Genetic differentiation of the sherry yeasts Saccharomyces cerevisiae

Molecular genetic study of the yeast Saccharomyces cerevisiae isolated at various stages of sherry making (young wine, solera, and criadera) in various winemaking regions of Spain demonstrated that sherry yeasts diverged from the primary winemaking yeasts according to several physiological and molecular markers. All sherry strains independently of the place and time of their isolation carry a 24-bp deletion in the ITS 1 region of ribosomal DNA, whereas the yeasts of the primary winemaking lack this deletion. Molecular karyotypes of the sherry yeast from different populations were found very similar.     

Genetic Differentiation of the Sherry Yeasts <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Molecular and genetic studies of the yeast Saccharomyces cerevisiae isolated at distinct stages of sherry making (young wine, solera, and criadera) in various winemaking regions of Spain demonstrated that sherry yeasts diverged from primary winemaking yeasts according to several physiological and molecular markers. All sherry strains, regardless of the place and time of their isolation, carry a 24-bp deletion in the ITS1 region of ribosomal DNA, whereas the yeasts of primary winemaking lack this deletion. Molecular karyotypes of sherry yeasts from different populations were found to be very similar.     

Analysis of yeast populations during alcoholic fermentation: a six year follow-up study

Wine yeasts were isolated from fermenting Garnatxa and Xarel.lo musts fermented in a newly built and operated winery between 1995 and 2000. The species of non-Saccharomyces yeasts and the Saccharomyces cerevisiae strains were identified by ribosomal DNA and mitochondrial DNA RFLP analysis respectively. Non-Saccharomyces yeasts, particularly Hanseniaspora uvarum and Candida stellata, dominated the first stages of fermentation. However Saccharomyces cerevisiae was present at the beginning of the fermentation and was the main yeast in the musts in one vintage (1999). In all the cases, S. cerevisiae took over the process in the middle and final stages of fermentation. The analysis of the S. cerevisiae strains showed that indigenous strains competed with commercial strains inoculated in other fermentation tanks of the cellar. The continuous use of commercial yeasts reduced the diversity and importance of the indigenous S. cerevisiae strains.     

Meiotic recombination at the ends of chromosomes in Saccharomyces cerevisiae

Meiotic reciprocal recombination (crossing over) was examined in the outermost 60-80 kb of almost all Saccharomyces cerevisiae chromosomes. These sequences included both repetitive gene-poor subtelomeric heterochromatin-like regions and their adjacent unique gene-rich euchromatin-like regions. Subtelomeric sequences underwent very little crossing over, exhibiting approximately two- to threefold fewer crossovers per kilobase of DNA than the genomic average. Surprisingly, the adjacent euchromatic regions underwent crossing over at twice the average genomic rate and contained at least nine new recombination "hot spots." These results prompted an analysis of existing genetic mapping data, which showed that meiotic reciprocal recombination rates were on average greater near chromosome ends exclusive of the subtelomeres. Thus, the distribution of crossovers in S. cerevisiae appears to resemble that found in several higher eukaryotes where the outermost chromosomal regions show increased crossing over.     

Effect of the use of commercial Saccharomyces strains in a newly established winery in Ronda (Málaga, Spain)

An ecological study of the yeasts present in a spontaneous and an inoculated fermentation in red wine was carried out in 2005 vintage in a winery located in the Denomination of Origin "Sierras de Málaga" (Málaga, southern of Spain). The winery operated by the first time with the 2003 vintage and since then, has used commercial yeast inocula to start alcoholic fermentation. Yeast isolates were identified by PCR-RFLP analysis of the 5.8S-ITS region from the ribosomal DNA and by mitochondrial DNA RFLP analysis. Except for non-Saccharomyces yeasts found in the fresh must before fermentation, all the isolates were found to be commercial Saccharomyces cerevisiae strains employed by the winery during the successive vintages; thus, no indigenous Saccharomyces yeasts were isolated during fermentation. The same four restriction patterns were found in non inoculated and inoculated vats, although with different frequencies. The use of commercial yeast starter in a new established winery seems to have prevented the development of a resident indigenous Saccharomyces flora.     

The Awa1 gene is required for the foam-forming phenotype and cell surface hydrophobicity of sake yeast

Sake, a traditional alcoholic beverage in Japan, is brewed with sake yeasts, which are classified as Saccharomyces cerevisiae. Almost all sake yeasts form a thick foam layer on sake mash during the fermentation process because of their cell surface hydrophobicity, which increases the cells' affinity for bubbles. To reduce the amount of foam, nonfoaming mutants were bred from foaming sake yeasts. Nonfoaming mutants have hydrophilic cell surfaces and no affinity for bubbles. We have cloned a gene from a foam-forming sake yeast that confers foaming ability to a nonfoaming mutant. This gene was named AWA1 and structures of the gene and its product were analyzed. The N- and C-terminal regions of Awa1p have the characteristic sequences of a glycosylphosphatidylinositol anchor protein. The entire protein is rich in serine and threonine residues and has a lot of repetitive sequences. These results suggest that Awa1p is localized in the cell wall. This was confirmed by immunofluorescence microscopy and Western blotting analysis using hemagglutinin-tagged Awa1p. Moreover, an awa1 disruptant of sake yeast was hydrophilic and showed a nonfoaming phenotype in sake mash. We conclude that Awa1p is a cell wall protein and is required for the foam-forming phenotype and the cell surface hydrophobicity of sake yeast.     

Heterogeneity in the ribosomal RNA genes of the yeast Yarrowia lipolytica; cloning and analysis of two size classes of repeats

Southern blotting of DNA from the ascomycetous yeast Yarrowia lipolytica revealed two major size classes of DNA units coding for rRNAs, which differ in length by about 1000 bp. We have cloned an rDNA unit of each size class. R-looping experiments revealed that the rRNA genes of both units are uninterrupted; subsequent heteroduplex analysis showed that the size difference both units is located within the nontranscribed spacer. Sequence analysis revealed that a major part of these spacers consists of a complex pattern of repetitions in periodicities of up to about 150 bp and that the difference between both rDNA units are located mainly in this repetitive region. Apart from different lengths of the repetitive regions, both rDNA units also reveal extended microheterogeneity within their homologous parts. Furthermore, no gene for 5S rRNA was observed in the spacer region. Therefore, the organization of the spacer of Yarrowia rDNA is clearly different from that of Saccharomyces cerevisiae.     

Phenotypic and genotypic diversity of wine yeasts used for acidic musts

The aim of this study was to examine the physiological and genetic stability of the industrial wine yeasts Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum under acidic stress during fermentation. The yeasts were sub-cultured in aerobic or fermentative conditions in media with or without l-malic acid. Changes in the biochemical profiles, karyotypes, and mitochondrial DNA profiles were assessed after minimum 50 generations. All yeast segregates showed a tendency to increase the range of compounds used as sole carbon sources. The wild strains and their segregates were aneuploidal or diploidal. One of the four strains of S. cerevisiae did not reveal any changes in the electrophoretic profiles of chromosomal and mitochondrial DNA, irrespective of culture conditions. The extent of genomic changes in the other yeasts was strain-dependent. In the karyotypes of the segregates, the loss of up to 2 and the appearance up to 3 bands was noted. The changes in their mtDNA patterns were much broader, reaching 5 missing and 10 additional bands. The only exception was S. bayanus var. uvarum Y.00779, characterized by significantly greater genome plasticity only under fermentative stress. Changes in karyotypes and mtDNA profiles prove that fermentative stress is the main driving force of the adaptive evolution of the yeasts. l-malic acid does not influence the extent of genomic changes and the resistance of wine yeasts exhibiting increased demalication activity to acidic stress is rather related to their ability to decompose this acid. The phenotypic changes in segregates, which were found even in yeasts that did not reveal deviations in their DNA profiles, show that phenotypic characterization may be misleading in wine yeast identification. Because of yeast gross genomic diversity, karyotyping even though it does not seem to be a good discriminative tool, can be useful in determining the stability of wine yeasts. Restriction analysis of mitochondrial DNA appears to be a more sensitive method allowing for an early detection of genotypic changes in yeasts. Thus, if both of these methods are applied, it is possible to conduct the quick routine assessment of wine yeast stability in pure culture collections depositing industrial strains.     

Diversity in organization and the origin of gene orders in the mitochondrial DNA molecules of the genus Saccharomyces

Sequencing of the Saccharomyces cerevisiae nuclear and mitochondrial genomes provided a new background for studies on the evolution of the genomes. In this study, mitochondrial genomes of a number of Saccharomyces yeasts were mapped by restriction enzyme analysis, the orders of the genes were determined, and two of the genes were sequenced. The genome organization, i.e., the size, presence of intergenic sequences, and gene order, as well as polymorphism within the coding regions, indicate that Saccharomyces mtDNA molecules are dynamic structures and have undergone numerous changes during their evolution. Since the separation and sexual isolation of different yeast lineages, the coding parts have been accumulating point mutations, presumably in a linear manner with the passage of time. However, the accumulation of other changes may not have been a simple function of time. Larger mtDNA molecules belonging to Saccharomyces sensu stricto yeasts have acquired extensive intergenic sequences, including guanosine-cytosine-rich clusters, and apparently have rearranged the gene order at higher rates than smaller mtDNAs belonging to the Saccharomyces sensu lato yeasts. While within the sensu stricto group transposition has been a predominant mechanism for the creation of novel gene orders, the sensu lato yeasts could have used both transposition- and inversion-based mechanisms.     

Mitochondrial DNA loss caused by ethanol in Saccharomyces flor yeasts.

Saccharomyces flor yeasts proliferate at the surface of sherry wine, which contains over 15% (vol) ethanol. Since ethanol is a powerful inducer of respiration-deficient mutants, this alcohol has been proposed to be the source of the high diversity found in the mitochondrial genomes of flor yeasts and other wine yeasts. Southern blot analysis suggests that mitochondrial DNA (mtDNA) polymorphic changes are due to minor lesions in the mitochondrial genome. As determined in this work by pulsed-field gel electrophoresis, restriction analysis, and Southern blot analysis, ethanol-induced petite mutants completely lack mtDNA (rho zero). Ethanol-induced changes in the mitochondrial genome that could explain the observed mtDNA polymorphism in flor yeasts were not found. The transfer of two different mtDNA variants from flor yeasts to a laboratory strain conferred in both cases an increase in ethanol tolerance in the recipient strain, suggesting that mtDNAs are probably subjected to positive selection pressure concerning their ability to confer ethanol tolerance.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe. The cytochrome b gene has an intron closely related to the first two introns in the Saccharomyces cerevisiae cox1 gene

The DNA sequence of the cob region of the Schizosaccharomyces pombe mitochondrial DNA has been determined. The cytochrome b structural gene is interrupted by an intron of 2526 base-pairs, which has an open reading frame of 2421 base-pairs in phase with the upstream exon. The position of the intron differs from those found in the cob genes of Saccharomyces cerevisiae, Aspergillus nidulans or Neurospora crassa. The Sch. pombe cob intron has the potential of assuming an RNA secondary structure almost identical to that proposed for the first two cox1 introns (group II) in S. cerevisiae and the p1-cox1 intron in Podospora anserina. It has most of the consensus nucleotides in the central core structure described for this group of introns and its comparison with other group II introns allows the identification of an additional conserved nucleotide stretch. A comparison of the predicted protein sequences of group II intronic coding regions reveals three highly conserved blocks showing pairwise amino acid identities of 34 to 53%. These regions comprise over 50% of the coding length of the intron but do not include the 5' region, which has strong secondary structural features. In addition to the potential intron folding, long helical structures involving repetitive sequences can be formed in the flanking cob exon regions. A comparison of the Sch. pombe cytochrome b sequence with those available from other organisms indicates that Sch. pombe is evolutionarily distant from both budding yeasts and filamentous fungi. As was seen for the Sch. pombe cox1 gene (Lang, 1984), the cob exons are translated using the universal genetic code and this distinguishes Sch. pombe mitochondria from all other fungal and animal mitochondrial systems.     

Molecular typing of wine yeast strains Saccharomyces bayanus var. uvarum using microsatellite markers

The Saccharomyces bayanus var. uvarum yeasts are associated with spontaneous fermentation of must. Some strains were shown to be enological yeasts of interest in different winemaking processes. The molecular typing of S. bayanus var. uvarum at the strain level has become significant for wine microbiologists. Four microsatellite loci were defined from the exploration of genomic DNA sequence of S. bayanus var. uvarum. The 40 strains studied were homozygote for the locus considered. The discriminating capacity of the microsatellite method was found to be equal to that of karyotypes analysis. Links between 37 indigenous strains with the same geographic origin could be established through the analysis of microsatellite patterns. The analysis of microsatellite polymorphism is a reliable method for wine S. bayanus var. uvarum strains and their hybrids with Saccharomyces cerevisiae identification in taxonomic, ecological studies and winemaking applications.     

Sequence-specific biosensors report drug-induced changes in epigenetic silencing in living cells

Treatment with demethylating drugs can induce demethylation and reactivation of abnormally silenced tumor suppressor genes in cancer cells, but it can also induce potentially deleterious loss of methylation of repetitive elements. To enable the observation of unwanted drug effects related to loss of methylation of repetitive DNA, we have developed a novel biosensor capable of reporting changes in DNA accessibility via luminescence, in living cells. The biosensor design comprises two independent modules, each with a polydactyl zinc finger domain fused to a half intein and to a split-luciferase domain that can be joined by conditional protein splicing after binding to adjacent DNA targets. We show that an artificial zinc finger design specifically targeting DNA sequences near the promoter region of the L1PA2 subfamily of Line-1 retroelements is able to generate luminescent signals, reporting loss of epigenetic silencing and increased DNA accessibility of retroelements in human cells treated with the demethylating drugs decitabine or 5-azacytidine.     

Distribution of L-azetidine-2-carboxylate N-acetyltransferase in yeast

The budding yeast Saccharomyces cerevisiae Sigma1278b contains the MPR1 gene encoding N-acetyltransferase, which detoxifies the L-proline analog L-azetidine-2-carboxylate (AZC). Of 131 yeasts tested, AZC acetyltransferase activity was detected in 17 strains of 41 strains that showed AZC resistance. Degenerate-PCR analysis revealed that two strains, i.e., Candida saitoana AKU4533 and Wickerhamia fluorescens AKU4722, contained a DNA fragment highly homologous to MPR1. This indicates that AZC acetyltransferases are widely distributed in yeasts.     

Evidence for multiple interspecific hybridization in Saccharomyces sensu stricto species

Fluorescent amplified fragment length polymorphism analysis demonstrates a high level of gene exchange between Saccharomyces sensu stricto species, with some strains having undergone multiple interspecific hybridization events with subsequent changes in genome complexity. Two lager strains were shown to be hybrids between Saccharomyces cerevisiae and the alloploid species Saccharomyces pastorianus. The genome structure of CBS 380(T), the type strain of Saccharomyces bayanus, is also consistent with S. pastorianus gene transfer. The results indicate that the cider yeast, CID1, possesses nuclear DNA from three separate species. Mating experiments show that there are no barriers to interspecific conjugation of haploid cells. Furthermore, the allopolyploid strains were able to undergo further hybridizations with other Saccharomyces sensu stricto yeasts. These results demonstrate that introgression between the Saccharomyces sensu stricto species is likely.     

AtMCM10 promotes DNA replication-coupled nucleosome assembly in Arabidopsis

Minichromosome Maintenance protein 10 (MCM10) is essential for DNA replication initiation and DNA elongation in yeasts and animals. Although the functions of MCM10 in DNA replication and repair have been well documented, the detailed mechanisms for MCM10 in these processes are not well known. Here, we identified AtMCM10 gene through a forward genetic screening for releasing a silenced marker gene. Although plant MCM10 possesses a similar crystal structure as animal MCM10, AtMCM10 is not essential for plant growth or development in Arabidopsis. AtMCM10 can directly bind to histone H3-H4 and promotes nucleosome assembly in vitro. The nucleosome density is decreased in Atmcm10, and most of the nucleosome density decreased regions in Atmcm10 are also regulated by newly synthesized histone chaperone Chromatin Assembly Factor-1 (CAF-1). Loss of both AtMCM10 and CAF-1 is embryo lethal, indicating that AtMCM10 and CAF-1 are indispensable for replication-coupled nucleosome assembly. AtMCM10 interacts with both new and parental histones. Atmcm10 mutants have lower H3.1 abundance and reduced H3K27me1/3 levels with releasing some silenced transposons. We propose that AtMCM10 deposits new and parental histones during nucleosome assembly, maintaining proper epigenetic modifications and genome stability during DNA replication.