DsdA (<scp>D</scp>‐serine deaminase): a new heterologous MX cassette for gene disruption and selection in <i>Saccharomyces cerevisiae</i>

Vorachek-Warren, Mara K.; McCusker, John H.

doi:10.1002/yea.1074

Cited by 32 publications

(17 citation statements)

References 27 publications

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“…Parent strains were made haploid by deleting the HO locus using the D-serine deaminase dsdAMX4 cassette, which confers ability to grow on D-serine as a sole nitrogen source (Vorachek-Warren and McCusker 2004). Genes to be tested were then knocked out with the MX hygromycin B cassette (Goldstein and McCusker 1999), and reciprocal hemizygotes were constructed by crossing the knockout to the other parent strain.…”

Section: Reciprocal Hemizygosity Analysismentioning

confidence: 99%

Small- and Large-Effect Quantitative Trait Locus Interactions Underlie Variation in Yeast Sporulation Efficiency

Lorenz

Cohen

2012

Genetics

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Quantitative trait loci (QTL) with small effects on phenotypic variation can be difficult to detect and analyze. Because of this a large fraction of the genetic architecture of many complex traits is not well understood. Here we use sporulation efficiency in Saccharomyces cerevisiae as a model complex trait to identify and study small-effect QTL. In crosses where the large-effect quantitative trait nucleotides (QTN) have been genetically fixed we identify small-effect QTL that explain approximately half of the remaining variation not explained by the major effects. We find that small-effect QTL are often physically linked to large-effect QTL and that there are extensive genetic interactions between small-and large-effect QTL. A more complete understanding of quantitative traits will require a better understanding of the numbers, effect sizes, and genetic interactions of small-effect QTL. COMPLEX traits exhibit non-Mendelian inheritance patterns, which arise from the segregation of multiple quantitative trait loci (QTL) (Lander and Schork 1994). A QTL is a region of the genome containing an allelic difference that causes a change in phenotype. Many medically and agriculturally important traits exhibit complex genetic architecture, including phenotypes ranging from diabetes and cancer penetrance to meat quality and frost tolerance in crops (Glazier et al. 2002;Heuven et al. 2009;Dumont et al. 2009;Gaudet et al. 2010). QTL with relatively large effects are the easiest to identify and analyze, yet most QTL have small average effects on complex traits (Mackay 2001). Thus, while theory and experiment suggest that a large fraction of the variation of many phenotypes will be explained by QTL with smaller effect sizes (Fisher 1930;Lango Allen et al. 2010;Yang et al. 2011), our current understanding of complex traits is based primarily on analyses of QTL with the largest effect sizes. Because small-effect QTL are necessarily more difficult to detect and analyze, a large fraction of the genetic architecture of most complex traits is not well understood. A more complete model of complex traits should include an understanding of the numbers, effect sizes, and interactions of small-effect QTL.To identify and study small-effect QTL, we used sporulation efficiency in the yeast Saccharomyces cerevisiae as a model complex trait (Gerke et al. 2006). This system offers several advantages for the study of QTL with relatively small-effect sizes. Sporulation efficiency is a highly heritable trait in yeast (Gerke et al. 2006). The measurements of sporulation efficiency can be performed in controlled environments that provide the statistical power to detect QTL with small-effect sizes. With this system, we previously identified four quantitative trait nucleotides (QTN) that have large effects on sporulation efficiency (Gerke et al. 2009). Here we describe crosses designed to uncover additional QTL that account for phenotypic variation not explained by the major-effect QTN. For the purposes of this study we define these addition...

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Section: Reciprocal Hemizygosity Analysismentioning

confidence: 99%

Small- and Large-Effect Quantitative Trait Locus Interactions Underlie Variation in Yeast Sporulation Efficiency

Lorenz

Cohen

2012

Genetics

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“…D-Serine ammonia lyase (DSDA, EC 4.2.1.14, formerly D-serine dehydratase), for example, catalyzes the deamination of D-serine, D-threonine, and D-allothreonine into their respective keto acids, ammonia, and water (Marceau et al 1988). The dsdA gene has been used as a selection marker gene in yeast transformation (Vorachek-Warren and McCusker 2004). Erikson et al (2005) demonstrated that the Escherichia coli dsdA can be used as a novel plant selectable marker with D-serine as the selection agent in Arabidopsis.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the E. coli d-serine ammonia lyase gene (Ec. dsdA) for use as a selectable marker in maize transformation

Lai

Privalle

Mei

et al. 2011

In Vitro Cell.Dev.Biol.-Plant

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In this study, the D-serine ammonia lyase (dsdA) gene from Escherichia coli was evaluated as a selectable marker for maize transformation. Plants are incapable of utilizing the D-form of most amino acids, and D-serine has recently been demonstrated to be phytoinhibitory to plant growth. D-Serine ammonia lyase detoxifies D-serine via a substrate-specific reaction to pyruvate, ammonia, and water. D-Serine inhibits germination of isolated maize immature embryos and growth of embryogenic callus from wild-type plants at concentrations about approx. 2-15 mM. Transgenic plants were recovered in the presence of D-serine in tissue culture media with dsdA as the selection marker at efficiencies comparable to using a mutated acetohydroxy acid synthase selection marker gene and selection in the presence of imidazolinone herbicides. Immature embryos infected with an Agrobacterium strain containing an acetohydroxy acid synthase gene construct without dsdA did not yield any transgenic events on the selection medium with 10 mM D-serine, indicating that D-serine provided selection tight enough to prevent escapes. Molecular analysis confirmed the integration of the dsdA gene into the genome of the transgenic plants. No adverse phenotypes were observed in the greenhouse, and expression of the dsdA marker had no affect on agronomic characteristics or grain yield in multi-location field trials. Seed compositional analysis demonstrated no significant differences in the contents of seed protein, starch, fatty acids, fiber, phytic acid, and free amino acids between transgenic and nontransgenic control plants. These data indicate that the dsdA gene is properly expressed in maize and the D-serine ammonia lyase (DSDA) enzyme functions appropriately to metabolize D-serine during in vitro selection. Preliminary safety assessments indicated that no adverse affects would be expected if humans were exposed to the DSDA protein in the diet from an allergenicity or toxicity perspective. The dsdA gene in combination with phytoinhibitory levels of Dserine represents a new and effective selectable marker system for maize transformation.

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“…Heterothallic strains were generated by deleting HO using dsdAMX (Vorachek-Warren and McCusker 2004), natMX (Goldstein and McCusker 1999) for BC187, dissecting tetrads and selecting MAT a or MAT α haploids. Only MAT α haploids were obtained for YJM210, DBVPG1106, and UWOPS87 and only MAT a for Y12.…”

Section: Methodsmentioning

confidence: 99%

A Noncomplementation Screen for Quantitative Trait Alleles inSaccharomyces cerevisiae

Huh¹,

Riles²,

Reyes³

et al. 2012

G3 Genes|Genomes|Genetics

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Both linkage and linkage disequilibrium mapping provide well-defined approaches to mapping quantitative trait alleles. However, alleles of small effect are particularly difficult to refine to individual genes and causative mutations. Quantitative noncomplementation provides a means of directly testing individual genes for quantitative trait alleles in a fixed genetic background. Here, we implement a genome-wide noncomplementation screen for quantitative trait alleles that affect colony color or size by using the yeast deletion collection. As proof of principle, we find a previously known allele of CYS4 that affects colony color and a novel allele of CTT1 that affects resistance to hydrogen peroxide. To screen nearly 4700 genes in nine diverse yeast strains, we developed a high-throughput robotic plating assay to quantify colony color and size. Although we found hundreds of candidate alleles, reciprocal hemizygosity analysis of a select subset revealed that many of the candidates were false positives, in part the result of background-dependent haploinsufficiency or second-site mutations within the yeast deletion collection. Our results highlight the difficulty of identifying small-effect alleles but support the use of noncomplementation as a rapid means of identifying quantitative trait alleles of large effect.

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DsdA (D‐serine deaminase): a new heterologous MX cassette for gene disruption and selection in Saccharomyces cerevisiae

Cited by 32 publications

References 27 publications

Small- and Large-Effect Quantitative Trait Locus Interactions Underlie Variation in Yeast Sporulation Efficiency

Small- and Large-Effect Quantitative Trait Locus Interactions Underlie Variation in Yeast Sporulation Efficiency

Evaluation of the E. coli d-serine ammonia lyase gene (Ec. dsdA) for use as a selectable marker in maize transformation

A Noncomplementation Screen for Quantitative Trait Alleles inSaccharomyces cerevisiae

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