Functional analysis of a genome requires accurate gene structure information and a complete gene inventory. A dual experimental strategy was used to verify and correct the initial genome sequence annotation of the reference plant Arabidopsis. Sequencing full-length cDNAs and hybridizations using RNA populations from various tissues to a set of high-density oligonucleotide arrays spanning the entire genome allowed the accurate annotation of thousands of gene structures. We identified 5817 novel transcription units, including a substantial amount of antisense gene transcription, and 40 genes within the genetically defined centromeres. This approach resulted in completion of approximately 30% of the Arabidopsis ORFeome as a resource for global functional experimentation of the plant proteome.

Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis, and an opportunistic human pathogen of global importance. We have sequenced its ∼20-megabase genome, which contains ∼6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages. The genome is rich in transposons, many of which cluster at candidate centromeric regions. The presence of these transposons may drive karyotype instability and phenotypic variation. C. neoformans encodes unique genes that may contribute to its unusual virulence properties, and comparison of two phenotypically distinct strains reveals variation in gene content in addition to sequence polymorphisms between the genomes.

Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.

We sequenced the genome of Saccharomyces cerevisiae strain YJM789, which was derived from a yeast isolated from the lung of an AIDS patient with pneumonia. The strain is used for studies of fungal infections and quantitative genetics because of its extensive phenotypic differences to the laboratory reference strain, including growth at high temperature and deadly virulence in mouse models. Here we show that the Ϸ12-Mb genome of YJM789 contains Ϸ60,000 SNPs and Ϸ6,000 indels with respect to the reference S288c genome, leading to protein polymorphisms with a few known cases of phenotypic changes. Several ORFs are found to be unique to YJM789, some of which might have been acquired through horizontal transfer. Localized regions of high polymorphism density are scattered over the genome, in some cases spanning multiple ORFs and in others concentrated within single genes. The sequence of YJM789 contains clues to pathogenicity and spurs the development of more powerful approaches to dissecting the genetic basis of complex hereditary traits.comparative genomics ͉ genome architecture ͉ introgression ͉ lateral gene transfer T here is extensive genetic and phenotypic diversity within species. Determining which of the vast amounts of sequence differences that are found among individuals of a species contribute to heritable traits will allow diseases to be tackled at the molecular level and aid in the development of novel therapies. Saccharomyces cerevisiae, commonly known as baker's or brewer's yeast, plays a central role in food production and is one of the most studied genetic model species. It is not only widely used in biotechnology but also is a powerful model system that has been applied to identify multigenetic factors of hereditary traits (1-7). The genome sequence of one laboratory strain, a derivative of S288c, was the first genome of a free-living eukaryotic organism to be sequenced (8). Over the last 10 years, this genome has served as the reference for the S. cerevisiae species and has catalyzed the development of whole-genome approaches to biology (9, 10). Despite frequent laboratory use of alternative strains, sequence information for S. cerevisiae beyond the domesticated strain S288c has been fragmentary. S288c, which originated from a strain isolated from a rotten fig, was chosen for sequencing because it possesses properties that make it easy to work with, such as minimal colony morphology switching, consistent growth rates in glucose media, and no flocculence (11). At several loci, S288c contains polymorphisms not found in natural isolates, which could be hallmarks of domestication (12,13). A growing number of S. cerevisiae infections in humans have recently been reported (14). As a result, S. cerevisiae is also regarded as an emerging opportunistic pathogen that can cause clinically relevant infections in different patient types and body sites (15)(16)(17). One clinical strain (YJM145), derived from a yeast isolated from an AIDS patient with S. cerevisiae pneumonia (18), has been studied extensively as ...

Bioactive compounds are widely used to modulate protein function and can serve as important leads for drug development. Identifying the in vivo targets of these compounds remains a challenge. Using yeast, we integrated three genome-wide gene-dosage assays to measure the effect of small molecules in vivo. A single TAG microarray was used to resolve the fitness of strains derived from pools of (i) homozygous deletion mutants, (ii) heterozygous deletion mutants and (iii) genomic library transformants. We demonstrated, with eight diverse reference compounds, that integration of these three chemogenomic profiles improves the sensitivity and specificity of small-molecule target identification. We further dissected the mechanism of action of two protein phosphatase inhibitors and in the process developed a framework for the rational design of multidrug combinations to sensitize cells with specific genotypes more effectively. Finally, we applied this platform to 188 novel synthetic chemical compounds and identified both potential targets and structure-activity relationships.

Knowing gene structure is vital to understanding gene function, and accurate genome annotation is essential for understanding cellular function. To this end, we have developed a genome-wide assay for mapping introns in Saccharomyces cerevisiae. Using high-density tiling arrays, we compared wild-type yeast to a mutant deficient for intron degradation. Our method identified 76% of the known introns, confirmed 18 previously predicted introns, and revealed 9 formerly undiscovered introns. Furthermore, we discovered that all 13 meiosis-specific intronic yeast genes undergo regulated splicing, which provides posttranscriptional regulation of the genes involved in yeast cell differentiation. Moreover, we found that Ϸ16% of intronic genes in yeast are incompletely spliced during exponential growth in rich medium, which suggests that meiosis is not the only biological process regulated by splicing. Our tiling-array assay provides a snapshot of the spliced transcriptome in yeast. This robust methodology can be used to explore environmentally distinct splicing responses and should be readily adaptable to the study of other organisms, including humans. meiosis ͉ regulated splicing ͉ Saccharomyces cerevisiae I ntronic sequences provide numerous functional elements that direct pre-mRNA processing and alternative splicing. In the relatively simple eukaryote Saccharomyces cerevisiae, introns direct splicing (1), can increase gene expression (2), and, in specific cases, may contain small nucleolar RNAs (3). Additionally, introns in yeast can modulate translation posttranscriptionally through a process known as regulated splicing (4-7). During regulated splicing, yeast cells under certain conditions can limit intron splicing in specific genes, which, in turn, disrupts translation through frame-shifting and/or introduction of nonsense codons (4-7). Accurate mapping of introns is an essential first step to understanding RNA splicing and function.S. cerevisiae is an easily manipulatable eukaryote with a relatively small extensively studied genome that shares many core spliceosome functions with humans (1). Only 5% of S. cerevisiae genes are interrupted by introns (8, 9), and all introns are constitutively removed before translation (10). Because its genome is relatively small and well characterized, yeast serves as an ideal model organism for new technologies.Tiling DNA microarrays, comprised of overlapping, end-toend, or closely spaced DNA probes, have been used to map cellular transcription in a variety of organisms. Tiling-array data have improved gene annotation and revealed extensive transcription of noncoding RNAs (11-13). We used a high-density yeast-tiling array with overlapping probes, which provides a per-strand resolution of eight nucleotides, to research premRNA processing in yeast. The closely spaced probes allowed for accurate measurement of small transcriptional features, such as single exons and small introns.In this paper, we describe the results of a high-resolution microarray investigation of yeast splicing during...

The purpose of introns in the architecturally simple genome of Saccharomyces cerevisiae is not well understood. To assay the functional relevance of introns, a series of computational analyses and several detailed deletion studies were completed on the intronic genes of S. cerevisiae. Mining existing data from genomewide studies on yeast revealed that intron-containing genes produce more RNA and more protein and are more likely to be haplo-insufficient than nonintronic genes. These observations for all intronic genes held true for distinct subsets of genes including ribosomal, nonribosomal, duplicated, and nonduplicated. Corroborating the result of computational analyses, deletion of introns from three essential genes decreased cellular RNA levels and caused measurable growth defects. These data provide evidence that introns improve transcriptional and translational yield and are required for competitive growth of yeast. T HE genes of complex organisms depend on intronsto provide regulatory sequences that allow for accurate pre-mRNA processing and alternative splicing. In multicellular organisms most genes contain at least one intron, usually more. In humans, for instance, 94% of the genes are interrupted by, on average, seven introns (Lander et al. 2001;Venter et al. 2001). Although splicing is closely coupled to several other processes during gene expression, it is still widely thought that the primary fitness benefits that introns confer to a species are through improved evolution via exon shuffling and increased proteome complexity by alternative splicing. On the basis of our observations we propose that introns confer an additional advantage: they improve the transcriptional and translational output of the genes they populate.The spliceosome, which removes introns from the coding mRNA, is a large cellular complex containing hundreds of proteins and at least five small nuclear RNAs. It is closely coupled to, and in some cases directly interacts with, the proteins responsible for transcription, capping, polyadenylation, RNA export, and nonsensemediated decay (Maniatis and Reed 2002). Given the extensive coupling of splicing with mRNA metabolism, it is not surprising that removing the introns from genes in higher eukaryotes (where intron-containing genes predominate) disrupts mRNA synthesis and often lowers cytoplasmic mRNA levels. The question arises: Are the introns directly responsible for increasing gene expression or does their removal act indirectly, by simply derailing the mRNA synthesis assembly line? Some examples in metazoans support a direct role in expression: introns containing transcriptional enhancers have been identified (Sleckman et al. 1996) and one group showed that removing introns from a gene disrupts nucleosome binding (Liu et al. 1995). There is, however, no consensus that introns serve to increase gene expression. To investigate the role that introns may play in cellular fitness we studied their genetic contribution to the fitness of Saccharomyces cerevisiae.In contrast to multi...

We develop a novel synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and encoded metabolites.