Publications by authors named "Adrian C Scott"

8 Publications

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

J Inherit Metab Dis 2020 07 27;43(4):758-769. Epub 2020 Feb 27.

Pacific Northwest Research Institute, Seattle, Washington.

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.
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http://dx.doi.org/10.1002/jimd.12227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7444316PMC
July 2020

Data-driven multiscale modeling reveals the role of metabolic coupling for the spatio-temporal growth dynamics of yeast colonies.

BMC Mol Cell Biol 2019 Dec 19;20(1):59. Epub 2019 Dec 19.

Luxembourg Centre for Systems Biomedicine, University of Luxembourg, 2, avenue de l'Université, Esch-sur-Alzette, L-4365, Luxembourg.

Background: Multicellular entities like mammalian tissues or microbial biofilms typically exhibit complex spatial arrangements that are adapted to their specific functions or environments. These structures result from intercellular signaling as well as from the interaction with the environment that allow cells of the same genotype to differentiate into well-organized communities of diversified cells. Despite its importance, our understanding how this cell-cell and metabolic coupling lead to functionally optimized structures is still limited.

Results: Here, we present a data-driven spatial framework to computationally investigate the development of yeast colonies as such a multicellular structure in dependence on metabolic capacity. For this purpose, we first developed and parameterized a dynamic cell state and growth model for yeast based on on experimental data from homogeneous liquid media conditions. The inferred model is subsequently used in a spatially coarse-grained model for colony development to investigate the effect of metabolic coupling by calibrating spatial parameters from experimental time-course data of colony growth using state-of-the-art statistical techniques for model uncertainty and parameter estimations. The model is finally validated by independent experimental data of an alternative yeast strain with distinct metabolic characteristics and illustrates the impact of metabolic coupling for structure formation.

Conclusions: We introduce a novel model for yeast colony formation, present a statistical methodology for model calibration in a data-driven manner, and demonstrate how the established model can be used to generate predictions across scales by validation against independent measurements of genetically distinct yeast strains.
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http://dx.doi.org/10.1186/s12860-019-0234-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6923950PMC
December 2019

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

G3 (Bethesda) 2018 01 4;8(1):239-251. Epub 2018 Jan 4.

Pacific Northwest Research Institute, Seattle, Washington 98122

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. We also identify a major-effect QTL associated with growth on salt that maps to the gene encoding the sodium exporter, We demonstrate that the salt phenotype is largely driven by variation in the promoter, which harbors a deletion that removes binding sites for the Mig1 and Nrg1 transcriptional repressors. Thus, our results identify natural variation associated with both coding and regulatory regions of the genome that underlie strong growth phenotypes.
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http://dx.doi.org/10.1534/g3.117.300392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765352PMC
January 2018

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

Genetics 2015 Jan 13;199(1):247-62. Epub 2014 Nov 13.

Pacific Northwest Diabetes Research Institute, Seattle, Washington 98122

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. Yeast strains with loss-of-function mutations in the yeast ortholog (GAL7) of the human disease gene (GALT) fail to grow in the presence of even small amounts of galactose due to accumulation of the same toxic intermediates that poison human cells. To isolate and individually genotype large numbers of the very rare (∼0.1%) galactose-tolerant recombinant progeny from a cross between two gal7Δ parents, we developed a new method, called "FACS-QTL." FACS-QTL improves upon the currently used approaches of bulk segregant analysis and extreme QTL mapping by requiring less genome engineering and strain manipulation as well as maintaining individual genotype information. Our results identified multiple distinct solutions by which the monogenic trait could be suppressed, including genetic and nongenetic mechanisms as well as frequent aneuploidy. Taken together, our results imply that the modifiers of monogenic traits are likely to be genetically complex and heterogeneous.
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http://dx.doi.org/10.1534/genetics.114.170563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4286688PMC
January 2015

BEST: barcode enabled sequencing of tetrads.

J Vis Exp 2014 May 1(87). Epub 2014 May 1.

Pacific Northwest Diabetes Research Institute;

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. The haploid colonies are then assigned sister spore relationships, i.e. information about which spores originated from the same tetrad, using molecular barcodes read during genotyping. By removing the bottleneck of manual dissection, hundreds or even thousands of tetrads can be isolated in minutes. Here we present a detailed description of the experimental procedures required to perform BEST in the yeast Saccharomyces cerevisiae, starting with a heterozygous diploid strain through the isolation of colonies derived from the haploid meiotic progeny.
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http://dx.doi.org/10.3791/51401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172027PMC
May 2014

Quantitative analysis of colony morphology in yeast.

Biotechniques 2014 Jan;56(1):18-27

Pacific Northwest Diabetes Research Institute, Seattle, WA; Molecular and Cellular Biology Program, University of Washington, Seattle, WA.

Microorganisms often form multicellular structures such as biofilms and structured colonies that can influence the organism's virulence, drug resistance, and adherence to medical devices. Phenotypic classification of these structures has traditionally relied on qualitative scoring systems that limit detailed phenotypic comparisons between strains. Automated imaging and quantitative analysis have the potential to improve the speed and accuracy of experiments designed to study the genetic and molecular networks underlying different morphological traits. For this reason, we have developed a platform that uses automated image analysis and pattern recognition to quantify phenotypic signatures of yeast colonies. Our strategy enables quantitative analysis of individual colonies, measured at a single time point or over a series of time-lapse images, as well as the classification of distinct colony shapes based on image-derived features. Phenotypic changes in colony morphology can be expressed as changes in feature space trajectories over time, thereby enabling the visualization and quantitative analysis of morphological development. To facilitate data exploration, results are plotted dynamically through an interactive Yeast Image Analysis web application (YIMAA; http://yimaa.cs.tut.fi) that integrates the raw and processed images across all time points, allowing exploration of the image-based features and principal components associated with morphological development.
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http://dx.doi.org/10.2144/000114123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3996921PMC
January 2014

Aneuploidy underlies a multicellular phenotypic switch.

Proc Natl Acad Sci U S A 2013 Jul 28;110(30):12367-72. Epub 2013 Jun 28.

Institute for Systems Biology, Seattle, WA 98109, USA.

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.
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http://dx.doi.org/10.1073/pnas.1301047110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3725063PMC
July 2013

High-throughput tetrad analysis.

Nat Methods 2013 Jul 12;10(7):671-5. Epub 2013 May 12.

Institute for Systems Biology, Seattle, Washington, USA.

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. Maintaining tetrad information allows accurate inference of missing genetic markers and full genotypes of missing (and presumably nonviable) individuals. An individual researcher was able to isolate over 3,000 yeast tetrads in 3 h, an output equivalent to that of almost 1 month of manual dissection. BEST is transferable to other microorganisms for which meiotic mapping is significantly more laborious.
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http://dx.doi.org/10.1038/nmeth.2479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696418PMC
July 2013