Yeast最新文献

Yeasts are naturally diverse, genetically tractable, and easy to grow such that researchers can investigate any number of genotypes, environments, or interactions thereof. However, studies of yeast transcriptomes have been limited by the processing capabilities of traditional RNA sequencing techniques. Here we optimize a powerful, high-throughput single-cell RNA sequencing (scRNAseq) platform, SPLiT-seq (Split Pool Ligation-based Transcriptome sequencing), for yeasts and apply it to 43,388 cells of multiple species and ploidies. This platform utilizes a combinatorial barcoding strategy to enable massively parallel RNA sequencing of hundreds of yeast genotypes or growth conditions at once. This method can be applied to most species or strains of yeast for a fraction of the cost of traditional scRNAseq approaches. Thus, our technology permits researchers to leverage "the awesome power of yeast" by allowing us to survey the transcriptome of hundreds of strains and environments in a short period of time and with no specialized equipment. The key to this method is that sequential barcodes are probabilistically appended to cDNA copies of RNA while the molecules remain trapped inside of each cell. Thus, the transcriptome of each cell is labeled with a unique combination of barcodes. Since SPLiT-seq uses the cell membrane as a container for this reaction, many cells can be processed together without the need to physically isolate them from one another in separate wells or droplets. Further, the first barcode in the sequence can be chosen intentionally to identify samples from different environments or genetic backgrounds, enabling multiplexing of hundreds of unique perturbations in a single experiment. In addition to greater multiplexing capabilities, our method also facilitates a deeper investigation of biological heterogeneity, given its single-cell nature. For example, in the data presented here, we detect transcriptionally distinct cell states related to cell cycle, ploidy, metabolic strategies, and so forth, all within clonal yeast populations grown in the same environment. Hence, our technology has two obvious and impactful applications for yeast research: the first is the general study of transcriptional phenotypes across many strains and environments, and the second is investigating cell-to-cell heterogeneity across the entire transcriptome.

Transcription enables the production of RNA from a DNA template. Due to the highly dynamic nature of transcription, live-cell imaging methods play a crucial role in measuring the kinetics of this process. For instance, transcriptional bursts have been visualized using fluorescent phage-coat proteins that associate tightly with messenger RNA (mRNA) stem loops formed on nascent transcripts. To convert the signal emanating from a transcription site into meaningful estimates of transcription dynamics, the influence of various parameters on the measured signal must be evaluated. Here, the effect of gene length on the intensity of the transcription site focus was analyzed. Intuitively, a longer gene can support a larger number of transcribing polymerases, thus leading to an increase in the measured signal. However, measurements of transcription induced by hyper-osmotic stress responsive promoters display independence from gene length. A mathematical model of the stress-induced transcription process suggests that the formation of gene loops that favor the recycling of polymerase from the terminator to the promoter can explain the observed behavior. One experimentally validated prediction from this model is that the amount of mRNA produced from a short gene should be higher than for a long one as the density of active polymerase on the short gene will be increased by polymerase recycling. Our data suggest that this recycling contributes significantly to the expression output from a gene and that polymerase recycling is modulated by the promoter identity and the cellular state.

Nitrogen catabolite repression (NCR) is a means for yeast to adapt its transcriptome to changing nitrogen sources in its environment. In conditions of derepression (under poor nitrogen conditions, upon rapamycin treatment, or when glutamine production is inhibited), two transcriptional activators of the GATA family are recruited to NCR-sensitive promoters and activate transcription of NCR-sensitive genes. Earlier observations have involved the Spt-Ada-Gcn5 acetyltransferase (SAGA) chromatin remodeling complex in these transcriptional regulations. In this report, we provide an illustration of the varying NCR-sensitive responses and question whether differing SAGA recruitment could explain this diversity of responses.

Mitochondria fulfil many essential roles and have their own genome, which is expressed as polycistronic transcripts that undergo co- or posttranscriptional processing and splicing. Due to the inherent complexity and limited technical accessibility of the mitochondrial transcriptome, fundamental questions regarding mitochondrial gene expression and splicing remain unresolved, even in the model eukaryote Saccharomyces cerevisiae. Long-read sequencing could address these fundamental questions. Therefore, a method for the enrichment of mitochondrial RNA and sequencing using Nanopore technology was developed, enabling the resolution of splicing of polycistronic genes and the quantification of spliced RNA. This method successfully captured the full mitochondrial transcriptome and resolved RNA splicing patterns with single-base resolution and was applied to explore the transcriptome of S. cerevisiae grown with glucose or ethanol as the sole carbon source, revealing the impact of growth conditions on mitochondrial RNA expression and splicing. This study uncovered a remarkable difference in the turnover of Group II introns between yeast grown in either mostly fermentative or fully respiratory conditions. Whether this accumulation of introns in glucose medium has an impact on mitochondrial functions remains to be explored. Combined with the high tractability of the model yeast S. cerevisiae, the developed method enables to monitor mitochondrial transcriptome responses in a broad range of relevant contexts, including oxidative stress, apoptosis and mitochondrial diseases.

The fission yeast species Schizosaccharomyces japonicus is currently divided into two varieties-S. japonicus var. japonicus and S. japonicus var. versatilis. Here we examine the var. versatilis isolate CBS5679. The CBS5679 genome shows 88% identity to the reference genome of S. japonicus var. japonicus at the coding sequence level, with phylogenetic analyses suggesting that it has split from the S. japonicus lineage 25 million years ago. The CBS5679 genome contains a reciprocal translocation between chromosomes 1 and 2, together with several large inversions. The products of genes linked to the major translocation are associated with 'metabolism' and 'cellular assembly' ontology terms. We further show that CBS5679 does not generate viable progeny with the reference strain of S. japonicus. Although CBS5679 shares closer similarity to the 'type' strain of var. versatilis as compared to S. japonicus, it is not identical to the type strain, suggesting population structure within var. versatilis. We recommend that the taxonomic status of S. japonicus var. versatilis is raised, with it being treated as a separate species, Schizosaccharomyces versatilis.

Schizosaccharomyces japonicus belongs to the single-genus class Schizosaccharomycetes, otherwise known as "fission yeasts." As part of a composite model system with its widely studied S. pombe sister species, S. japonicus has provided critical insights into the workings and the evolution of cell biological mechanisms. Furthermore, its divergent biology makes S. japonicus a valuable model organism in its own right. However, the currently available genome assembly contains gaps and has been unable to resolve centromeres and other repeat-rich chromosomal regions. Here we present a telomere-to-telomere long-read genome assembly of the S. japonicus genome. This includes the three megabase-length chromosomes, with centromeres hundreds of kilobases long, rich in 5S ribosomal RNA genes, transfer RNA genes, long terminal repeats, and short repeats. We identify a gene-sparse region on chromosome 2 that resembles a 331 kb centromeric duplication. We revise the genome size of S. japonicus to at least 16.6 Mb and possibly up to 18.12 Mb, at least 30% larger than previous estimates. Our whole genome assembly will support the growing S. japonicus research community and facilitate research in new directions, including centromere and DNA repeat evolution, and yeast comparative genomics.

Schizosaccharomyces japonicus Yukawa et Maki (1931) and Schizosaccharomyces versatilis Wickerham et Duprat (1945) have been treated as varieties of S. japonicus or as conspecific, based on various approaches including mating trials and nDNA/nDNA optical reassociation studies. However, the type strains of S. japonicus and S. versatilis differ by five substitutions (99.15% identity) and one 1-bp indel in the sequences of the D1/D2 domain of the 26S rRNA gene, and 23 substitutions (96.3% identity) and 31-bp indels in the sequences of internal transcribed spacer (ITS) of rRNA, suggesting that they may not be conspecific. To reassess their taxonomic status, we conducted mating trials and whole-genome analyses. Mating trials using the type strains showed a strong but incomplete prezygotic sterility barrier, yielding interspecies mating products at two orders of magnitude lower efficiency than intraspecies matings. These mating products, which were exclusively allodiploid hybrids, were unable to undergo the haplontic life cycle of the parents. We generated chromosome-level gap-less genome assemblies for both type strains. Whole genome sequences yielded an average nucleotide identity (ANI) of 86.4%, indicating clear separation of S. japonicus and S. versatilis. Based on these findings, we propose the reinstatement of S. versatilis as a distinct species (holotype strain: CBS 103^T and ex-types: NRRL Y-1026, NBRC 1607, ATCC 9987, PYCC 7100; Mycobank no.: 847838).

Auxotrophic strains starving for their cognate nutrient, termed auxotrophic starvation, are characterized by a shorter lifespan, higher glucose wasting phenotype, and inability to accomplish cell cycle arrest when compared to a "natural starvation," where a cell is starving for natural environmental growth-limiting nutrients such as phosphate. Since evidence of this physiological response is limited to only a subset of auxotrophs, we evaluated a panel of auxotrophic mutants to determine whether these responses are characteristic of a broader range of amino acid auxotrophs. Based on the starvation survival kinetics, the panel of strains was grouped into three categories-short-lived strains, strains with survival similar to a prototrophic wild type strain, and long-lived strains. Among the short-lived strains, we observed that the tyrosine, asparagine, threonine, and aspartic acid auxotrophs rapidly decline in viability, with all strains unable to arrest cell cycle progression. The three basic amino acid auxotrophs had a survival similar to a prototrophic strain starving in minimal media. The leucine, tryptophan, methionine, and cysteine auxotrophs displayed the longest lifespan. We also demonstrate how the phenomenon of glucose wasting is limited to only a subset of the tested auxotrophs, namely the asparagine, leucine, and lysine auxotrophs. Furthermore, we observed pleiotropic phenotypes associated with a subgroup of auxotrophs, highlighting the importance of considering unintended phenotypic effects when using auxotrophic strains especially in chronological aging experiments.