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Background

Fusarium graminearum (Fg), a major fungal pathogen of cultivated cereals, is responsible for billions of dollars in agriculture losses. There is a growing interest in understanding the transcriptional regulation of this organism, especially the regulation of genes underlying its pathogenicity. The generation of whole genome sequence assemblies for Fg and three closely related Fusarium species provides a unique opportunity for such a study.

Results

Applying comparative genomics approaches, we developed a computational pipeline to systematically discover evolutionarily conserved regulatory motifs in the promoter, downstream and the intronic regions of Fg genes, based on the multiple alignments of sequenced Fusarium genomes. Using this method, we discovered 73 candidate regulatory motifs in the promoter regions. Nearly 30% of these motifs are highly enriched in promoter regions of Fg genes that are associated with a specific functional category. Through comparison to Saccharomyces cerevisiae (Sc) and Schizosaccharomyces pombe (Sp), we observed conservation of transcription factors (TFs), their binding sites and the target genes regulated by these TFs related to pathways known to respond to stress conditions or phosphate metabolism. In addition, this study revealed 69 and 39 conserved motifs in the downstream regions and the intronic regions, respectively, of Fg genes. The top intronic motif is the splice donor site. For the downstream regions, we noticed an intriguing absence of the mammalian and Sc poly-adenylation signals among the list of conserved motifs.

Conclusion

This study provides the first comprehensive list of candidate regulatory motifs in Fg, and underscores the power of comparative genomics in revealing functional elements among related genomes. The conservation of regulatory pathways among the Fusarium genomes and the two yeast species reveals their functional significance, and provides new insights in their evolutionary importance among Ascomycete fungi.

Genomic clones capable of complementing a previously isolated arginine auxotrophic mutant strain of the filamentous yeast Trichosporon cutaneum DSM 70698 have been identified by DNA-mediated transformation, and a complementing 4,082-bp subfragment was sequenced. This analysis revealed an intact gene (arg4) showing a high degree of homology with the Saccharomyces cerevisiae CPA2 gene encoding the large subunit of carbamoyl-phosphate synthetase (CPS-A). The inferred amino acid sequence of the T. cutaneum argA-encoded protein contains 1,168 residues showing 62% identity with the sequence of the S. cerevisiae CPA2 protein, and the comparison of the two sequences uncovered a putative intron sequence of 81 nucleotides close to the 5' end of the coding region of the T. cutaneum argA gene. The presence of this intron was confirmed by nuclease protection studies and by direct DNA sequence analysis of a cDNA fragment which had been obtained by PCR amplification. The T. cutaneum intron shares the general characteristics of introns found in yeasts and filamentous fungi. A major transcript of around 4 kb was found in Northern (RNA) blots. The T. cutaneum argA coding region was expressed in Escherichia coli under the control of the regulatable tac promoter. A roughly 130-kDa protein which was found to cross-react with an anti-rat CPS antibody in Western blots (immunoblots) was observed. Two putative ATP-binding domains were identified, one in the amino-terminal half of the argA-encoded protein and the other in the carboxy-terminal half. These domains are highly conserved among the known CPS-A sequences from S. cerevisiae, E. coli, and the rat. From these results we conclude that the T. cutaneum argA gene encodes the large subunit of CPS. This is the first gene to be identified and analyzed in the T. cutaneum DSM 70698 strain.

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Transposable genetic elements are ubiquitous, yet their presence or absence at any given position within a genome can vary between individual cells, tissues, or strains. Transposable elements have profound impacts on host genomes by altering gene expression, assisting in genomic rearrangements, causing insertional mutations, and serving as sources of phenotypic variation. Characterizing a genome's full complement of transposons requires whole genome sequencing, precluding simple studies of the impact of transposition on interindividual variation. Here, we describe a global mapping approach for identifying transposon locations in any genome, using a combination of transposon-specific DNA extraction and microarray-based comparative hybridization analysis. We use this approach to map the repertoire of endogenous transposons in different laboratory strains of Saccharomyces cerevisiae and demonstrate that transposons are a source of extensive genomic variation. We also apply this method to mapping bacterial transposon insertion sites in a yeast genomic library. This unique whole genome view of transposon location will facilitate our exploration of transposon dynamics, as well as defining bases for individual differences and adaptive potential.

Synopsis

Transposons, or mobile DNA sequences—first described by Barbara McClintock—are interesting and important residents of all genomes. They are involved in gene creation and regulation, chromosome evolution, and generation of mutations, events that can occur on hugely varying time scales, from millions of years to mere days in the lab. Some transposons have even been “tamed” by geneticists for use as tools for marking genes and making mutations. In yeast, genome sequencing has given us a snapshot of transposons present in one strain at one particular time. The authors developed a method to easily, accurately, and globally track transposons in order to study how their locations change in different strains or during an experiment. The method involves finding pieces of DNA that contain the ends of transposons along with neighboring DNA and attaching these segments to magnetic beads. A magnet is then used to separate the selected DNAs away from the rest of the genome. The transposon-associated DNA is labeled with dyes and applied to a microarray, a glass slide with over 40,000 unique sequence features of yeast DNA attached. Each feature that lights up with the dye marks a transposon location. This new technique allows investigators to easily identify specific strains, to accurately monitor mobile portions of the genome, and to determine the role of transposons in phenotypic differences.

Human intervention has subjected the yeast Saccharomyces cerevisiae to multiple rounds of independent domestication and thousands of generations of artificial selection. As a result, this species comprises a genetically diverse collection of natural isolates as well as domesticated strains that are used in specific industrial applications. However the scope of genetic diversity that was captured during the domesticated evolution of the industrial representatives of this important organism remains to be determined. To begin to address this, we have produced whole-genome assemblies of six commercial strains of S. cerevisiae (four wine and two brewing strains). These represent the first genome assemblies produced from S. cerevisiae strains in their industrially-used forms and the first high-quality assemblies for S. cerevisiae strains used in brewing. By comparing these sequences to six existing high-coverage S. cerevisiae genome assemblies, clear signatures were found that defined each industrial class of yeast. This genetic variation was comprised of both single nucleotide polymorphisms and large-scale insertions and deletions, with the latter often being associated with ORF heterogeneity between strains. This included the discovery of more than twenty probable genes that had not been identified previously in the S. cerevisiae genome. Comparison of this large number of S. cerevisiae strains also enabled the characterization of a cluster of five ORFs that have integrated into the genomes of the wine and bioethanol strains on multiple occasions and at diverse genomic locations via what appears to involve the resolution of a circular DNA intermediate. This work suggests that, despite the scrutiny that has been directed at the yeast genome, there remains a significant reservoir of ORFs and novel modes of genetic transmission that may have significant phenotypic impact in this important model and industrial species.

Author Summary

The yeast S. cerevisiae has been associated with human activity for thousands of years in industries such as baking, brewing, and winemaking. During this time, humans have effectively domesticated this microorganism, with different industries selecting for specific desirable phenotypic traits. This has resulted in the species S. cerevisiae comprising a genetically diverse collection of individual strains that are often suited to very specific roles (e.g. wine strains produce wine but not beer and vice versa). In order to understand the genetic differences that underpin these diverse industrial characteristics, we have sequenced the genomes of six industrial strains of S. cerevisiae that comprise four strains used in commercial wine production and two strains used in beer brewing. By comparing these genome sequences to existing S. cerevisiae genome sequences from laboratory, pathogenic, bioethanol, and “natural” isolates, we were able to identify numerous genetic differences among these strains including the presence of novel open reading frames and genomic rearrangements, which may provide the basis for the phenotypic differences observed among these strains.

The first completed eukaryotic genome sequence was that of the yeast Saccharomyces cerevisiae, and the Saccharomyces Genome Database (SGD; http://www.yeastgenome.org/) is the original model organism database. SGD remains the authoritative community resource for the S. cerevisiae reference genome sequence and its annotation, and continues to provide comprehensive biological information correlated with S. cerevisiae genes and their products. A diverse set of yeast strains have been sequenced to explore commercial and laboratory applications, and a brief history of those strains is provided. The publication of these new genomes has motivated the creation of new tools, and SGD will annotate and provide comparative analyses of these sequences, correlating changes with variations in strain phenotypes and protein function. We are entering a new era at SGD, as we incorporate these new sequences and make them accessible to the scientific community, all in an effort to continue in our mission of educating researchers and facilitating discovery.

Database URL: http://www.yeastgenome.org/

Paired end mapping of chromosomal fragments has been used in human cells to identify numerous structural variations in chromosomes of individuals and of cancer cell lines; however, the molecular, biological and bioinformatics methods for this technology are still in development. Here, we present a parallel bioinformatics approach to analyze chromosomal paired-end tag (ChromPET) sequence data and demonstrate its application in identifying gene rearrangements in the model organism Saccharomyces cerevisiae. We detected several expected events, including a chromosomal rearrangement of the nonessential arm of chromosome V induced by selective pressure, rearrangements introduced during strain construction and gene conversion at the MAT locus. In addition, we discovered several unannotated Ty element insertions that are present in the reference yeast strain, but not in the reference genome sequence, suggesting a few revisions are necessary in the latter. These data demonstrate that application of the chromPET technique to a genetically tractable organism like yeast provides an easy screen for studying the mechanisms of chromosomal rearrangements during the propagation of a species.

Background

Environmental stresses and inhibitors encountered by Saccharomyces cerevisiae strains are the main limiting factors in bioethanol fermentation. Strains with different genetic backgrounds usually show diverse stress tolerance responses. An understanding of the mechanisms underlying these phenotypic diversities within S. cerevisiae populations could guide the construction of strains with desired traits.

Results

We explored the genetic characteristics of the bioethanol S. cerevisiae strain YJS329 and elucidated how genetic variations in its genome were correlated with specified traits compared to similar traits in the S288c-derived strain, BYZ1. Karyotypic electrophoresis combined with array-comparative genomic hybridization indicated that YJS329 was a diploid strain with a relatively constant genome as a result of the fewer Ty elements and lack of structural polymorphisms between homologous chromosomes that it contained. By comparing the sequence with the S288c genome, a total of 64,998 SNPs, 7,093 indels and 11 unique genes were identified in the genome of YJS329-derived haploid strain YJSH1 through whole-genome sequencing. Transcription comparison using RNA-Seq identified which of the differentially expressed genes were the main contributors to the phenotypic differences between YJS329 and BYZ1. By combining the results obtained from the genome sequences and the transcriptions, we predicted how the SNPs, indels and chromosomal copy number variations may affect the mRNA expression profiles and phenotypes of the yeast strains. Furthermore, some genetic breeding strategies to improve the adaptabilities of YJS329 were designed and experimentally verified.

Conclusions

Through comparative functional genomic analysis, we have provided some insights into the mechanisms underlying the specific traits of the bioenthanol strain YJS329. The work reported here has not only enriched the available genetic resources of yeast but has also indicated how functional genomic studies can be used to improve genetic breeding in yeast.

Background

Experimental evolution of microbial populations provides a unique opportunity to study evolutionary adaptation in response to controlled selective pressures. However, until recently it has been difficult to identify the precise genetic changes underlying adaptation at a genome-wide scale. New DNA sequencing technologies now allow the genome of parental and evolved strains of microorganisms to be rapidly determined.

Results

We sequenced >93.5% of the genome of a laboratory-evolved strain of the yeast Saccharomyces cerevisiae and its ancestor at >28× depth. Both single nucleotide polymorphisms and copy number amplifications were found, with specific gains over array-based methodologies previously used to analyze these genomes. Applying a segmentation algorithm to quantify structural changes, we determined the approximate genomic boundaries of a 5× gene amplification. These boundaries guided the recovery of breakpoint sequences, which provide insights into the nature of a complex genomic rearrangement.

Conclusions

This study suggests that whole-genome sequencing can provide a rapid approach to uncover the genetic basis of evolutionary adaptations, with further applications in the study of laboratory selections and mutagenesis screens. In addition, we show how single-end, short read sequencing data can provide detailed information about structural rearrangements, and generate predictions about the genomic features and processes that underlie genome plasticity.

Saccharomyces bayanus is a yeast species described as one of the two parents of the hybrid brewing yeast S. pastorianus. Strains CBS380T and NBRC1948 have been retained successively as pure-line representatives of S. bayanus. In the present study, sequence analyses confirmed and upgraded our previous finding: S. bayanus type strain CBS380T harbours a mosaic genome. The genome of strain NBRC1948 was also revealed to be mosaic. Both genomes were characterized by amplification and sequencing of different markers, including genes involved in maltotriose utilization or genes detected by array-CGH mapping. Sequence comparisons with public Saccharomyces spp. nucleotide sequences revealed that the CBS380T and NBRC1948 genomes are composed of: a predominant non-cerevisiae genetic background belonging to S. uvarum, a second unidentified species provisionally named S. lagerae, and several introgressed S. cerevisiae fragments. The largest cerevisiae-introgressed DNA common to both genomes totals 70kb in length and is distributed in three contigs, cA, cB and cC. These vary in terms of length and presence of MAL31 or MTY1 (maltotriose-transporter gene). In NBRC1948, two additional cerevisiae-contigs, cD and cE, totaling 12kb in length, as well as several smaller cerevisiae fragments were identified. All of these contigs were partially detected in the genomes of S. pastorianus lager strains CBS1503 (S. monacensis) and CBS1513 (S. carlsbergensis) explaining the noticeable common ability of S. bayanus and S. pastorianus to metabolize maltotriose. NBRC1948 was shown to be inter-fertile with S. uvarum CBS7001. The cross involving these two strains produced F1 segregants resembling the strains CBS380T or NRRLY-1551. This demonstrates that these S. bayanus strains were the offspring of a cross between S. uvarum and a strain similar to NBRC1948. Phylogenies established with selected cerevisiae and non-cerevisiae genes allowed us to decipher the complex hybridisation events linking S. lagerae/S. uvarum/S. cerevisiae with their hybrid species, S. bayanus/pastorianus.

Background

Understanding the adaptive changes that alter the function of proteins during evolution is an important question for biology and medicine. The increasing number of completely sequenced genomes from closely related organisms, as well as individuals within species, facilitates systematic detection of recent selection events by means of comparative genomics.

Results

We have used genome-wide strain-specific single nucleotide polymorphism data from 64 strains of budding yeast (Saccharomyces cerevisiae or Saccharomyces paradoxus) to determine whether adaptive positive selection is correlated with protein regions showing propensity for different classes of structure conformation. Data from phylogenetic and population genetic analysis of 3,746 gene alignments consistently shows a significantly higher degree of positive Darwinian selection in intrinsically disordered regions of proteins compared to regions of alpha helix, beta sheet or tertiary structure. Evidence of positive selection is significantly enriched in classes of proteins whose functions and molecular mechanisms can be coupled to adaptive processes and these classes tend to have a higher average content of intrinsically unstructured protein regions.

Conclusions

We suggest that intrinsically disordered protein regions may be important for the production and maintenance of genetic variation with adaptive potential and that they may thus be of central significance for the evolvability of the organism or cell in which they occur.

We discovered on the chromosome of Saccharomyces cerevisiae Σ1278b novel genes involved in l-proline analogue l-azetidine-2-carboxylic acid resistance which are not present in the standard laboratory strains. The 5.4 kb-DNA fragment was cloned from the genomic library of the l-azetidine-2-carboxylic acid-resistant mutant derived from a cross between S. cerevisiae strains S288C and Σ1278b. The nucleotide sequence of a 4.5-kb segment exhibited no identity with the sequence in the genome project involving strain S288C. Deletion analysis indicated that one open reading frame encoding a predicted protein of 229 amino acids is indispensable for l-azetidine-2-carboxylic acid resistance. The protein sequence was found to be a member of the N-acetyltransferase superfamily. Genomic Southern analysis and gene disruption showed that two copies of the novel gene with one amino acid change at position 85 required for l-azetidine-2-carboxylic acid resistance were present on chromosomes X and XIV of Σ1278b background strains. When this novel MPR1 or MPR2 gene (sigma 1278b gene for l-proline analogue resistance) was introduced into the other S. cerevisiae strains, all of the recombinants were resistant to l-azetidine-2-carboxylic acid, indicating that both MPR1 and MPR2 are expressed and have a global function in S. cerevisiae.

Variation in gene expression levels on a genomic scale has been detected among different strains, among closely related species, and within populations of genetically identical cells. What are the driving forces that lead to expression divergence in some genes and conserved expression in others? Here we employ flux balance analysis to address this question for metabolic genes. We consider the genome-scale metabolic model of Saccharomyces cerevisiae, and its entire space of optimal and near-optimal flux distributions. We show that this space reveals underlying evolutionary constraints on expression regulation, as well as on the conservation of the underlying gene sequences. Genes that have a high range of optimal flux levels tend to display divergent expression levels among different yeast strains and species. This suggests that gene regulation has diverged in those parts of the metabolic network that are less constrained. In addition, we show that genes that are active in a large fraction of the space of optimal solutions tend to have conserved sequences. This supports the possibility that there is less selective pressure to maintain genes that are relevant for only a small number of metabolic states.

Synopsis

The regulation of gene product activity allows cells to efficiently cope with various tasks under varying conditions. Given that, one may have expected that striving for efficiency would cause genetically similar cells to have similar regulation. However, in reality, high variations in gene expression levels are detected between different strains and even between genetically identical cells taken from the same culture. What are the driving forces that lead to expression divergence in some genes and conserved expression in others?

To address this question, the authors study the conservation of regulation in yeast metabolism, using a computational model. They find that genes coding for reactions whose flux rates are narrowly constrained by the cellular need to maximize growth rate tend to have strictly conserved regulation and expression. However, when a wide range of flux rates is compatible with high cellular growth rates, the corresponding regulation and expression patterns are free to diverge. Furthermore, enzymes that participate in a large number of alternative metabolic behaviors tend to be encoded by genes with a highly conserved sequence. Taken together, these findings support the hypothesis that maintaining large variability in the overall expression and metabolic repertoire of the cell is under marked evolutionary selection.

Identifying the nucleotides that cause gene expression variation is a critical step in dissecting the genetic basis of complex traits. Here, we focus on polymorphisms that are predicted to alter transcription factor binding sites (TFBSs) in the yeast, Saccharomyces cerevisiae. We assembled a confident set of transcription factor motifs using recent protein binding microarray and ChIP-chip data and used our collection of motifs to predict a comprehensive set of TFBSs across the S. cerevisiae genome. We used a population genomics analysis to show that our predictions are accurate and significantly improve on our previous annotation. Although predicting gene expression from sequence is thought to be difficult in general, we identified a subset of genes for which changes in predicted TFBSs correlate well with expression divergence between yeast strains. Our analysis thus demonstrates both the accuracy of our new TFBS predictions and the feasibility of using simple models of gene regulation to causally link differences in gene expression to variation at individual nucleotides.

Background

Genes are created by a variety of evolutionary processes, some of which generate duplicate copies of an entire gene, while others rearrange pre-existing genetic elements or co-opt previously non-coding sequence to create genes with 'novel' sequences. These novel genes are thought to contribute to distinct phenotypes that distinguish organisms. The creation, evolution, and function of duplicated genes are well-studied; however, the genesis and early evolution of novel genes are not well-characterized. We developed a computational approach to investigate these issues by integrating genome-wide comparative phylogenetic analysis with functional and interaction data derived from small-scale and high-throughput experiments.

Results

We examine the function and evolution of new genes in the yeast Saccharomyces cerevisiae. We observed significant differences in the functional attributes and interactions of genes created at different times and by different mechanisms. Novel genes are initially less integrated into cellular networks than duplicate genes, but they appear to gain functions and interactions more quickly than duplicates. Recently created duplicated genes show evidence of adapting existing functions to environmental changes, while young novel genes do not exhibit enrichment for any particular functions. Finally, we found a significant preference for genes to interact with other genes of similar age and origin.

Conclusions

Our results suggest a strong relationship between how and when genes are created and the roles they play in the cell. Overall, genes tend to become more integrated into the functional networks of the cell with time, but the dynamics of this process differ significantly between duplicate and novel genes.

The ability of cells to respond to environmental changes and adapt their metabolism enables cell survival under stressful conditions. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) is particularly well adapted to the harsh conditions of anaerobic wine fermentation. However, S. cerevisiae gene function has not been previously systematically interrogated under conditions of industrial fermentation. We performed a genome-wide study of essential and nonessential S. cerevisiae gene requirements during grape juice fermentation to identify deletion strains that are either depleted or enriched within the viable fermentative population. Genes that function in autophagy and ubiquitin-proteasome degradation are required for optimal survival during fermentation, whereas genes that function in ribosome assembly and peroxisome biogenesis impair fitness during fermentation. We also uncover fermentation phenotypes for 139 uncharacterized genes with no previously known cellular function. We demonstrate that autophagy is induced early in wine fermentation in a nitrogen-replete environment, suggesting that autophagy may be triggered by other forms of stress that arise during fermentation. These results provide insights into the complex fermentation process and suggest possible means for improvement of industrial fermentation strains.

The phenome is the complete set of phenotypes resulting from genetic variation in populations of an organism. Saturation of a phenome implies the identification and phenotypic description of mutations in all genes in an organism, potentially constrained to those encoding proteins. The human genome is believed to contain 20-25,000 protein coding genes, but only a small fraction of these have documented mutant phenotypes, thus the human phenome is far from complete. In model organisms, genetic saturation entails the identification of multiple mutant alleles of a gene or locus, allowing a consistent description of mutational phenotypes for that gene. Saturation of several model organisms has been attempted, usually by targeting annotated coding genes with insertional transposons (Drosophila melanogaster, Mus musculus) or by sequence directed deletion (Saccharomyces cerevisiae) or using libraries of antisense oligonucleotide probes injected directly into animals (Caenorhabditis elegans, Danio rerio). This paper reviews the general state of the human phenome, and discusses theoretical and practical considerations toward a saturation analysis in humans. Throughout, emphasis is placed on high penetrance genetic variation, of the kind typically asociated with monogenic versus complex traits.

In yeast, the chromatin remodeler Isw1 shifts nucleosomes from mid-coding, to more 5’ regions of genes and may regulate transcriptional elongation.

Background

The positions of nucleosomes along eukaryotic DNA are defined by the local DNA sequence and are further tuned by the activity of chromatin remodelers. While the genome-wide effect of most remodelers has not been described, recent studies in Saccharomyces cerevisiae have shown that Isw2 prevents ectopic expression of anti-sense and suppressed transcripts at gene ends.

Results

We examined the genome-wide function of the Isw2 homologue, Isw1, by mapping nucleosome positioning in S. cerevisiae and Saccharomyces paradoxus strains deleted of ISW1. We found that Isw1 functions primarily within coding regions of genes, consistent with its putative role in transcription elongation. Upon deletion of ISW1, mid-coding nucleosomes were shifted upstream (towards the 5' ends) in about half of the genes. Isw1-dependent shifts were correlated with trimethylation of H3K79 and were enriched at genes with internal cryptic initiation sites.

Conclusions

Our results suggest a division of labor between Isw1 and Isw2, whereby Isw2 maintains repressive chromatin structure at gene ends while Isw1 has a similar function at mid-coding regions. The differential specificity of the two remodelers may be specified through interactions with particular histone marks.

A novel cell cycle gene was identified by a computer search for genes partly homologous to known CDC genes, CDC6 of Saccharomyces cerevisiae and CDC18 of Schizosaccharomyces pombe, using the nucleotide sequence data base for S. cerevisiae produced by the Yeast Sequencing Project. The protein sequence coded by the cloned gene was found to be identical to that of purified ORC1 protein. Disruption of the gene and subsequent tetrad analysis revealed that the gene was essential for growth. The function of the gene product was analyzed by depleting the protein from the cell using a mutant haploid strain containing the disrupted ORC1 gene on the chromosome and a galactose-inducible gene coding for HA-tagged ORC1 protein on a single copy plasmid. The HA-tagged protein was expressed during growth in the presence of galactose but began to decrease rapidly upon depletion of galactose. Analysis of the cell cycle progression of the mutant cells by FACS after the removal of galactose from the medium, and microscope observations of cells and their nuclei revealed that the normal progression of 2N cells was immediately impeded as the ORC1 protein started to decrease. This was blocked completely in the cells that had progressed to the S phase under conditions deficient in ORC1 protein followed by cell death. Two-dimensional gel analysis of the replication intermediates after the galactose removal revealed that the depletion of ORC1 protein caused a decrease in the frequency of initiation of chromosomal replication, eventually resulting in the inhibition of replication as a whole. The function of the ORC1 protein in the cell cycle progression of S. cerevisiae is discussed in light of current information on ORC.

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Saccharomyces cerevisiae has been a powerful model for uncovering the landscape of binary gene interactions through whole-genome screening. Complex heterozygous interactions are potentially important to human genetic disease as loss-of-function alleles are common in human genomes. We have been using complex haploinsufficiency (CHI) screening with the actin gene to identify genes related to actin function and as a model to determine the prevalence of CHI interactions in eukaryotic genomes. Previous CHI screening between actin and null alleles for non-essential genes uncovered ∼240 deleterious CHI interactions. In this report, we have extended CHI screening to null alleles for essential genes by mating a query strain to sporulations of heterozygous knock-out strains. Using an act1Δ query, knock-outs of 60 essential genes were found to be CHI with actin. Enriched in this collection were functional categories found in the previous screen against non-essential genes, including genes involved in cytoskeleton function and chaperone complexes that fold actin and tubulin. Novel to this screen was the identification of genes for components of the TFIID transcription complex and for the proteasome. We investigated a potential role for the proteasome in regulating the actin cytoskeleton and found that the proteasome physically associates with actin filaments in vitro and that some conditional mutations in proteasome genes have gross defects in actin organization. Whole-genome screening with actin as a query has confirmed that CHI interactions are important phenotypic drivers. Furthermore, CHI screening is another genetic tool to uncover novel functional connections. Here we report a previously unappreciated role for the proteasome in affecting actin organization and function.

Author Summary

Individuals inherit two copies of each gene, one from each parent, and frequently the two copies are different from each other. Sometimes one copy is completely defective, but since there is one normal copy there may be no negative consequences. Our research is focused on understanding the consequence of inheriting one bad copy for two or more different genes. Geneticists refer to inheriting one bad copy of a gene as a haploinsufficiency and inheriting one bad copy of multiple genes as a complex haploinsufficiency. Using yeast as a model system, we have addressed, in a systematic way, what occurs if a cell inherits one bad copy of the gene called actin and one bad copy of one of the ∼1,000 essential genes in the yeast genome. We discovered that complex haploinsufficiency between actin and one of 60 different essential genes leads to reduced cell viability. These 60 genes are highly enriched for certain functional groups, including those involved in protein degradation and gene expression. It is expected that approaches such as these in model organisms will be applicable to understanding the potential for deleterious complex haploinsufficient interactions in the human genome.

A temperature-conditional mit- mutant of Saccharomyces cerevisiae has been characterized; the mutant strain h45 cannot grow at 36 degrees C on nonfermentable substrates yet appears to be normal at 28 degrees C. The mutation in strain h45 maps genetically to the oli1 region of the mitochondrial DNA (mtDNA) genome, and prevents the synthesis at 36 degrees C of the oli1 gene product, subunit 9 of the mitochondrial ATPase complex. Since the level of oli1 mRNA in mutant h45 is close to normal at 36 degrees C, it is concluded that there is a specific block in translation of this mRNA at the non-permissive temperature. DNA sequence analysis of mtDNA from strain h45 reveals an additional T residue inserted 88 bp upstream of the oli1 coding region, in the A,T-rich sequence that is transcribed into the 5'-untranslated region of the oli1 mRNA. Sequence data on two revertants show that one returns to wild-type parental (J69-1B) mtDNA sequence, whilst the other contains an inserted A residue adjacent to the T inserted in the original h45 mutant. The results are discussed in terms of the stability of folds in RNA upstream of putative ribosome-binding sites in mitochondrial mRNA, and the potential action of nuclear-coded proteins that might be activators of the translation of specific mitochondrial mRNAs in yeast mitochondria.

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Gene tagging facilitates systematic genomic and proteomic analyses but chromosomal tagging typically disrupts gene regulatory sequences. Here we describe a seamless gene tagging approach that preserves endogenous gene regulation and is potentially applicable in any species with efficient DNA double-strand break repair by homologous recombination. We implement seamless tagging in Saccharomyces cerevisiae and demonstrate its application for protein tagging while preserving simultaneously upstream and downstream gene regulatory elements. Seamless tagging is compatible with high-throughput strain construction using synthetic genetic arrays (SGA), enables functional analysis of transcription antisense to open reading frames and should facilitate systematic and minimally-invasive analysis of gene functions.

High-quality, well-annotated genome sequences and standardized laboratory strains fuel experimental and evolutionary research. We present improved genome sequences of three species of Saccharomyces sensu stricto yeasts: S. bayanus var. uvarum (CBS 7001), S. kudriavzevii (IFO 1802T and ZP 591), and S. mikatae (IFO 1815T), and describe their comparison to the genomes of S. cerevisiae and S. paradoxus. The new sequences, derived by assembling millions of short DNA sequence reads together with previously published Sanger shotgun reads, have vastly greater long-range continuity and far fewer gaps than the previously available genome sequences. New gene predictions defined a set of 5261 protein-coding orthologs across the five most commonly studied Saccharomyces yeasts, enabling a re-examination of the tempo and mode of yeast gene evolution and improved inferences of species-specific gains and losses. To facilitate experimental investigations, we generated genetically marked, stable haploid strains for all three of these Saccharomyces species. These nearly complete genome sequences and the collection of genetically marked strains provide a valuable toolset for comparative studies of gene function, metabolism, and evolution, and render Saccharomyces sensu stricto the most experimentally tractable model genus. These resources are freely available and accessible through www.SaccharomycesSensuStricto.org.

Background

Proteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray.

Results

Whole genome microarray analysis revealed that 63% of the coding sequences (CDSs) present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes), and the flagellar glycosylation island (FGI). These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5) has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI.

Conclusion

Proteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism outbreaks.

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.

Saccharomyces cerevisiae is one of the premier model systems for studying the genomics and evolution of transposable elements. The availability of the S. cerevisiae genome led to unprecedented insights into its five known transposable element families (the LTR retrotransposons Ty1-Ty5) in the years shortly after its completion. However, subsequent advances in bioinformatics tools for analysing transposable elements and the recent availability of genome sequences for multiple strains and species of yeast motivates new investigations into Ty evolution in S. cerevisiae. Here we provide a comprehensive phylogenetic and population genetic analysis of all Ty families in S. cerevisiae based on a systematic re-annotation of Ty elements in the S288c reference genome. We show that previous annotation efforts have underestimated the total copy number of Ty elements for all known families. In addition, we identify a new family of Ty3-like elements related to the S. paradoxus Ty3p which is composed entirely of degenerate solo LTRs. Phylogenetic analyses of LTR sequences identified three families with short-branch, recently active clades nested among long branch, inactive insertions (Ty1, Ty3, Ty4), one family with essentially all recently active elements (Ty2) and two families with only inactive elements (Ty3p and Ty5). Population genomic data from 38 additional strains of S. cerevisiae show that the majority of Ty insertions in the S288c reference genome are fixed in the species, with insertions in active clades being predominantly polymorphic and insertions in inactive clades being predominantly fixed. Finally, we use comparative genomic data to provide evidence that the Ty2 and Ty3p families have arisen in the S. cerevisiae genome by horizontal transfer. Our results demonstrate that the genome of a single individual contains important information about the state of TE population dynamics within a species and suggest that horizontal transfer may play an important role in shaping the genomic diversity of transposable elements in unicellular eukaryotes.