Spatiotemporal patterns of gene expression during development of a complex colony morphology.

Clonal communities of single celled organisms, such as bacterial or fungal colonies and biofilms, are spatially structured, with subdomains of cells experiencing differing environmental conditions. In the development of such communities, cell specialization is not only important to respond and adapt to the local environment but has the potential to increase the fitness of the clonal community through division of labor. Here, we examine colony development in a yeast strain (F13) that produces colonies with a highly structured "ruffled" phenotype in the colony periphery and an unstructured "smooth" phenotype in the colony center.

Constructing and interpreting a large-scale variant effect map for an ultrarare disease gene: Comprehensive prediction of the functional impact of PSAT1 genotypes.

Reduced activity of the enzymes encoded by PHGDH, PSAT1, and PSPH causes a set of ultrarare, autosomal recessive diseases known as serine biosynthesis defects. These diseases present in a broad phenotypic spectrum: at the severe end is Neu-Laxova syndrome, in the intermediate range are infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end is childhood disease with intellectual disability. However, L-serine supplementation, especially if started early, can ameliorate and in some cases even prevent symptoms.

The functional impact of 1,570 individual amino acid substitutions in human OTC.

Deleterious mutations in the X-linked gene encoding ornithine transcarbamylase (OTC) cause the most common urea cycle disorder, OTC deficiency. This rare but highly actionable disease can present with severe neonatal onset in males or with later onset in either sex. Individuals with neonatal onset appear normal at birth but rapidly develop hyperammonemia, which can progress to cerebral edema, coma, and death, outcomes ameliorated by rapid diagnosis and treatment.

Predicting the functional effect of compound heterozygous genotypes from large scale variant effect maps.

Background: Pathogenic variants in , and cause a set of rare, autosomal recessive diseases known as serine biosynthesis defects. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu-Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately in the form of infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, as childhood disease with intellectual disability. However, because L-serine supplementation, especially if started early, can ameliorate and in some cases even prevent symptoms, knowledge of pathogenic variants is highly actionable.

A method for quantifying sporulation efficiency and isolating meiotic progeny in non-GMO strains of Saccharomyces cerevisiae.

Meiotic mapping, a linkage-based method for analyzing the recombinant progeny of a cross, has long been a cornerstone of genetic research. The yeast Saccharomyces cerevisiae is a powerful system because it is possible to isolate and cultivate the four products (spores) of a single meiotic event. However, the throughput of this process has historically been limited by the process of identifying tetrads in a heterogeneous population of vegetative cells, tetrads, and dyads followed by manual separation (dissection) of the spores contained in a tetrad.

A yeast-based complementation assay elucidates the functional impact of 200 missense variants in human PSAT1.

Defects in serine biosynthesis resulting from loss of function mutations in PHGDH, PSAT1, and PSPH cause a set of rare, autosomal recessive diseases known as Neu-Laxova syndrome (NLS) or serine-deficiency disorders. The diseases present with a broad range of phenotypes including lethality, severe neurological manifestations, seizures, and intellectual disability. However, because L-serine supplementation, especially if started prenatally, can ameliorate and in some cases even prevent symptoms, knowledge of pathogenic variants is medically actionable.

Computational Inference Software for Tetrad Assembly from Randomly Arrayed Yeast Colonies.

We describe an information-theory-based method and associated software for computationally identifying sister spores derived from the same meiotic tetrad. The method exploits specific DNA sequence features of tetrads that result from meiotic centromere and allele segregation patterns. Because the method uses only the genomic sequence, it alleviates the need for tetrad-specific barcodes or other genetic modifications to the strains.

Selection of Candida albicans trisomy during oropharyngeal infection results in a commensal-like phenotype.

When the fungus Candida albicans proliferates in the oropharyngeal cavity during experimental oropharyngeal candidiasis (OPC), it undergoes large-scale genome changes at a much higher frequency than when it grows in vitro. Previously, we identified a specific whole chromosome amplification, trisomy of Chr6 (Chr6x3), that was highly overrepresented among strains recovered from the tongues of mice with OPC. To determine the functional significance of this trisomy, we assessed the virulence of two Chr6 trisomic strains and a Chr5 trisomic strain in the mouse model of OPC.

A polyploid admixed origin of beer yeasts derived from European and Asian wine populations.

Strains of Saccharomyces cerevisiae used to make beer, bread, and wine are genetically and phenotypically distinct from wild populations associated with trees. The origins of these domesticated populations are not always clear; human-associated migration and admixture with wild populations have had a strong impact on S. cerevisiae population structure.

Physical basis for long-distance communication along meiotic chromosomes.

Viable gamete formation requires segregation of homologous chromosomes connected, in most species, by cross-overs. DNA double-strand break (DSB) formation and the resulting cross-overs are regulated at multiple levels to prevent overabundance along chromosomes. Meiotic cells coordinate these events between distant sites, but the physical basis of long-distance chromosomal communication has been unknown.

Natural Variation in and Underlie Condition-Specific Growth Defects in .

Despite their ubiquitous use in laboratory strains, naturally occurring loss-of-function mutations in genes encoding core metabolic enzymes are relatively rare in wild isolates of Here, we identify a naturally occurring serine auxotrophy in a sake brewing strain from Japan. Through a cross with a honey wine (white tecc) brewing strain from Ethiopia, we map the minimal medium growth defect to , which encodes 3-phosphoserine aminotransferase and is orthologous to the human disease gene, To investigate the impact of this polymorphism under conditions of abundant external nutrients, we examine growth in rich medium alone or with additional stresses, including the drugs caffeine and rapamycin and relatively high concentrations of copper, salt, and ethanol. Consistent with studies that found widespread effects of different auxotrophies on RNA expression patterns in rich media, we find that the loss-of-function allele dominates the quantitative trait locus (QTL) landscape under many of these conditions, with a notable exacerbation of the effect in the presence of rapamycin and caffeine.

Transcriptional Profiling of Biofilm Regulators Identified by an Overexpression Screen in .

Biofilm formation by microorganisms is a major cause of recurring infections and removal of biofilms has proven to be extremely difficult given their inherent drug resistance . Understanding the biological processes that underlie biofilm formation is thus extremely important and could lead to the development of more effective drug therapies, resulting in better infection outcomes. Using the yeast as a biofilm model, overexpression screens identified , , , , , and as regulators of biofilm formation.

Dissecting Gene Expression Changes Accompanying a Ploidy-Based Phenotypic Switch.

Aneuploidy, a state in which the chromosome number deviates from a multiple of the haploid count, significantly impacts human health. The phenotypic consequences of aneuploidy are believed to arise from gene expression changes associated with the altered copy number of genes on the aneuploid chromosomes. To dissect the mechanisms underlying altered gene expression in aneuploids, we used RNA-seq to measure transcript abundance in colonies of the haploid Saccharomyces cerevisiae strain F45 and two aneuploid derivatives harboring disomies of chromosomes XV and XVI.

Independent Origins of Yeast Associated with Coffee and Cacao Fermentation.

Modern transportation networks have facilitated the migration and mingling of previously isolated populations of plants, animals, and insects. Human activities can also influence the global distribution of microorganisms. The best-understood example is yeasts associated with winemaking.

Aneuploidy: Tolerating Tolerance.

Individuals, and cells, vary in their ability to tolerate aneuploidy, an unbalanced chromosome complement. Tolerance mechanisms can be karyotype-specific or general. General tolerance mechanisms may allow cells to benefit from the phenotypic plasticity conferred by access to multiple aneuploid states.

Allelic variation, aneuploidy, and nongenetic mechanisms suppress a monogenic trait in yeast.

Clinically relevant features of monogenic diseases, including severity of symptoms and age of onset, can vary widely in response to environmental differences as well as to the presence of genetic modifiers affecting the trait's penetrance and expressivity. While a better understanding of modifier loci could lead to treatments for Mendelian diseases, the rarity of individuals harboring both a disease-causing allele and a modifying genotype hinders their study in human populations. We examined the genetic architecture of monogenic trait modifiers using a well-characterized yeast model of the human Mendelian disease classic galactosemia.

BEST: barcode enabled sequencing of tetrads.

Tetrad analysis is a valuable tool for yeast genetics, but the laborious manual nature of the process has hindered its application on large scales. Barcode Enabled Sequencing of Tetrads (BEST)1 replaces the manual processes of isolating, disrupting and spacing tetrads. BEST isolates tetrads by virtue of a sporulation-specific GFP fusion protein that permits fluorescence-activated cell sorting of tetrads directly onto agar plates, where the ascus is enzymatically digested and the spores are disrupted and randomly arrayed by glass bead plating.

Genomic sequence diversity and population structure of Saccharomyces cerevisiae assessed by RAD-seq.

The budding yeast Saccharomyces cerevisiae is important for human food production and as a model organism for biological research. The genetic diversity contained in the global population of yeast strains represents a valuable resource for a number of fields, including genetics, bioengineering, and studies of evolution and population structure. Here, we apply a multiplexed, reduced genome sequencing strategy (restriction site-associated sequencing or RAD-seq) to genotype a large collection of S.

Aneuploidy underlies a multicellular phenotypic switch.

Although microorganisms are traditionally used to investigate unicellular processes, the yeast Saccharomyces cerevisiae has the ability to form colonies with highly complex, multicellular structures. Colonies with the "fluffy" morphology have properties reminiscent of bacterial biofilms and are easily distinguished from the "smooth" colonies typically formed by laboratory strains. We have identified strains that are able to reversibly toggle between the fluffy and smooth colony-forming states.

High-throughput tetrad analysis.

Tetrad analysis has been a gold-standard genetic technique for several decades. Unfortunately, the need to manually isolate, disrupt and space tetrads has relegated its application to small-scale studies and limited its integration with high-throughput DNA sequencing technologies. We have developed a rapid, high-throughput method, called barcode-enabled sequencing of tetrads (BEST), that uses (i) a meiosis-specific GFP fusion protein to isolate tetrads by FACS and (ii) molecular barcodes that are read during genotyping to identify spores derived from the same tetrad.

Phylogenetic ubiquity and shuffling of the bacterial RecBCD and AddAB recombination complexes.

RecBCD and AddAB are bacterial enzymes that share similar helicase and nuclease activities and initiate repair of DNA double-strand breaks by homologous recombination. Examination of the phylogenetic distribution of AddAB and RecBCD revealed that one or the other complex is present in most sequenced bacteria. In addition, horizontal gene transfer (HGT) events involving addAB and recBCD appear to be common, with the genes encoding one complex frequently replacing those encoding the other.

Ctp1 and Exonuclease 1, alternative nucleases regulated by the MRN complex, are required for efficient meiotic recombination.

Double-strand breaks (DSBs) in DNA are lethal unless repaired. Faithful repair requires processing of the DSB ends and interaction with intact homologous DNA, which can produce genetic recombinants. To determine the role of nucleases in DSB end-processing and joint molecule resolution, we studied recombination at the site of a single DSB, generated by induction of the I-SceI endonuclease, during meiosis of fission yeast lacking Rec12 (Spo11 homolog) and, hence, other DSBs.

Indistinguishable landscapes of meiotic DNA breaks in rad50+ and rad50S strains of fission yeast revealed by a novel rad50+ recombination intermediate.

The fission yeast Schizosaccharomyces pombe Rec12 protein, the homolog of Spo11 in other organisms, initiates meiotic recombination by creating DNA double-strand breaks (DSBs) and becoming covalently linked to the DNA ends of the break. This protein-DNA linkage has previously been detected only in mutants such as rad50S in which break repair is impeded and DSBs accumulate. In the budding yeast Saccharomyces cerevisiae, the DSB distribution in a rad50S mutant is markedly different from that in wild-type (RAD50) meiosis, and it was suggested that this might also be true for other organisms.

Helicobacter pylori AddAB helicase-nuclease and RecA promote recombination-related DNA repair and survival during stomach colonization.

Helicobacter pylori colonization of the human stomach is characterized by profound disease-causing inflammation. Bacterial proteins that detoxify reactive oxygen species or recognize damaged DNA adducts promote infection, suggesting that H. pylori requires DNA damage repair for successful in vivo colonization.

The fission yeast BLM homolog Rqh1 promotes meiotic recombination.

RecQ helicases are found in organisms as diverse as bacteria, fungi, and mammals. These proteins promote genome stability, and mutations affecting human RecQ proteins underlie premature aging and cancer predisposition syndromes, including Bloom syndrome, caused by mutations affecting the BLM protein. In this study we show that mutants lacking the Rqh1 protein of the fission yeast Schizosaccharomyces pombe, a RecQ and BLM homolog, have substantially reduced meiotic recombination, both gene conversions and crossovers.