type sequence and the SB-type sequence. Existing bottom-fermenting yeast strains share a common ancestor in which the chimeric chromosome X was generated. The knowledge gained in this study sheds light on the evolution of bottom-fermenting yeasts.
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28. |
Pal S,
Park DH,
Plapp BV,
( 2009 ) Activity of yeast alcohol dehydrogenases on benzyl alcohols and benzaldehydes: characterization of ADH1 from Saccharomyces carlsbergensis and transition state analysis. PMID : 19022233 : DOI : 10.1016/j.cbi.2008.10.037 PMC : PMC2712655 Abstract >>
The substrate specificities of yeast alcohol dehydrogenases I and II from Saccharomyces cerevisiae (SceADH1 and SceADH2) and Saccharomyces carlsbergensis (ScbADH1) were studied. For this work, the gene for the S. carlsbergensis ADH1 was cloned, sequenced and expressed. The amino acid sequence of ScbADH1 differs at four positions as compared to SceADH1, including substitutions of two glutamine residues with glutamic acid residues, and has the same sequence as the commercial yeast enzyme, which apparently is prepared from S. carlsbergensis. The electrophoretic mobilities of ScbADH1, SceADH2 and commercial ADH are similar. The kinetics and specificities of ScbADH1 and SceADH1 acting on branched, long-chain and benzyl alcohols are very similar, but the catalytic efficiency of SceADH2 is about 10-100-fold higher on these substrates. A three-dimensional structure of SceADH1 shows that the substrate binding pocket has Met-270, whereas SceADH2 has Leu-270, which allows larger substrates to bind. The reduction of a series of p-substituted benzaldehydes catalyzed by SceADH2 is significantly enhanced by electron-withdrawing groups, whereas the oxidation of p-substituted aromatic alcohols may be only slightly affected by the substituents. The substituent effects on catalysis generally reflect the effects on the equilibrium constant for the reaction, where electron-withdrawing substituents favor alcohol. The results are consistent with a transition state that is electronically similar to the alcohol, supporting previous results obtained with commercial yeast ADH.
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29. |
Huang CH,
Lee FL,
Tai CJ,
( 2009 ) The beta-tubulin gene as a molecular phylogenetic marker for classification and discrimination of the Saccharomyces sensu stricto complex. PMID : 19112604 : DOI : 10.1007/s10482-008-9296-1 Abstract >>
The Saccharomyces sensu stricto complex comprises seven very closely related species. In this study, we compared the use of two different phylogenetic markers, the 26S rDNA and beta-tubulin genes, for discriminating phylogenetic relationships among Saccharomyces sensu stricto strains using sequencing as well as RFLP methods. The average sequence similarity for the beta-tubulin gene (90.0%) among seven strains was significantly less than that for 26S rDNA (98.6%). This result demonstrates that beta-tubulin gene sequences provided higher resolution than 26S rDNA sequences. Species-specific restriction profiles of the Saccharomyces strains were obtained by cutting them with the Tsp509I enzyme. Our data indicate that phylogenetic relationships between these strains are best resolved using sequencing or RFLP analysis of the beta-tubulin gene.
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30. |
Ogata T,
Izumikawa M,
Kohno K,
Shibata K,
( 2008 ) Chromosomal location of Lg-FLO1 in bottom-fermenting yeast and the FLO5 locus of industrial yeast. PMID : 18564345 : DOI : 10.1111/j.1365-2672.2008.03852.x Abstract >>
To determine the chromosomal location and entire sequence of Lg-FLO1, the expression of which causes the flocculation of bottom-fermenting yeast. Two cosmid clones carrying DNA from a bottom-fermenting yeast chromosome VIII right-arm end were selected by colony hybridization. Sequencing revealed that the clones contained DNA derived from a Saccharomyces cerevisiae type chromosome VIII and a Saccharomyces bayanus type chromosome VIII, both from bottom-fermenting yeast. Lg-FLO1 is located on the S. cerevisiae type chromosome VIII at the same position as the FLO5 gene of the laboratory yeast S. cerevisiae S288c. The unique chromosome VIII structure of bottom-fermenting yeast is conserved among other related strains. FLO5 and Lg-FLO1 promoter sequences are identical except for the presence of three 42 bp repeats in the latter, which are associated with gene activity. Flocculin genes might have been generated by chromosomal recombination at these repeats. This is the first report of the exact chromosomal location and entire sequence of Lg-FLO1. This information will be useful in the brewing industry for the identification of normal bottom-fermenting yeast. Moreover, variations in the FLO5 locus among strains are thought to reflect yeast evolution.
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31. |
Rainieri S,
Kodama Y,
Nakao Y,
Pulvirenti A,
Giudici P,
( 2008 ) The inheritance of mtDNA in lager brewing strains. PMID : 18318709 : DOI : 10.1111/j.1567-1364.2008.00363.x Abstract >>
In this work, we compared the mtDNA of a number of interspecific Saccharomyces hybrids (Saccharomyces cerevisiae x Saccharomyces uvarum and S. cerevisiae x Saccharomyces bayanus) to the mtDNA of 22 lager brewing strains that are thought to be the result of a natural hybridization between S. cerevisiae and another Saccharomyces yeast, possibly belonging to the species S. bayanus. We detected that in hybrids constructed in vitro, the mtDNA could be inherited from either parental strain. Conversely, in the lager strains tested, the mtDNA was never of the S. cerevisiae type. Moreover, the nucleotide sequence of lager brewing strains COXII gene was identical to S. bayanus strain NBRC 1948 COXII gene. MtDNA restriction analysis carried out with three enzymes confirmed this finding. However, restriction analysis with a fourth enzyme (AvaI) provided restriction patterns for lager strains that differed from those of S. bayanus strain NBRC 1948. Our results raise the hypothesis that the human-driven selection carried out on existing lager yeasts has favored only those bearing optimal fermentation characteristics at low temperatures, which harbor the mtDNA of S. bayanus.
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32. |
Yoshida S,
Hashimoto K,
Tanaka-Kanai K,
Yoshimoto H,
Kobayashi O,
( 2007 ) Identification and characterization of amidase- homologous AMI1 genes of bottom-fermenting yeast. PMID : 17924455 : DOI : 10.1002/yea.1551 Abstract >>
It has been proposed that a bottom-fermenting yeast strain of Saccharomyces pastorianus is a natural hybrid between S. cerevisiae and S. bayanus and possesses at least two types of genome. In the process of conducting expressed sequence tag (EST) analysis, we isolated bottom-fermenting yeast-specific (BFY) genes that have no significant homology with sequences in the S288C database. One of the BFY genes, AMI1, encodes a protein with homology to an amidase conserved among plants, Bacillus subtilis, Neurospora crassa, Schizosaccharomyces pombe and Saccharomyces species, with the exception of S. cerevisiae S288C. In the bottom-fermenting yeast, three alleles of AMI1 (one AMI1-A and two AMI1-B alleles) were found on different chromosomes. AMI1-A on chromosome XIII is most homologous to the S. bayanus AMI1 gene, while AMI1-B on chromosome X is most homologous to the Saccharomyces paradoxus AMI1 gene. Overproduction of AMI1 in S. cerevisiae resulted in a slow-growth phenotype. Although a hydropathy plot shows that Ami1p has a putative signal sequence, it was located in the cell, not secreted into the medium. By metabolome analysis of intracellular compounds, the amount of histidine and arginine is increased, and the amount of threonine, lysine and nicotinic acid is decreased in the Ami1p-overproducing strain as compared with the control, suggesting that Ami1p may hydrolyse some amides related to amino acid and niacin metabolism in the cell.
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33. |
Spitaels F,
Wieme AD,
Janssens M,
Aerts M,
Daniel HM,
Van Landschoot A,
De Vuyst L,
Vandamme P,
( 2014 ) The microbial diversity of traditional spontaneously fermented lambic beer. PMID : 24748344 : DOI : 10.1371/journal.pone.0095384 PMC : PMC3991685 Abstract >>
Lambic sour beers are the products of a spontaneous fermentation that lasts for one to three years before bottling. The present study determined the microbiota involved in the fermentation of lambic beers by sampling two fermentation batches during two years in the most traditional lambic brewery of Belgium, using culture-dependent and culture-independent methods. From 14 samples per fermentation, over 2000 bacterial and yeast isolates were obtained and identified. Although minor variations in the microbiota between casks and batches and a considerable species diversity were found, a characteristic microbial succession was identified. This succession started with a dominance of Enterobacteriaceae in the first month, which were replaced at 2 months by Pediococcus damnosus and Saccharomyces spp., the latter being replaced by Dekkera bruxellensis at 6 months fermentation duration.
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34. |
Bateman DA,
Wickner RB,
( 2012 ) [PSI+] Prion transmission barriers protect Saccharomyces cerevisiae from infection: intraspecies 'species barriers'. PMID : 22095075 : DOI : 10.1534/genetics.111.136655 PMC : PMC3276615 Abstract >>
[PSI+] is a prion of Sup35p, an essential translation termination and mRNA turnover factor. The existence of lethal [PSI+] variants, the absence of [PSI+] in wild strains, the mRNA turnover function of the Sup35p prion domain, and the stress reaction to prion infection suggest that [PSI+] is a disease. Nonetheless, others have proposed that [PSI+] and other yeast prions benefit their hosts. We find that wild Saccharomyces cerevisiae strains are polymorphic for the sequence of the prion domain and particularly in the adjacent M domain. Here we establish that these variations within the species produce barriers to prion transmission. The barriers are partially asymmetric in some cases, and evidence for variant specificity in barriers is presented. We propose that, as the PrP 129M/V polymorphism protects people from Creutzfeldt-Jakob disease, the Sup35p polymorphisms were selected to protect yeast cells from prion infection. In one prion incompatibility group, the barrier is due to N109S in the Sup35 prion domain and several changes in the middle (M) domain, with either the single N109S mutation or the group of M changes (without the N109S) producing a barrier. In another, the barrier is due to a large deletion in the repeat domain. All are outside the region previously believed to determine transmission compatibility. [SWI+], a prion of the chromatin remodeling factor Swi1p, was also proposed to benefit its host. We find that none of 70 wild strains carry this prion, suggesting that it is not beneficial.
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35. |
Ikushima S,
Tateishi Y,
Kanai K,
Shimada E,
Tanaka M,
Ishiguro T,
Mizutani S,
Kobayashi O,
( 2012 ) Multi-locus genotyping of bottom fermenting yeasts by single nucleotide polymorphisms indicative of brewing characteristics. PMID : 22227119 : DOI : 10.1016/j.jbiosc.2011.12.002 Abstract >>
Yeast plays a capital role in brewing fermentation and has a direct impact on flavor and aroma. For the evaluation of competent brewing strains during quality control or development of novel strains it is standard practice to perform fermentation tests, which are costly and time-consuming. Here, we have categorized DNA markers which enable to distinguish and to screen brewing strains more efficiently than ever before. Sequence analysis at 289 loci in the genomes of six bottom fermenting Saccharomyces pastorianus strains revealed that 30 loci contained single nucleotide polymorphisms (SNPs). By determining the nucleotide sequences at the SNP-loci in 26 other S. pastorianus strains and 20 strains of the top fermenting yeast Saccharomyces cerevisiae, almost all these strains could be discriminated solely on the basis of the SNPs. By comparing the fermentative phenotypes of these strains we found that some DNA markers showed a strong association with brewing characteristics, such as the production of ethyl acetate and hydrogen sulphide (H2S). Therefore, the DNA markers we identified will facilitate quality control and the efficient development of brewing yeast strains.
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36. |
( 1997 ) Examination of the intron in the meiosis-specific recombination gene REC114 in Saccharomyces. PMID : 9267437 : DOI : 10.1007/s004380050513 Abstract >>
REC114 is one of 10 genes known to be required for the initiation of meiotic recombination in Saccharomyces cerevisiae. It is transcribed only in meiosis, and our previous sequence analysis suggested the presence of an intron in the 3' end of the gene. Hypotheses in the literature have suggested, because of its unusual location, either that the putative intron in REC114 is likely to be necessary for expression, or that there may actually be no intron present. This work demonstrates that REC114 does have an intron and is one of only three genes in yeast with introns located in the 3' end. Furthermore, the 3' splice site utilized in REC114 is a very rare AAG sequence; only three other genes in yeast use this nonconsensus sequence. The splicing of REC114 does not require MER1, a gene known to be involved in meiosis-specific RNA processing. In fact, an intronless copy of REC114 can complement a null rec114 mutation. Thus, it does not appear that the intron is essential for expression of REC114. Although the intron is not absolutely required for meiotic function, it is conserved in evolution; two other species of yeast contain an intron at the same location in their REC114 genes.
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37. |
( 1996 ) Nucleotide sequences of alcohol acetyltransferase genes from lager brewing yeast, Saccharomyces carlsbergensis. PMID : 8771714 : DOI : 10.1002/(SICI)1097-0061(199605)12:6%3C593::AID-YEA593%3E3.0.CO;2-B Abstract >>
The nucleotide sequences of alcohol acetyltransferase genes isolated from lager brewing yeast, Saccharomyces carlsbergensis have been determined. S. carlsbergensis has one ATF1 gene and another homologous gene, the Lg-ATF1 gene. There was a high degree of homology between the amino acid sequences deduced for the ATF1 protein and the Lg-ATF1 protein (75.7%), but the N-terminal region has a relatively low degree of homology. Southern analysis and contour-clamped homogeneous electric field analysis of Saccharomyces strains suggest that the ATF1 gene is located on chromosome XV in S. cerevisiae and that the Lg-ATF1 gene might originate from the "non-S. cerevisiae' genome of S. carlsbergensis, which is similar to that of S. bayanus and S. pastorianus.
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38. |
( 1997 ) Isolation of early meiotic recombination genes analogous to S. cerevisiae REC104 from the yeasts S. paradoxus and S. pastorianus. PMID : 9000375 : Abstract >>
The REC104 gene of Saccharomyces cerevisiae is required to initiate recombination in meiosis. Mutations in REC104 eliminate meiotic recombination and lead to the production of inviable spores. To determine if analogous genes exist in other yeasts, clones that hybridized to a REC104 probe were isolated from the yeasts S. paradoxus and S. pastorianus. When transformed into a rec104 strain, the REC104 analogs from these two yeasts restored spore viability and meiotic recombination to the same level as a REC104 gene cloned from S. cerevisiae. Compared to S. cerevisiae, the S. paradoxus gene codes for 79% identical amino acids and has 86% nucleic-acid identity in the promoter region and 84% in the coding region. The S. pastorianus gene codes for 63% identical amino acids and has 59% and 71% identity in the promoter and the coding regions, respectively.
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39. |
( 1994 ) Yeast acyl-CoA-binding protein: acyl-CoA-binding affinity and effect on intracellular acyl-CoA pool size. PMID : 8093000 : DOI : 10.1042/bj3020479 PMC : PMC1137253 Abstract >>
Acyl-CoA-binding protein (ACBP) is a 10 kDa protein characterized in vertebrates. We have isolated two ACBP homologues from the yeast Saccharomyces carlsbergensis, named yeast ACBP types 1 and 2. Both proteins contain 86 amino acid residues and are identical except for four conservative substitutions. In comparison with human ACBP, yeast ACBPs exhibit 48% (type 1) and 49% (type 2) conservation of amino acid residues. The amino acid sequence of S. carlsbergensis ACBP type 1 was found to be identical with the one ACBP present in Saccharomyces cerevisiae. A recombinant form of this protein was expressed in Escherichia coli and S. cerevisiae, purified, and its acyl-CoA-binding properties were characterized by isoelectric focusing and microcalorimetric analyses. The yeast ACBP was found to bind acyl-CoA esters with high affinity (Kd 0.55 x 10(-10) M). Overexpression of yeast ACBP in S. cerevisiae resulted in a significant expansion of the intracellular acyl-CoA pool. Finally, Southern-blotting analysis of the two genes encoding ACBP types 1 and 2 in S. carlsbergensis strongly indicated that this species is a hybrid between S. cerevisiae and Saccharomyces monacensis.
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40. |
( 1994 ) Saccharomyces carlsbergensis contains two functional MET2 alleles similar to homologues from S. cerevisiae and S. monacensis. PMID : 8125336 : DOI : 10.1016/0378-1119(94)90727-7 Abstract >>
The brewing yeast, Saccharomyces, carlsbergensis, is allopolyploid, derived from two diverged genomes. To obtain information about the possible origin of this yeast, we cloned two different S. carlsbergensis MET2 genes (encoding homoserine acetyltransferase). One has a nucleotide (nt) sequence identical or very similar to MET2 of Saccharomyces cerevisiae. The other has a different sequence, but was functional in S. cerevisiae. This allele was sequenced and revealed a coding region of 486 amino acids (aa). The nt sequence of the coding region showed 82% homology to S. cerevisiae MET2, while the derived aa sequences were 94% identical. Hybridization experiments to genomic DNA of different yeast strains revealed that the divergent MET2 gene had higher sequence homology to segments from type strains of S. monacensis, S. bayanus and S. uvarum than to MET2 from S. cerevisiae. Sequencing of 330 bp of a PCR-amplified fragment of MET2 from these organisms shows that the non-S. cerevisiae-like sequence from S. carlsbergensis is identical to the corresponding sequence in S. monacensis, while it is 93% homologous with S. bayanus and S. uvarum. Our results are consistent with the proposal that S. carlsbergensis originated as a hybrid between S. monacensis and S. cerevisiae. The complete identity of the MET2 fragments from S. monacensis and the S. carlsbergensis-specific MET2 allele suggests that the hybridization must have been a quite recent event.
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41. |
Fox KM,
Karplus PA,
( 1994 ) Old yellow enzyme at 2 A resolution: overall structure, ligand binding, and comparison with related flavoproteins. PMID : 7881908 : Abstract >>
Old yellow enzyme (OYE) was the first flavoenzyme purified, but its function is still unknown. Nevertheless, the NADPH oxidase activity, the flavin mononucleotide environment and the ligand-binding properties of OYE have been extensively studied by biochemical and spectroscopic approaches. Full interpretation of these data requires structural information. The crystal structures of oxidized and reduced OYE at 2 A resolution reveal an alpha/beta-barrel topology clearly related to trimethylamine dehydrogenase. Complexes of OYE with p-hydroxybenzaldehyde, beta-estradiol, and an NADPH analog show all three binding at a common site, stacked on the flavin. The putative NADPH binding mode is novel as it involves primary recognition of the nicotinamide mononucleotide portion. This work shows that the striking spectral changes seen upon phenol binding are due to close physical association of the flavin and phenolate. It also identifies the structural class of OYE and suggests that if NADPH is its true substrate, then OYE has adopted NADPH dependence during evolution.
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42. |
Citron BA,
Donelson JE,
( 1984 ) Sequence of the Saccharomyces GAL region and its transcription in vivo. PMID : 6715281 : PMC : PMC215408 Abstract >>
In Saccharomyces, the enzymes used to convert galactose to glucose are specified by three coordinately expressed, tightly linked genes, GAL7, GAL10, and GAL1. These genes are induced by galactose and are controlled by the positive regulator gene gal4 and the negative regulator gene gal80. GAL81 mutations, which are known to alter the gal4 protein, produce a constitutive phenotype. We have cloned fragments of Saccharomyces carlsbergensis DNA that span 26.3 kilobases surrounding the three clustered GAL genes. About 5 kilobases of the sequence was determined, which includes the entire GAL1 gene, the two intercistronic regions, and portions of the coding sequences of GAL10 and GAL7. Some amino acid homology between the GAL1 gene product, galactokinase, and the Escherichia coli galactokinase was detected. By using various Saccharomyces DNA fragments, the accumulation of GAL1 and GAL10 RNA in yeast cells after induction with galactose was studied. Our results, using wild-type, gal4-, gal80-, and GAL81-1- yeast cells, support the hypothesis that control is exerted at the transcriptional level.
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43. |
( 1994 ) Two divergent MET10 genes, one from Saccharomyces cerevisiae and one from Saccharomyces carlsbergensis, encode the alpha subunit of sulfite reductase and specify potential binding sites for FAD and NADPH. PMID : 7928966 : DOI : 10.1128/jb.176.19.6050-6058.1994 PMC : PMC196824 Abstract >>
The yeast assimilatory sulfate reductase is a complex enzyme that is responsible for conversion of sulfite into sulfide. To obtain information on the nature of this enzyme, we isolated and sequenced the MET10 gene of Saccharomyces cerevisiae and a divergent MET10 allele from Saccharomyces carlsbergensis. The polypeptides deduced from the identically sized open reading frames (1,035 amino acids) of both MET10 genes have molecular masses of around 115 kDa and are 88% identical to each other. The transcript of S. cerevisiae MET10 has a size comparable to that of the open reading frame and is transcriptionally repressed by methionine in a way similar to that seen for other MET genes of S. cerevisiae. Distinct homology was found between the putative MET10-encoded polypeptide and flavin-interacting parts of the sulfite reductase flavoprotein subunit (encoded by cysJ) from Escherichia coli and several other flavoproteins. A significant N-terminal homology to pyruvate flavodoxin oxidoreductase (encoded by nifJ) from Klebsiella pneumoniae, together with a lack of obvious flavin mononucleotide-binding motifs in the MET10 deduced amino acid sequence, suggests that the yeast assimilatory sulfite reductase is a distinct type of sulfite reductase.
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44. |
( 2013 ) Structural and biochemical characterization of the N-terminal domain of flocculin Lg-Flo1p from Saccharomyces pastorianus reveals a unique specificity for phosphorylated mannose. PMID : 23281814 : DOI : 10.1111/febs.12102 Abstract >>
The mechanism of yeast flocculation is generally considered to be mediated through the interaction of cell surface flocculins and mannan carbohydrates. In the present study, the crystal structure of the soluble 25-kDa lectin domain of flocculin 1 from brewer's yeast (Lg-Flo1p) was resolved to 2.5 ?, and its binding specificity towards oligosaccharides was investigated by fluorescence spectroscopy. Lg-Flo1p displays broad specificity towards sugars and has a 14-fold higher affinity for mannose 1-phosphate and glucose 1-phosphate compared to their unphosphorylated counterparts. Based on the results of a structural analysis, we propose that this higher affinity is the result of a charge interaction with a lysine residue in a carbohydrate-binding loop region, NAKAL, unique to NewFlo type flocculins. This raises the possibility of a unique mechanism of flocculation in NewFlo type yeast, which recognizes phosphorylated cell surface mannans. Structural data have been deposited in the Protein Data Bank under accession number 4GQ7.
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45. |
( 1997 ) Constitutive mutations of the Saccharomyces cerevisiae MAL-activator genes MAL23, MAL43, MAL63, and mal64. PMID : 9258674 : PMC : PMC1208075 Abstract >>
We report the sequence of several MAL-activator genes, including inducible, constitutive, and noninducible alleles of MAL23, MAL43, MAL63, and mal64. Constitutive alleles of MAL23 and MAL43 vary considerably from inducible alleles in their C-terminal domain, with many of the alterations clustered and common to both alleles. The 27 alterations from residues 238-461 of Mal43-C protein are sufficient for constitutivity, but the minimal number of alterations needed for the constitutive phenotype could not be determined. The sequence of mal64, a nonfunctional homologue of MAL63, revealed that Mal64p is 85% identical to Mal63p. Two mutations that activate mal64 and cause constitutivity are nonsense mutations resulting in truncated proteins of 306 and 282 residues. We conclude that the C-terminal region of the MAL-activator, from residues 283-470, contains a maltose-responsive negative regulatory domain, and that extensive mutation or deletion of the entire region causes loss of the negative regulatory function. Additionally, certain sequence elements in the region appear to be necessary for efficient induction of the full-length Mal63 activator protein. These studies highlight the role of ectopic recombination as an important mechanism of mutagenesis of the telomere-associated family of MAL loci.
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46. |
( 1998 ) On the active site of Old Yellow Enzyme. Role of histidine 191 and asparagine 194. PMID : 9830019 : DOI : 10.1074/jbc.273.49.32753 Abstract >>
Old Yellow Enzyme (OYE) binds phenolic ligands forming long wavelength (500-800 nm) charge-transfer complexes. The enzyme is reduced by NADPH, and oxygen, quinones, and alpha,beta-unsaturated aldehydes and ketones can act as electron acceptors to complete catalytic turnover. Solution of the crystal structure of OYE1 from brewer's bottom yeast (Fox, K. M., and Karplus, P. A. (1994) Structure 2, 1089-1105) made it possible to identify histidine 191 and asparagine 194 as amino acid residues that hydrogen-bond with the phenolic ligands, stabilizing the anionic form involved in charge-transfer interaction with the FMN prosthetic group. His-191 and Asn-194 are also predicted to interact with the nicotinamide ring of NADPH in the active site. Mutations of His-191 to Asn, Asn-194 to His, and a double mutation, H191N/N194H, were made of OYE1. It was not possible to isolate the N191H mutant enzyme, but the other two mutant forms had the expected effect on phenolic ligand binding, i.e. decreased binding affinity and decreased charge-transfer absorbance. Reduction of the H191N mutant enzyme by NADPH was similar to that of OYE1, but the reduction rate constant for NADH was greatly decreased. The double mutant enzyme had an increased rate constant for reduction by NADPH, but the reduction rate constant with NADH was lower by a factor of 15. The reactivity of OYE1 and the mutant enzymes with oxygen was similar, but the reactivity of 2-cyclohexenone was greatly decreased by the mutations. The crystal structures of the two mutant forms showed only minor changes from that of the wild type enzyme.
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( 1997 ) Saccharomyces carlsbergensis contains two functional genes encoding the acyl-CoA binding protein, one similar to the ACB1 gene from S. cerevisiae and one identical to the ACB1 gene from S. monacensis. PMID : 9434347 : DOI : 10.1002/(SICI)1097-0061(199712)13:15<1409::AID-YEA188>3.0.CO;2-A Abstract >>
Saccharomyces carlsbergensis is an amphiploid, and it has previously been suggested that the genomes of S. carlsbergensis originate from S. cerevisiae and S. monacensis. We have cloned the ACB1 genes encoding the acyl-CoA binding protein (ACBP) from S. carlsbergensis, S. cerevisiae and S. monacensis. Two genes were found in S. carlsbergensis and named ACB1 type 1 and type 2, respectively. The type 1 gene is identical to the S. cerevisiae ACB1 gene except for three substitutions, one single base pair deletion and one double base pair insertion, all located in the promoter region. The type 2 gene is completely identical to the S. monacensis ACB1 gene. These findings substantiate the notion that S. carlsbergensis is a hybrid between S. cerevisiae and S. monacensis. Both ACB1 type 1 and type 2 are actively transcribed in S. carlsbergensis and transcription is initiated at sites identical to those used for transcriptional initiation of the ACB1 genes in S. cerevisiae and S. monacensis, respectively. Two polyadenylation sites, spaced 225 bp apart, are present in the S. cerevisiae ACB1 gene. The upstream polyadenylation site is used exclusively during exponential growth, whereas both sites are utilized during later stages of growth.
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