A procedure is described for the efficient preparation of isotopically enriched RNAs of defined sequence. Uniformly labelled nucleotide 5'triphosphates (NTPs) were prepared from E.coli grown on 13C and/or 15N isotopically enriched media. These procedures routinely yield 180 mumoles of labelled NTPs per gram of 13C enriched glucose. The labelled NTPs were then used to synthesize RNA oligomers by in vitro transcription. Several 13C and/or 15N labelled RNAs have been synthesized for the sequence r(GGCGCUUGCGUC). Under conditions of high salt or low salt, this RNA forms either a symmetrical duplex with two U.U base pairs or a hairpin containing a CUUG loop respectively. These procedures were used to synthesize uniformly labelled RNAs and a RNA labelled only on the G and C residues. The ability to generate milligram quantities of isotopically labelled RNAs allows application of multi-dimensional heteronuclear magnetic resonance experiments that enormously simplify the resonance assignment and solution structure determination of RNAs. Examples of several such heteronuclear NMR experiments are shown.
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. Using a combination of flow cytometry and high-throughput restriction-site associated DNA tag sequencing, we show that this switch is correlated with a change in chromosomal copy number. Furthermore, the gain of a single chromosome is sufficient to switch a strain from the fluffy to the smooth state, and its subsequent loss to revert the strain back to the fluffy state. Because copy number imbalance of six of the 16 S. cerevisiae chromosomes and even a single gene can modulate the switch, our results support the hypothesis that the state switch is produced by dosage-sensitive genes, rather than a general response to altered DNA content. These findings add a complex, multicellular phenotype to the list of molecular and cellular traits known to be altered by aneuploidy and suggest that chromosome missegregation can provide a quick, heritable, and reversible mechanism by which organisms can toggle between phenotypes. colony morphology | copy number variation | phenotypic switching | bet-hedging
SUMMARY 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. Humans began making wine in the Middle East over 9,000 years ago [1, 2]. Selecting favorable fermentation products created specialized strains of Saccharomyces cerevisiae [3, 4] that were transported along with the grapevines. Today, S. cerevisiae strains residing in vineyards around the world are genetically similar, and their population structure suggests a common origin that followed the path of human migration [3–7]. Like wine, coffee and cacao depend on microbial fermentation [8, 9] and have been globally dispersed by humans. Theobroma cacao originated in the Amazon and Orinoco Basins of Colombia and Venezuela [10], was cultivated in Central America by the Mesoamerican peoples, and introduced to Europeans by Cortés in 1530 [11]. Coffea, native to Ethiopia, was disseminated by Arab traders throughout the Middle East and North Africa in the 6th century and was introduced to European consumers in the 17th century [12]. Here, we test whether the yeasts associated with coffee and cacao are genetically similar, crop-specific populations or genetically diverse, geography-specific populations. Our results uncovered populations that, while defined by niche and geography, also bear signatures of admixture between major populations in events independent of the transport of the plants. Thus, human-associated fermentations and migration may have affected the distribution of yeast involved in the production of coffee and chocolate.
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. Here, we describe a functional assay that leverages the evolutionary conservation of an enzyme in the serine biosynthesis pathway, phosphoserine aminotransferase, and the ability of the human protein‐coding sequence (PSAT1) to functionally replace its yeast ortholog (SER1). Results from our quantitative, yeast‐based assay agree well with clinical annotations and expectations based on the disease literature. Using this assay, we have measured the functional impact of the 199 PSAT1 variants currently listed in ClinVar, gnomAD, and the literature. We anticipate that the assay could be used to comprehensively assess the functional impact of all SNP‐accessible amino acid substitution mutations in PSAT1, a resource that could aid variant interpretation and identify potential NLS carriers.
Tetrad analysis has been a gold standard genetic technique for several decades. Unfortunately, the manual nature of the process has relegated its application to small-scale studies and limited its integration with rapidly evolving DNA sequencing technologies. We have developed a rapid, high-throughput method, called Barcode Enabled Sequencing of Tetrads (BEST), that replaces the manual processes of isolating, disrupting and spacing tetrads. BEST uses a meiosis-specific GFP fusion protein to isolate tetrads by fluorescence-activated cell sorting and molecular barcodes that are read during genotyping to identify spores derived from the same tetrad. Maintaining tetrad information allows accurate inference of missing genetic markers and full genotypes of missing (and presumably nonviable) individuals. By removing the bottleneck of manual dissection, hundreds or even thousands of tetrads can be isolated in minutes. We demonstrate the approach in Saccharomyces cerevisiae, but BEST is readily transferable to microorganisms in which meiotic mapping is significantly more laborious.
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