José García-Martínez

CYGD: the Comprehensive Yeast Genome Database

The Comprehensive Yeast Genome Database (CYGD) compiles a comprehensive data resource for information on the cellular functions of the yeast Saccharomyces cerevisiae and related species, chosen as the best understood model organism for eukaryotes. The database serves as a common resource generated by a European consortium, going beyond the provision of sequence information and functional annotations on individual genes and proteins. In addition, it provides information on the physical and functional interactions among proteins as well as other genetic elements. These cellular networks include metabolic and regulatory pathways, signal transduction and transport processes as well as co-regulated gene clusters. As more yeast genomes are published, their annotation becomes greatly facilitated using S.cerevisiae as a reference. CYGD provides a way of exploring related genomes with the aid of the S.cerevisiae genome as a backbone and SIMAP, the Similarity Matrix of Proteins. The comprehensive resource is available under http://mips.gsf.de/genre/proj/yeast/.

Chimeric genomes of natural hybrids of Saccharomyces cerevisiae and Saccharomyces kudriavzevii.

ABSTRACT Recently, a new type of hybrid resulting from the hybridization between Saccharomyces cerevisiae and Saccharomyces kudriavzevii was described. These strains exhibit physiological properties of potential biotechnological interest. A preliminary characterization of these hybrids showed a trend to reduce the S. kudriavzevii fraction of the hybrid genome. We characterized the genomic constitution of several wine S. cerevisiae × S. kudriavzevii strains by using a combined approach based on the restriction fragment length polymorphism analysis of gene regions, comparative genome hybridizations with S. cerevisiae DNA arrays, ploidy analysis, and gene dose determination by quantitative real-time PCR. The high similarity in the genome structures of the S. cerevisiae × S. kudriavzevii hybrids under study indicates that they originated from a single hybridization event. After hybridization, the hybrid genome underwent extensive chromosomal rearrangements, including chromosome losses and the generation of chimeric chromosomes by the nonreciprocal recombination between homeologous chromosomes. These nonreciprocal recombinations between homeologous chromosomes occurred in highly conserved regions, such as Ty long terminal repeats (LTRs), rRNA regions, and conserved protein-coding genes. This study supports the hypothesis that chimeric chromosomes may have been generated by a mechanism similar to the recombination-mediated chromosome loss acting during meiosis in Saccharomyces hybrids. As a result of the selective processes acting during fermentation, hybrid genomes maintained the S. cerevisiae genome but reduced the S. kudriavzevii fraction.

Comprehensive Transcriptional Analysis of the Oxidative Response in Yeast

The oxidative stress response in Saccharomyces cerevisiae has been analyzed by parallel determination of mRNA levels and transcription rates for the entire genome. A mathematical algorithm has been adapted for a dynamic situation such as the response to stress, to calculate theoretical mRNA decay rates from the experimental data. Yeast genes have been grouped into 25 clusters according to mRNA level and transcription rate kinetics, and average mRNA decay rates have been calculated for each cluster. In most of the genes, changes in one or both experimentally determined parameters occur during the stress response. 24% of the genes are transcriptionally induced without an increase in mRNA levels. The lack of parallelism between the evolution of the mRNA amount and transcription rate predicts changes in mRNA stability during stress. Genes for ribosomal proteins and rRNA processing enzymes are abundant among those whose mRNAs are predicted to destabilize. The number of genes whose mRNAs are predicted to stabilize is lower, although some protein folding or proteasomal genes are among the latter. We have confirmed the mathematical predictions for several genes pertaining to different clusters by experimentally determining mRNA decay rates using the regulatable tetO promoter in transcriptional expression conditions not affected by the oxidative stress. This study indicates that the oxidative stress response in yeast cells is not only conditioned by gene transcription but also by the mRNA decay dynamics and that this complex response may be particularly relevant to explain the temporary down-regulation of protein synthesis occurring during stress.

Transcriptional Response of Saccharomyces cerevisiae to Different Nitrogen Concentrations during Alcoholic Fermentation

ABSTRACT Gene expression profiles of a wine strain of Saccharomyces cerevisiae PYCC4072 were monitored during alcoholic fermentations with three different nitrogen supplies: (i) control fermentation (with enough nitrogen to complete sugar fermentation), (ii) nitrogen-limiting fermentation, and (iii) the addition of nitrogen to the nitrogen-limiting fermentation (refed fermentation). Approximately 70% of the yeast transcriptome was altered in at least one of the fermentation stages studied, revealing the continuous adjustment of yeast cells to stressful conditions. Nitrogen concentration had a decisive effect on gene expression during fermentation. The largest changes in transcription profiles were observed when the early time points of the N-limiting and control fermentations were compared. Despite the high levels of glucose present in the media, the early responses of yeast cells to low nitrogen were characterized by the induction of genes involved in oxidative glucose metabolism, including a significant number of mitochondrial associated genes resembling the yeast cell response to glucose starvation. As the N-limiting fermentation progressed, a general downregulation of genes associated with catabolism was observed. Surprisingly, genes encoding ribosomal proteins and involved in ribosome biogenesis showed a slight increase during N starvation; besides, genes that comprise the RiBi regulon behaved distinctively under the different experimental conditions. Here, for the first time, the global response of nitrogen-depleted cells to nitrogen addition under enological conditions is described. An important gene expression reprogramming occurred after nitrogen addition; this reprogramming affected genes involved in glycolysis, thiamine metabolism, and energy pathways, which enabled the yeast strain to overcome the previous nitrogen starvation stress and restart alcoholic fermentation.

DNA chips for yeast biotechnology. The case of wine yeasts

The yeast Saccharomyces cerevisiae is one of the most popular model organisms. It was the first eukaryote whose genome was sequenced. Since then many functional analysis projects have tried to find the function of many genes and to understand its metabolism in a holistic way. Apart from basic science this microorganism is of great interest in several biotechnology processes, such as winemaking. Only global studies of the cell as a whole can help us to understand many of the technical problems facing winemaking. DNA chip technology is one of the most promising tools for the analysis of cell physiology. Yeast has been the model organism for the development of this technique. Many of the studies can be applied to improve our knowledge of wine strains. Nevertheless wine strains are quite different in some aspects from the laboratory reference strains so a particular study of wine strains and especially during the winemaking process is needed. During the past two years some groups have started this study and the first results have been published. We review here the current state of the knowledge of wine yeast and the capacity of DNA chip technology for its improvement.

Heat Shock Response in Yeast Involves Changes in Both Transcription Rates and mRNA Stabilities

We have analyzed the heat stress response in the yeast Saccharomyces cerevisiae by determining mRNA levels and transcription rates for the whole transcriptome after a shift from 25°C to 37°C. Using an established mathematical algorithm, theoretical mRNA decay rates have also been calculated from the experimental data. We have verified the mathematical predictions for selected genes by determining their mRNA decay rates at different times during heat stress response using the regulatable tetO promoter. This study indicates that the yeast response to heat shock is not only due to changes in transcription rates, but also to changes in the mRNA stabilities. mRNA stability is affected in 62% of the yeast genes and it is particularly important in shaping the mRNA profile of the genes belonging to the environmental stress response. In most cases, changes in transcription rates and mRNA stabilities are homodirectional for both parameters, although some interesting cases of antagonist behavior are found. The statistical analysis of gene targets and sequence motifs within the clusters of genes with similar behaviors shows that both transcriptional and post-transcriptional regulons apparently contribute to the general heat stress response by means of transcriptional factors and RNA binding proteins.

Saccharomyces cerevisiae Glutaredoxin 5-deficient Cells Subjected to Continuous Oxidizing Conditions Are Affected in the Expression of Specific Sets of Genes

The Saccharomyces cerevisiae GRX5 gene codes for a mitochondrial glutaredoxin involved in the synthesis of iron/sulfur clusters. Its absence prevents respiratory growth and causes the accumulation of iron inside cells and constitutive oxidation of proteins. Null Δgrx5 mutants were used as an example of continuously oxidized cells, as opposed to situations in which oxidative stress is instantaneously caused by addition of external oxidants. Whole transcriptome analysis was carried out in the mutant cells. The set of genes whose expression was affected by the absence of Grx5 does not significantly overlap with the set of genes affected in respiratory petite mutants. Many Aft1-dependent genes involved in iron utilization that are up-regulated in a frataxin mutant were also up-regulated in the absence of Grx5. BIO5 is another Aft1-dependent gene induced both upon iron deprivation and in Δgrx5 cells; this links iron and biotin metabolism. Other genes are specifically affected under the oxidative conditions generated by the grx5 mutation. One of these is MLP1, which codes for a homologue of the Slt2 kinase. Cells lacking MLP1 and GRX5 are hypersensitive to oxidative stress caused by external agents and exhibit increased protein oxidation in relation to single mutants. This in turn points to a role for Mlp1 in protection against oxidative stress. The genes of the Hap4 regulon, which are involved in respiratory metabolism, are down-regulated in Δgrx5 cells. This effect is suppressed by HAP4 overexpression. Inhibition of respiratory metabolism during continuous moderately oxidative conditions could be a protective response by the cell.

Regulon-specific control of transcription elongation across the yeast genome.

Transcription elongation by RNA polymerase II was often considered an invariant non-regulated process. However, genome-wide studies have shown that transcriptional pausing during elongation is a frequent phenomenon in tightly-regulated metazoan genes. Using a combination of ChIP-on-chip and genomic run-on approaches, we found that the proportion of transcriptionally active RNA polymerase II (active versus total) present throughout the yeast genome is characteristic of some functional gene classes, like those related to ribosomes and mitochondria. This proportion also responds to regulatory stimuli mediated by protein kinase A and, in relation to cytosolic ribosomal-protein genes, it is mediated by the silencing domain of Rap1. We found that this inactive form of RNA polymerase II, which accumulates along the full length of ribosomal protein genes, is phosphorylated in the Ser5 residue of the CTD, but is hypophosphorylated in Ser2. Using the same experimental approach, we show that the in vivo–depletion of FACT, a chromatin-related elongation factor, also produces a regulon-specific effect on the expression of the yeast genome. This work demonstrates that the regulation of transcription elongation is a widespread, gene class–dependent phenomenon that also affects housekeeping genes.

The Sas3p and Gcn5p histone acetyltransferases are recruited to similar genes

BackgroundSpecific histone modifications can perform several cellular functions, for example, as signals to recruit trans-acting factors and as modulators of chromatin structure. Acetylation of Lys14 of histone H3 is the main target of many histone acetyltransferases in vitro and may play a central role in the stability of the nucleosome. This study is focused on the genome-wide binding of Saccharomyces cerevisiae histone acetyltransferases that are specific for Lys14 of histone H3.ResultsWe have used a variation of the genome-wide location analysis method, based on a macroarray platform, to identify binding sites of yeast histone acetyltransferase catalytic subunits and to correlate their positions with acetylation of Lys14 of histone H3. Our results revealed that the histone acetyltransferases Sas3p and Gcn5p are recruited to a pool of intensely transcribed genes and that there is considerable overlap between the two cohorts of Sas3p and Gcn5p bound gene pools. We also demonstrate a positive correlation between binding sites of both proteins and the acetylation state of Lys14 of histone H3. Finally, a positive correlation between the decrease of H3 Lys14 acetylation in a GCN5 deleted strain and the Gcn5p genome occupancy is shown.ConclusionOur data support a model in which both Gcn5p and Sas3p act as general activators of an overlapping pool of intensely transcribed genes. Since both proteins preferentially acetylate Lys14 of histone H3, our data support the hypothesis that acetylation of this specific residue facilitates the action of the transcriptional apparatus.

A method for genome-wide analysis of DNA helical tension by means of psoralen–DNA photobinding

The helical tension of chromosomal DNA is one of the epigenetic landmarks most difficult to examine experimentally. The occurrence of DNA crosslinks mediated by psoralen photobinding (PB) stands as the only suitable probe for assessing this problem. PB is affected by chromatin structure when is done to saturation; but it is mainly determined by DNA helical tension when it is done to very low hit conditions. Hence, we developed a method for genome-wide analysis of DNA helical tension based on PB. We adjusted in vitro PB conditions that discern DNA helical tension and applied them to Saccharomyces cerevisiae cells. We selected the in vivo cross-linked DNA sequences and identified them on DNA arrays. The entire procedure was robust. Comparison of PB obtained in vivo with that obtained in vitro with naked DNA revealed that numerous chromosomal regions had deviated PB values. Similar analyses in yeast topoisomerase mutants uncovered further PB alterations across specific chromosomal domains. These results suggest that distinct chromosome compartments might confine different levels of DNA helical tension in yeast. Genome-wide analysis of psoralen–DNA PB can be, therefore, a useful approach to uncover a trait of the chromosome architecture not amenable to other techniques.