Publications by authors named "Maitreya J Dunham"

83 Publications

Multiplexing Mutation Rate Assessment: Determining Pathogenicity of Msh2 Variants in S. cerevisiae.

Genetics 2021 Apr 12. Epub 2021 Apr 12.

Genome Sciences Department, University of Washington, Seattle, WA 98195, USA.

Despite the fundamental importance of mutation rate as a driving force in evolution and disease risk, common methods to assay mutation rate are time consuming and tedious. Established methods such as fluctuation tests and mutation accumulation experiments are low-throughput and often require significant optimization to ensure accuracy. We established a new method to determine the mutation rate of many strains simultaneously by tracking mutation events in a chemostat continuous culture device and applying deep sequencing to link mutations to alleles of a DNA-repair gene. We applied this method to assay the mutation rate of hundreds of Saccharomyces cerevisiae strains carrying mutations in the gene encoding Msh2, a DNA repair enzyme in the mismatch repair pathway (MMR). Loss-of-function (LOF) mutations in MSH2 are associated with hereditary non-polyposis colorectal cancer (HNPCC), an inherited disorder that increases risk for many different cancers. However, the vast majority of MSH2 variants found in human populations have insufficient evidence to be classified as either pathogenic or benign. We first benchmarked our method against Luria-Delbrück fluctuation tests using a collection of published MSH2 missense variants. Our pooled screen successfully identified previously characterized non-functional alleles as high mutators. We then created an additional 185 human missense variants in the yeast ortholog, including both characterized and uncharacterized alleles curated from ClinVar and other clinical testing data. In a set of alleles of known pathogenicity, our assay recapitulated ClinVar's classification; we then estimated pathogenicity for 157 variants classified as uncertain or conflicting reports of significance. This method is capable of studying the mutation rate of many microbial species and can be applied to problems ranging from the generation of high-fidelity polymerases to measuring the frequency of antibiotic resistance emergence.
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http://dx.doi.org/10.1093/genetics/iyab058DOI Listing
April 2021

Transposable Element Mobilization in Interspecific Yeast Hybrids.

Genome Biol Evol 2021 Mar;13(3)

Department of Genome Sciences, University of Washington, Seattle, Washington, USA.

Barbara McClintock first hypothesized that interspecific hybridization could provide a "genomic shock" that leads to the mobilization of transposable elements (TEs). This hypothesis is based on the idea that regulation of TE movement is potentially disrupted in hybrids. However, the handful of studies testing this hypothesis have yielded mixed results. Here, we set out to identify if hybridization can increase transposition rate and facilitate colonization of TEs in Saccharomyces cerevisiae × Saccharomyces uvarum interspecific yeast hybrids. Saccharomyces cerevisiae have a small number of active long terminal repeat retrotransposons (Ty elements), whereas their distant relative S. uvarum have lost the Ty elements active in S. cerevisiae. Although the regulation system of Ty elements is known in S. cerevisiae, it is unclear how Ty elements are regulated in other Saccharomyces species, and what mechanisms contributed to the loss of most classes of Ty elements in S. uvarum. Therefore, we first assessed whether TEs could insert in the S. uvarum sub-genome of a S. cerevisiae × S. uvarum hybrid. We induced transposition to occur in these hybrids and developed a sequencing technique to show that Ty elements insert readily and nonrandomly in the S. uvarum genome. We then used an in vivo reporter construct to directly measure transposition rate in hybrids, demonstrating that hybridization itself does not alter rate of mobilization. However, we surprisingly show that species-specific mitochondrial inheritance can change transposition rate by an order of magnitude. Overall, our results provide evidence that hybridization can potentially facilitate the introduction of TEs across species boundaries and alter transposition via mitochondrial transmission, but that this does not lead to unrestrained proliferation of TEs suggested by the genomic shock theory.
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http://dx.doi.org/10.1093/gbe/evab033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952228PMC
March 2021

Dietary Change Enables Robust Growth-Coupling of Heterologous Methyltransferase Activity in Yeast.

ACS Synth Biol 2020 12 12;9(12):3408-3415. Epub 2020 Nov 12.

Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.

Genetic modifications of living organisms and proteins are made possible by a catalogue of molecular and synthetic biology tools, yet proper screening assays for genetic variants of interest continue to lag behind. Synthetic growth-coupling (GC) of enzyme activities offers a simple, inexpensive way to track such improvements. In this follow-up study we present the optimization of a recently established GC design for screening of heterologous methyltransferases (MTases) and related pathways in the yeast . Specifically, upon testing different media compositions and genetic backgrounds, improved GC of different heterologous MTase activities is obtained. Furthermore, we demonstrate the strength of the system by screening a library of catechol O-MTase variants converting protocatechuic acid into vanillic acid. We demonstrated high correlation ( = 0.775) between vanillic acid and cell density as a proxy for MTase activity. We envision that the improved MTase GC can aid evolution-guided optimization of biobased production processes for methylated compounds with yeast in the future.
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http://dx.doi.org/10.1021/acssynbio.0c00348DOI Listing
December 2020

Effect of the Ala234Asp replacement in mitochondrial branched-chain amino acid aminotransferase on the production of BCAAs and fusel alcohols in yeast.

Appl Microbiol Biotechnol 2020 Sep 10;104(18):7915-7925. Epub 2020 Aug 10.

Division of Biological Science, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0192, Japan.

In the yeast Saccharomyces cerevisiae, the mitochondrial branched-chain amino acid (BCAA) aminotransferase Bat1 plays an important role in the synthesis of BCAAs (valine, leucine, and isoleucine). Our upcoming study (Large et al. bioRχiv. 10.1101/2020.06.26.166157, Large et al. 2020) will show that the heterozygous tetraploid beer yeast strain, Wyeast 1056, which natively has a variant causing one amino acid substitution of Ala234Asp in Bat1 on one of the four chromosomes, produced higher levels of BCAA-derived fusel alcohols in the brewer's wort medium than a derived strain lacking this mutation. Here, we investigated the physiological role of the A234D variant Bat1 in S. cerevisiae. Both bat1∆ and bat1 cells exhibited the same phenotypes relative to the wild-type Bat1 strain-namely, a repressive growth rate in the logarithmic phase; decreases in intracellular valine and leucine content in the logarithmic and stationary growth phases, respectively; an increase in fusel alcohol content in culture medium; and a decrease in the carbon dioxide productivity. These results indicate that amino acid change from Ala to Asp at position 234 led to a functional impairment of Bat1, although homology modeling suggests that Asp234 in the variant Bat1 did not inhibit enzymatic activity directly. KEY POINTS: • Yeast cells expressing Bat1 exhibited a slower growth phenotype. • The Val and Leu levels were decreased in yeast cells expressing Bat1. • The A234D substitution causes a loss-of-function in Bat1. • The A234D substitution in Bat1 increased fusel alcohol production in yeast cells.
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http://dx.doi.org/10.1007/s00253-020-10800-yDOI Listing
September 2020

The environmental stress response causes ribosome loss in aneuploid yeast cells.

Proc Natl Acad Sci U S A 2020 07 6;117(29):17031-17040. Epub 2020 Jul 6.

David H. Koch Institute for Integrative Cancer Research, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139;

Aneuploidy, a condition characterized by whole chromosome gains and losses, is often associated with significant cellular stress and decreased fitness. However, how cells respond to the aneuploid state has remained controversial. In aneuploid budding yeast, two opposing gene-expression patterns have been reported: the "environmental stress response" (ESR) and the "common aneuploidy gene-expression" (CAGE) signature, in which many ESR genes are oppositely regulated. Here, we investigate this controversy. We show that the CAGE signature is not an aneuploidy-specific gene-expression signature but the result of normalizing the gene-expression profile of actively proliferating aneuploid cells to that of euploid cells grown into stationary phase. Because growth into stationary phase is among the strongest inducers of the ESR, the ESR in aneuploid cells was masked when stationary phase euploid cells were used for normalization in transcriptomic studies. When exponentially growing euploid cells are used in gene-expression comparisons with aneuploid cells, the CAGE signature is no longer evident in aneuploid cells. Instead, aneuploid cells exhibit the ESR. We further show that the ESR causes selective ribosome loss in aneuploid cells, providing an explanation for the decreased cellular density of aneuploid cells. We conclude that aneuploid budding yeast cells mount the ESR, rather than the CAGE signature, in response to aneuploidy-induced cellular stresses, resulting in selective ribosome loss. We propose that the ESR serves two purposes in aneuploid cells: protecting cells from aneuploidy-induced cellular stresses and preventing excessive cellular enlargement during slowed cell cycles by down-regulating translation capacity.
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http://dx.doi.org/10.1073/pnas.2005648117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382292PMC
July 2020

Fitness benefits of loss of heterozygosity in hybrids.

Genome Res 2019 10 23;29(10):1685-1692. Epub 2019 Sep 23.

Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA.

With two genomes in the same organism, interspecific hybrids have unique fitness opportunities and costs. In both plants and yeasts, wild, pathogenic, and domesticated hybrids may eliminate portions of one parental genome, a phenomenon known as loss of heterozygosity (LOH). Laboratory evolution of hybrid yeast recapitulates these results, with LOH occurring in just a few hundred generations of propagation. In this study, we systematically looked for alleles that are beneficial when lost in order to determine how prevalent this mode of adaptation may be and to determine candidate loci that might underlie the benefits of larger-scale chromosome rearrangements. These aims were accomplished by mating with the deletion collection to create hybrids such that each nonessential allele is deleted. Competitive fitness assays of these pooled, barcoded, hemizygous strains, and accompanying controls, revealed a large number of loci for which LOH is beneficial. We found that the fitness effects of hemizygosity are dependent on the species context, the selective environment, and the species origin of the deleted allele. Further, we found that hybrids have a wider distribution of fitness consequences versus matched hemizygous diploids. Our results suggest that LOH can be a successful strategy for adaptation of hybrids to new environments, and we identify candidate loci that drive the chromosomal rearrangements observed in evolution of yeast hybrids.
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http://dx.doi.org/10.1101/gr.245605.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771408PMC
October 2019

Interrogation of CYP2D6 Structural Variant Alleles Improves the Correlation Between CYP2D6 Genotype and CYP2D6-Mediated Metabolic Activity.

Clin Transl Sci 2020 01 25;13(1):147-156. Epub 2019 Oct 25.

Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, Montana, USA.

The cytochrome P450 2D6 (CYP2D6) gene locus is challenging to accurately genotype due to numerous single nucleotide variants and complex structural variation. Our goal was to determine whether the CYP2D6 genotype-phenotype correlation is improved when diplotype assignments incorporate structural variation, identified by the bioinformatics tool Stargazer, with next-generation sequencing data. Using CYP2D6 activity measured with substrates dextromethorphan and metoprolol, activity score explained 40% and 34% of variability in metabolite formation rates, respectively, when diplotype calls incorporated structural variation, increasing from 36% and 31%, respectively, when diplotypes did not incorporate structural variation. We also investigated whether the revised Clinical Pharmacogenetics Implementation Consortium (CPIC) recommendations for translating genotype to phenotype improve CYP2D6 activity predictions over the current system. Although the revised recommendations do not improve the correlation between activity score and CYP2D6 activity, perhaps because of low frequency of the CYP2D6*10 allele, the correlation with metabolizer phenotype group was significantly improved for both substrates. We also measured the function of seven rare coding variants: one (A449D) exhibited decreased (44%) and another (R474Q) increased (127%) activity compared with reference CYP2D6.1 protein. Allele-specific analysis found that A449D is part of a novel CYP2D6*4 suballele, CYP2D6*4.028. The novel haplotype containing R474Q was designated CYP2D6*138 by PharmVar; another novel haplotype containing R365H was designated CYP2D6*139. Accuracy of CYP2D6 phenotype prediction is improved when the CYP2D6 gene locus is interrogated using next-generation sequencing coupled with structural variation analysis. Additionally, revised CPIC genotype to phenotype translation recommendations provides an improvement in assigning CYP2D6 activity.
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http://dx.doi.org/10.1111/cts.12695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6951848PMC
January 2020

Temperature preference can bias parental genome retention during hybrid evolution.

PLoS Genet 2019 09 16;15(9):e1008383. Epub 2019 Sep 16.

Genome Sciences Department, University of Washington, Seattle, Washington, United States of America.

Interspecific hybridization can introduce genetic variation that aids in adaptation to new or changing environments. Here, we investigate how hybrid adaptation to temperature and nutrient limitation may alter parental genome representation over time. We evolved Saccharomyces cerevisiae x Saccharomyces uvarum hybrids in nutrient-limited continuous culture at 15°C for 200 generations. In comparison to previous evolution experiments at 30°C, we identified a number of responses only observed in the colder temperature regime, including the loss of the S. cerevisiae allele in favor of the cryotolerant S. uvarum allele for several portions of the hybrid genome. In particular, we discovered a genotype by environment interaction in the form of a loss of heterozygosity event on chromosome XIII; which species' haplotype is lost or maintained is dependent on the parental species' temperature preference and the temperature at which the hybrid was evolved. We show that a large contribution to this directionality is due to a temperature dependent fitness benefit at a single locus, the high affinity phosphate transporter gene PHO84. This work helps shape our understanding of what forces impact genome evolution after hybridization, and how environmental conditions may promote or disfavor the persistence of hybrids over time.
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http://dx.doi.org/10.1371/journal.pgen.1008383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6762194PMC
September 2019

Phenotypic and Genotypic Consequences of CRISPR/Cas9 Editing of the Replication Origins in the rDNA of .

Genetics 2019 09 10;213(1):229-249. Epub 2019 Jul 10.

Department of Genome Sciences, University of Washington, Seattle, Washington 98195

The complex structure and repetitive nature of eukaryotic ribosomal DNA (rDNA) is a challenge for genome assembly, thus the consequences of sequence variation in rDNA remain unexplored. However, renewed interest in the role that rDNA variation may play in diverse cellular functions, aside from ribosome production, highlights the need for a method that would permit genetic manipulation of the rDNA. Here, we describe a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based strategy to edit the rDNA locus in the budding yeast , developed independently but similar to one developed by others. Using this approach, we modified the endogenous rDNA origin of replication in each repeat by deleting or replacing its consensus sequence. We characterized the transformants that have successfully modified their rDNA locus and propose a mechanism for how CRISPR/Cas9-mediated editing of the rDNA occurs. In addition, we carried out extended growth and life span experiments to investigate the long-term consequences that altering the rDNA origin of replication have on cellular health. We find that long-term growth of the edited clones results in faster-growing suppressors that have acquired segmental aneusomy of the rDNA-containing region of chromosome XII or aneuploidy of chromosomes XII, II, or IV. Furthermore, we find that all edited isolates suffer a reduced life span, irrespective of their levels of extrachromosomal rDNA circles. Our work demonstrates that it is possible to quickly, efficiently, and homogeneously edit the rDNA origin via CRISPR/Cas9.
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http://dx.doi.org/10.1534/genetics.119.302351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727806PMC
September 2019

Applying Multiplex Assays to Understand Variation in Pharmacogenes.

Clin Pharmacol Ther 2019 08 30;106(2):290-294. Epub 2019 May 30.

Department of Genome Sciences, University of Washington, Seattle, Washington, USA.

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http://dx.doi.org/10.1002/cpt.1468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6663607PMC
August 2019

A combination of transcription factors mediates inducible interchromosomal contacts.

Elife 2019 05 13;8. Epub 2019 May 13.

Department of Genome Sciences, University of Washington, Seattle, United States.

The genome forms specific three-dimensional contacts in response to cellular or environmental conditions. However, it remains largely unknown which proteins specify and mediate such contacts. Here we describe an assay, MAP-C (Mutation Analysis in Pools by Chromosome conformation capture), that simultaneously characterizes the effects of hundreds of or -acting mutations on a chromosomal contact. Using MAP-C, we show that inducible interchromosomal pairing between alleles in saturated cultures of yeast is mediated by three transcription factors, Leu3, Sdd4 (Ypr022c), and Rgt1. The coincident, combined binding of all three factors is strongest at the locus and is also specific to saturated conditions. We applied MAP-C to further explore the biochemical mechanism of these contacts, and find they require the structured regulatory domain of Rgt1, but no known interaction partners of Rgt1. Altogether, our results demonstrate MAP-C as a powerful method for dissecting the mechanistic basis of chromosome conformation.
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http://dx.doi.org/10.7554/eLife.42499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548505PMC
May 2019

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

PLoS Biol 2019 03 5;17(3):e3000147. Epub 2019 Mar 5.

Pacific Northwest Research Institute, Seattle, Washington, United States of America.

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. We examined the population genetic history of beer strains and found that ale strains and the S. cerevisiae portion of allotetraploid lager strains were derived from admixture between populations closely related to European grape wine strains and Asian rice wine strains. Similar to both lager and baking strains, ale strains are polyploid, providing them with a passive means of remaining isolated from other populations and providing us with a living relic of their ancestral hybridization. To reconstruct their polyploid origin, we phased the genomes of two ale strains and found ale haplotypes to both be recombinants between European and Asian alleles and to also contain novel alleles derived from extinct or as yet uncharacterized populations. We conclude that modern beer strains are the product of a historical melting pot of fermentation technology.
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http://dx.doi.org/10.1371/journal.pbio.3000147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400334PMC
March 2019

Transposon insertional mutagenesis in reveals -acting effects influencing species-dependent essential genes.

Genome Res 2019 03 11;29(3):396-406. Epub 2019 Jan 11.

Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA.

To understand how complex genetic networks perform and regulate diverse cellular processes, the function of each individual component must be defined. Comprehensive phenotypic studies of mutant alleles have been successful in model organisms in determining what processes depend on the normal function of a gene. These results are often ported to newly sequenced genomes by using sequence homology. However, sequence similarity does not always mean identical function or phenotype, suggesting that new methods are required to functionally annotate newly sequenced species. We have implemented comparative analysis by high-throughput experimental testing of gene dispensability in , a sister species of We created haploid and heterozygous diploid Tn7 insertional mutagenesis libraries in to identify species-dependent essential genes, with the goal of detecting genes with divergent functions and/or different genetic interactions. Comprehensive gene dispensability comparisons with predicted diverged dispensability at 12% of conserved orthologs, and validation experiments confirmed 22 differentially essential genes. Despite their differences in essentiality, these genes were capable of cross-species complementation, demonstrating that acting factors that are background-dependent contribute to differential gene essentiality. This study shows that direct experimental testing of gene disruption phenotypes across species can inform comparative genomic analyses and improve gene annotations. Our method can be widely applied in microorganisms to further our understanding of genome evolution.
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http://dx.doi.org/10.1101/gr.232330.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396416PMC
March 2019

Expression and Functional Characterization of Breast Cancer-Associated Cytochrome P450 4Z1 in .

Drug Metab Dispos 2017 12 10;45(12):1364-1371. Epub 2017 Oct 10.

Departments of Medicinal Chemistry (M.G.M., S.R., J.P.K., L.P., A.N., B.G., R.A.T., A.E.R.), Genome Sciences (K.A.S., C.J.A., M.J.D., D.M.F.), and Bioengineering (D.M.F.), University of Washington, Seattle, Washington

CYP4Z1 is an "orphan" cytochrome P450 (P450) enzyme that has provoked interest because of its hypothesized role in breast cancer through formation of the signaling molecule 20-hydroxyeicosatetraenoic acid (20-HETE). We expressed human CYP4Z1 in and evaluated its catalytic capabilities toward arachidonic and lauric acids (AA and LA). Specific and sensitive mass spectrometry assays enabled discrimination of the regioselectivity of hydroxylation of these two fatty acids. CYP4Z1 generated 7-, 8-, 9-, 10-, and 11-hydroxy LA, whereas the 12-hydroxy metabolite was not detected. HET0016, the prototypic CYP4 inhibitor, only weakly inhibited laurate metabolite formation (IC ∼15 M). CYP4Z1 preferentially oxidized AA to the 14(S),15(R)-epoxide with high regioselectivity and stereoselectivity, a reaction that was also insensitive to HET0016, but neither 20-HETE nor 20-carboxy-AA were detectable metabolites. Docking of LA and AA into a CYP4Z1 homology model was consistent with this preference for internal fatty acid oxidation. Thus, human CYP4Z1 has an inhibitor profile and product regioselectivity distinct from most other CYP4 enzymes, consistent with CYP4Z1's lack of a covalently linked heme. These data suggest that, if CYP4Z1 modulates breast cancer progression, it does so by a mechanism other than direct production of 20-HETE.
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http://dx.doi.org/10.1124/dmd.117.078188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5697098PMC
December 2017

Identification of a novel interspecific hybrid yeast from a metagenomic spontaneously inoculated beer sample using Hi-C.

Yeast 2018 01 19;35(1):71-84. Epub 2017 Oct 19.

Department of Genome Sciences, University of Washington, Seattle, WA, USA.

Interspecific hybridization is a common mechanism enabling genetic diversification and adaptation; however, the detection of hybrid species has been quite difficult. The identification of microbial hybrids is made even more complicated, as most environmental microbes are resistant to culturing and must be studied in their native mixed communities. We have previously adapted the chromosome conformation capture method Hi-C to the assembly of genomes from mixed populations. Here, we show the method's application in assembling genomes directly from an uncultured, mixed population from a spontaneously inoculated beer sample. Our assembly method has enabled us to de-convolute four bacterial and four yeast genomes from this sample, including a putative yeast hybrid. Downstream isolation and analysis of this hybrid confirmed its genome to consist of Pichia membranifaciens and that of another related, but undescribed, yeast. Our work shows that Hi-C-based metagenomic methods can overcome the limitation of traditional sequencing methods in studying complex mixtures of genomes. Copyright © 2017 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/yea.3280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771821PMC
January 2018

Variant Interpretation: Functional Assays to the Rescue.

Am J Hum Genet 2017 Sep;101(3):315-325

Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA; Department of Bioengineering, University of Washington, Seattle, WA 98195, USA. Electronic address:

Classical genetic approaches for interpreting variants, such as case-control or co-segregation studies, require finding many individuals with each variant. Because the overwhelming majority of variants are present in only a few living humans, this strategy has clear limits. Fully realizing the clinical potential of genetics requires that we accurately infer pathogenicity even for rare or private variation. Many computational approaches to predicting variant effects have been developed, but they can identify only a small fraction of pathogenic variants with the high confidence that is required in the clinic. Experimentally measuring a variant's functional consequences can provide clearer guidance, but individual assays performed only after the discovery of the variant are both time and resource intensive. Here, we discuss how multiplex assays of variant effect (MAVEs) can be used to measure the functional consequences of all possible variants in disease-relevant loci for a variety of molecular and cellular phenotypes. The resulting large-scale functional data can be combined with machine learning and clinical knowledge for the development of "lookup tables" of accurate pathogenicity predictions. A coordinated effort to produce, analyze, and disseminate large-scale functional data generated by multiplex assays could be essential to addressing the variant-interpretation crisis.
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http://dx.doi.org/10.1016/j.ajhg.2017.07.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5590843PMC
September 2017

Chemostat Culture for Yeast Physiology and Experimental Evolution.

Cold Spring Harb Protoc 2017 Jul 5;2017(7):pdb.top077610. Epub 2017 Jul 5.

Department of Genome Sciences, University of Washington, Seattle, Washington 98195.

Continuous culture provides many benefits over the classical batch style of growing yeast cells. Steady-state cultures allow for precise control of growth rate and environment. Cultures can be propagated for weeks or months in these controlled environments, which is important for the study of experimental evolution. Despite these advantages, chemostats have not become a highly used system, in large part because of their historical impracticalities, including low throughput, large footprint, systematic complexity, commercial unavailability, high cost, and insufficient protocol availability. However, we have developed methods for building a relatively simple, low-cost, small footprint array of chemostats that can be run in multiples of 32. This "ministat array" can be applied to problems in yeast physiology and experimental evolution.
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http://dx.doi.org/10.1101/pdb.top077610DOI Listing
July 2017

Chemostat Culture for Yeast Experimental Evolution.

Cold Spring Harb Protoc 2017 Jul 5;2017(7):pdb.prot089011. Epub 2017 Jul 5.

Department of Genome Sciences, University of Washington, Seattle, Washington 98195

Experimental evolution is one approach used to address a broad range of questions related to evolution and adaptation to strong selection pressures. Experimental evolution of diverse microbial and viral systems has routinely been used to study new traits and behaviors and also to dissect mechanisms of rapid evolution. This protocol describes the practical aspects of experimental evolution with yeast grown in chemostats, including the setup of the experiment and sampling methods as well as best laboratory and record-keeping practices.
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http://dx.doi.org/10.1101/pdb.prot089011DOI Listing
July 2017

Chemostat Culture for Yeast Physiology.

Cold Spring Harb Protoc 2017 Jul 5;2017(7):pdb.prot089003. Epub 2017 Jul 5.

Department of Genome Sciences, University of Washington, Seattle, Washington 98195

The use of chemostat culture facilitates the careful comparison of different yeast strains growing in well-defined conditions. Variations in physiology can be measured by examining gene expression, metabolite levels, protein content, and cell morphology. In this protocol, we show how a combination of sample types can be collected during harvest from a single 20-mL chemostat in a ministat array, with special attention to coordinating the handling of the most time-sensitive sample types.
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http://dx.doi.org/10.1101/pdb.prot089003DOI Listing
July 2017

Assembly of a Mini-Chemostat Array.

Cold Spring Harb Protoc 2017 Jul 5;2017(7):pdb.prot088997. Epub 2017 Jul 5.

Department of Genome Sciences, University of Washington, Seattle, Washington 98195

Here, we describe instructions for the assembly of an array of miniature (20-mL) chemostats or "ministats" built from relatively inexpensive off-the-shelf parts. In experiments with yeast cultures, we have observed reproducibility in cellular physiology, gene expression patterns, and evolutionary outcomes with different ministats as well as between ministats and commercial large-volume platforms. Growth in continuous culture is a primary means for the characterization of yeast steady-state physiology, competition between strains, and long-term evolution experiments. We hope that these relatively inexpensive and high-throughput devices make the advantages of continuous culture growth more accessible to researchers.
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http://dx.doi.org/10.1101/pdb.prot088997DOI Listing
July 2017

The dynamic three-dimensional organization of the diploid yeast genome.

Elife 2017 05 24;6. Epub 2017 May 24.

Department of Genome Sciences, University of Washington, Seattle, United States.

The budding yeast is a long-standing model for the three-dimensional organization of eukaryotic genomes. However, even in this well-studied model, it is unclear how homolog pairing in diploids or environmental conditions influence overall genome organization. Here, we performed high-throughput chromosome conformation capture on diverged hybrid diploids to obtain the first global view of chromosome conformation in diploid yeasts. After controlling for the Rabl-like orientation using a polymer model, we observe significant homolog proximity that increases in saturated culture conditions. Surprisingly, we observe a localized increase in homologous interactions between the alleles specifically under galactose induction and saturated growth. This pairing is accompanied by relocalization to the nuclear periphery and requires Nup2, suggesting a role for nuclear pore complexes. Together, these results reveal that the diploid yeast genome has a dynamic and complex 3D organization.
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http://dx.doi.org/10.7554/eLife.23623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5476426PMC
May 2017

Experimental Evolution Reveals Favored Adaptive Routes to Cell Aggregation in Yeast.

Genetics 2017 06 26;206(2):1153-1167. Epub 2017 Apr 26.

Department of Genome Sciences, University of Washington School of Medicine, Seattle, Washington 98195

Yeast flocculation is a community-building cell aggregation trait that is an important mechanism of stress resistance and a useful phenotype for brewers; however, it is also a nuisance in many industrial processes, in clinical settings, and in the laboratory. Chemostat-based evolution experiments are impaired by inadvertent selection for aggregation, which we observe in 35% of populations. These populations provide a testing ground for understanding the breadth of genetic mechanisms uses to flocculate, and which of those mechanisms provide the biggest adaptive advantages. In this study, we employed experimental evolution as a tool to ask whether one or many routes to flocculation are favored, and to engineer a strain with reduced flocculation potential. Using a combination of whole genome sequencing and bulk segregant analysis, we identified causal mutations in 23 independent clones that had evolved cell aggregation during hundreds of generations of chemostat growth. In 12 of those clones, we identified a transposable element insertion in the promoter region of known flocculation gene , and, in an additional five clones, we recovered loss-of-function mutations in transcriptional repressor , which regulates and other related genes. Other causal mutations were found in genes that have not been previously connected to flocculation. Evolving a deletion strain revealed that this single deletion reduces flocculation occurrences to 3%, and demonstrated the efficacy of using experimental evolution as a tool to identify and eliminate the primary adaptive routes for undesirable traits.
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http://dx.doi.org/10.1534/genetics.116.198895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499169PMC
June 2017

A multiplex culture system for the long-term growth of fission yeast cells.

Yeast 2017 08 6;34(8):343-355. Epub 2017 Jun 6.

SyntheCell Team, Institute of Genetics and Development of Rennes, CNRS UMR 6290, Rennes, France.

Maintenance of long-term cultures of yeast cells is central to a broad range of investigations, from metabolic studies to laboratory evolution assays. However, repeated dilutions of batch cultures lead to variations in medium composition, with implications for cell physiology. In Saccharomyces cerevisiae, powerful miniaturized chemostat setups, or ministat arrays, have been shown to allow for constant dilution of multiple independent cultures. Here we set out to adapt these arrays for continuous culture of a morphologically and physiologically distinct yeast, the fission yeast Schizosaccharomyces pombe, with the goal of maintaining constant population density over time. First, we demonstrated that the original ministats are incompatible with growing fission yeast for more than a few generations, prompting us to modify different aspects of the system design. Next, we identified critical parameters for sustaining unbiased vegetative growth in these conditions. This requires deletion of the gsf2 flocculin-encoding gene, along with addition of galactose to the medium and lowering of the culture temperature. Importantly, we improved the flexibility of the ministats by developing a piezo-pump module for the independent regulation of the dilution rate of each culture. This made it possible to easily grow strains that have different generation times in the same assay. Our system therefore allows for maintaining multiple fission yeast cultures in exponential growth, adapting the dilution of each culture over time to keep constant population density for hundreds of generations. These multiplex culture systems open the door to a new range of long-term experiments using this model organism. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/yea.3237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542872PMC
August 2017

Loss of Heterozygosity Drives Adaptation in Hybrid Yeast.

Mol Biol Evol 2017 07;34(7):1596-1612

Department of Genome Sciences, University of Washington, Seattle, WA.

Hybridization is often considered maladaptive, but sometimes hybrids can invade new ecological niches and adapt to novel or stressful environments better than their parents. The genomic changes that occur following hybridization that facilitate genome resolution and/or adaptation are not well understood. Here, we examine hybrid genome evolution using experimental evolution of de novo interspecific hybrid yeast Saccharomyces cerevisiae × Saccharomyces uvarum and their parentals. We evolved these strains in nutrient-limited conditions for hundreds of generations and sequenced the resulting cultures identifying numerous point mutations, copy number changes, and loss of heterozygosity (LOH) events, including species-biased amplification of nutrient transporters. We focused on a particularly interesting example, in which we saw repeated LOH at the high-affinity phosphate transporter gene PHO84 in both intra- and interspecific hybrids. Using allele replacement methods, we tested the fitness of different alleles in hybrid and S. cerevisiae strain backgrounds and found that the LOH is indeed the result of selection on one allele over the other in both S. cerevisiae and the hybrids. This is an example where hybrid genome resolution is driven by positive selection on existing heterozygosity and demonstrates that even infrequent outcrossing may have lasting impacts on adaptation.
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http://dx.doi.org/10.1093/molbev/msx098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5455960PMC
July 2017

Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome.

Nat Genet 2017 Apr 6;49(4):643-650. Epub 2017 Mar 6.

US Meat Animal Research Center, ARS USDA, Clay Center, Nebraska, USA.

The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus) based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced what is, to our knowledge, the most continuous de novo mammalian assembly to date, with chromosome-length scaffolds and only 649 gaps. Our assembly represents a ∼400-fold improvement in continuity due to properly assembled gaps, compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, representing the largest repeat family and immune gene complex yet produced for an individual of a ruminant species.
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http://dx.doi.org/10.1038/ng.3802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909822PMC
April 2017

Differential paralog divergence modulates genome evolution across yeast species.

PLoS Genet 2017 02 14;13(2):e1006585. Epub 2017 Feb 14.

Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America.

Evolutionary outcomes depend not only on the selective forces acting upon a species, but also on the genetic background. However, large timescales and uncertain historical selection pressures can make it difficult to discern such important background differences between species. Experimental evolution is one tool to compare evolutionary potential of known genotypes in a controlled environment. Here we utilized a highly reproducible evolutionary adaptation in Saccharomyces cerevisiae to investigate whether experimental evolution of other yeast species would select for similar adaptive mutations. We evolved populations of S. cerevisiae, S. paradoxus, S. mikatae, S. uvarum, and interspecific hybrids between S. uvarum and S. cerevisiae for ~200-500 generations in sulfate-limited continuous culture. Wild-type S. cerevisiae cultures invariably amplify the high affinity sulfate transporter gene, SUL1. However, while amplification of the SUL1 locus was detected in S. paradoxus and S. mikatae populations, S. uvarum cultures instead selected for amplification of the paralog, SUL2. We measured the relative fitness of strains bearing deletions and amplifications of both SUL genes from different species, confirming that, converse to S. cerevisiae, S. uvarum SUL2 contributes more to fitness in sulfate limitation than S. uvarum SUL1. By measuring the fitness and gene expression of chimeric promoter-ORF constructs, we were able to delineate the cause of this differential fitness effect primarily to the promoter of S. uvarum SUL1. Our data show evidence of differential sub-functionalization among the sulfate transporters across Saccharomyces species through recent changes in noncoding sequence. Furthermore, these results show a clear example of how such background differences due to paralog divergence can drive changes in genome evolution.
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http://dx.doi.org/10.1371/journal.pgen.1006585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5308817PMC
February 2017

Evolution of protein phosphorylation across 18 fungal species.

Science 2016 10;354(6309):229-232

Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.

Living organisms have evolved protein phosphorylation, a rapid and versatile mechanism that drives signaling and regulates protein function. We report the phosphoproteomes of 18 fungal species and a phylogenetic-based approach to study phosphosite evolution. We observe rapid divergence, with only a small fraction of phosphosites conserved over hundreds of millions of years. Relative to recently acquired phosphosites, ancient sites are enriched at protein interfaces and are more likely to be functionally important, as we show for sites on H2A1 and eIF4E. We also observe a change in phosphorylation motif frequencies and kinase activities that coincides with the whole-genome duplication event. Our results provide an evolutionary history for phosphosites and suggest that rapid evolution of phosphorylation can contribute strongly to phenotypic diversity.
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http://dx.doi.org/10.1126/science.aaf2144DOI Listing
October 2016

High-Throughput Identification of Adaptive Mutations in Experimentally Evolved Yeast Populations.

PLoS Genet 2016 Oct 11;12(10):e1006339. Epub 2016 Oct 11.

Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America.

High-throughput sequencing has enabled genetic screens that can rapidly identify mutations that occur during experimental evolution. The presence of a mutation in an evolved lineage does not, however, constitute proof that the mutation is adaptive, given the well-known and widespread phenomenon of genetic hitchhiking, in which a non-adaptive or even detrimental mutation can co-occur in a genome with a beneficial mutation and the combined genotype is carried to high frequency by selection. We approximated the spectrum of possible beneficial mutations in Saccharomyces cerevisiae using sets of single-gene deletions and amplifications of almost all the genes in the S. cerevisiae genome. We determined the fitness effects of each mutation in three different nutrient-limited conditions using pooled competitions followed by barcode sequencing. Although most of the mutations were neutral or deleterious, ~500 of them increased fitness. We then compared those results to the mutations that actually occurred during experimental evolution in the same three nutrient-limited conditions. On average, ~35% of the mutations that occurred during experimental evolution were predicted by the systematic screen to be beneficial. We found that the distribution of fitness effects depended on the selective conditions. In the phosphate-limited and glucose-limited conditions, a large number of beneficial mutations of nearly equivalent, small effects drove the fitness increases. In the sulfate-limited condition, one type of mutation, the amplification of the high-affinity sulfate transporter, dominated. In the absence of that mutation, evolution in the sulfate-limited condition involved mutations in other genes that were not observed previously-but were predicted by the systematic screen. Thus, gross functional screens have the potential to predict and identify adaptive mutations that occur during experimental evolution.
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http://dx.doi.org/10.1371/journal.pgen.1006339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5065121PMC
October 2016

Neocentromeres Provide Chromosome Segregation Accuracy and Centromere Clustering to Multiple Loci along a Candida albicans Chromosome.

PLoS Genet 2016 Sep 23;12(9):e1006317. Epub 2016 Sep 23.

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America.

Assembly of kinetochore complexes, involving greater than one hundred proteins, is essential for chromosome segregation and genome stability. Neocentromeres, or new centromeres, occur when kinetochores assemble de novo, at DNA loci not previously associated with kinetochore proteins, and they restore chromosome segregation to chromosomes lacking a functional centromere. Neocentromeres have been observed in a number of diseases and may play an evolutionary role in adaptation or speciation. However, the consequences of neocentromere formation on chromosome missegregation rates, gene expression, and three-dimensional (3D) nuclear structure are not well understood. Here, we used Candida albicans, an organism with small, epigenetically-inherited centromeres, as a model system to study the functions of twenty different neocentromere loci along a single chromosome, chromosome 5. Comparison of neocentromere properties relative to native centromere functions revealed that all twenty neocentromeres mediated chromosome segregation, albeit to different degrees. Some neocentromeres also caused reduced levels of transcription from genes found within the neocentromere region. Furthermore, like native centromeres, neocentromeres clustered in 3D with active/functional centromeres, indicating that formation of a new centromere mediates the reorganization of 3D nuclear architecture. This demonstrates that centromere clustering depends on epigenetically defined function and not on the primary DNA sequence, and that neocentromere function is independent of its distance from the native centromere position. Together, the results show that a neocentromere can form at many loci along a chromosome and can support the assembly of a functional kinetochore that exhibits native centromere functions including chromosome segregation accuracy and centromere clustering within the nucleus.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035033PMC
http://dx.doi.org/10.1371/journal.pgen.1006317DOI Listing
September 2016

Characterization of a panARS-based episomal vector in the methylotrophic yeast Pichia pastoris for recombinant protein production and synthetic biology applications.

Microb Cell Fact 2016 Aug 11;15(1):139. Epub 2016 Aug 11.

Institute of Medical Biology (A-STAR), 8a Biomedical Grove, #06-06, Singapore, 138648, Singapore.

Background: Recombinant protein production in the methylotrophic yeast Pichia pastoris largely relies on integrative vectors. Although the stability of integrated expression cassettes is well appreciated for most applications, the availability of reliable episomal vectors for this host would represent a useful tool to expedite cloning and high-throughput screening, ameliorating also the relatively high clonal variability reported in transformants from integrative vectors caused by off-target integration in the P. pastoris genome. Recently, heterologous and endogenous autonomously replicating sequences (ARS) were identified in P. pastoris by genome mining, opening the possibility of expanding the available toolbox to include efficient episomal plasmids. The aim of this technical report is to validate a 452-bp sequence ("panARS") in context of P. pastoris expression vectors, and to compare their performance to classical integrative plasmids. Moreover, we aimed to test if such episomal vectors would be suitable to sustain in vivo recombination, using fragments for transformation, directly in P. pastoris cells.

Results: A panARS-based episomal vector was evaluated using blue fluorescent protein (BFP) as a reporter gene. Normalized fluorescence from colonies carrying panARS-BFP outperformed the level of signal obtained from integrative controls by several-fold, whereas endogenous sequences, identified from the P. pastoris genome, were not as efficient in terms of protein production. At the single cell level, panARS-BFP clones showed lower interclonal variability but higher intraclonal variation compared to their integrative counterparts, supporting the idea that heterologous protein production could benefit from episomal plasmids. Finally, efficiency of 2-fragment and 3-fragment in vivo recombination was tested using varying lengths of overlapping regions and molar ratios between fragments. Upon optimization, minimal background was obtained for in vivo assembled vectors, suggesting this could be a quick and efficient method to generate of episomal plasmids of interest.

Conclusions: An expression vector based on the panARS sequence was shown to outperform its integrative counterparts in terms of protein productivity and interclonal variability, facilitating recombinant protein expression and screening. Using optimized fragment lengths and ratios, it was possible to perform reliable in vivo recombination of fragments in P. pastoris. Taken together, these results support the applicability of panARS episomal vectors for synthetic biology approaches.
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http://dx.doi.org/10.1186/s12934-016-0540-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981965PMC
August 2016