The spectrum of Trp− mutants isolated as 5‐fluoroanthranilate‐resistant clones in Saccharomyces bayanus, S. mikatae and S. paradoxus

Jones, Elizabeth W.; Berget, Peter B.; Burnette, James M.; Anderson, Candice; Asafu-Adjei, Denise; Avetisian, Seda; Barrie, Fatmata; Chen, Ruby; Chu, Bur; Conroy, Samantha A.; Conroy, Sean; Dill, Allyson; Eimer, William A.; Garrity, Diane; Greenwood, Alexander I.; Hamilton, Tamara P.; Hucko, Simon; Jackson, C. R. S.; Livesey, Kristen M.; Monaco, Tiffany; Onorato, C.E.; Otsuka, Mai; Pai, Satyan; Schaeffer, G Bradley; Shung, Sharon; Spath, Samantha; Stahlman, J.; Sweeney, Blake A.; Wiltrout, Elizabeth; Yurovsky, Daniel; Zonneveld, Andrea

doi:10.1002/yea.1552

Cited by 14 publications

(7 citation statements)

References 8 publications

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“…The two species can make interspecific hybrids, allowing complementation tests with S. cerevisiae alleles. However, with a few exceptions (Serra et al 2003; Talarek et al 2004; Jones et al 2008; Zill and Rine 2008; Gallagher et al 2009; Zill et al 2010), little experimental work has been performed in S. bayanus and even less at genome scale (Bullard et al 2010; Tsankov et al 2010; Busby et al 2011; Guan et al 2011; Muller and Nieduszynski 2012). …”

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confidence: 99%

A New System for Comparative Functional Genomics of Saccharomyces Yeasts

et al. 2013

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Whole-genome sequencing, particularly in fungi, has progressed at a tremendous rate. More difficult, however, is experimental testing of the inferences about gene function that can be drawn from comparative sequence analysis alone. We present a genome-wide functional characterization of a sequenced but experimentally understudied budding yeast, Saccharomyces bayanus var. uvarum (henceforth referred to as S. bayanus), allowing us to map changes over the 20 million years that separate this organism from S. cerevisiae. We first created a suite of genetic tools to facilitate work in S. bayanus. Next, we measured the gene-expression response of S. bayanus to a diverse set of perturbations optimized using a computational approach to cover a diverse array of functionally relevant biological responses. The resulting data set reveals that gene-expression patterns are largely conserved, but significant changes may exist in regulatory networks such as carbohydrate utilization and meiosis. In addition to regulatory changes, our approach identified gene functions that have diverged. The functions of genes in core pathways are highly conserved, but we observed many changes in which genes are involved in osmotic stress, peroxisome biogenesis, and autophagy. A surprising number of genes specific to S. bayanus respond to oxidative stress, suggesting the organism may have evolved under different selection pressures than S. cerevisiae. This work expands the scope of genome-scale evolutionary studies from sequence-based analysis to rapid experimental characterization and could be adopted for functional mapping in any lineage of interest. Furthermore, our detailed characterization of S. bayanus provides a valuable resource for comparative functional genomics studies in yeast.

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confidence: 99%

A New System for Comparative Functional Genomics of Saccharomyces Yeasts

et al. 2013

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“…It is therefore highly likely that the FAA‐resistant and Trp − auxotrophic colonies might be derived from additional mutation(s) of H. polymorpha TRP3, TRP4 or TRP5, which could also confer resistance to 5‐FAA in the tryptophan biosynthetic pathway (Toyn et al , 2000). Similarly, a recent study reported that, among Trp − and FAA‐resistant mutants of diploid and/or haploid Saccharomyces ‐related species, trp3, trp4 and trp5 mutants were recovered; in contrast, no trp1 mutants were recovered by complementation test (Jones et al , 2008). Thus, recycling of the HpTRP1 gene appears impractical for multiple disruptions in H. polymorpha as opposed to S. cerevisiae and Y. lipolytica .…”

Section: Resultsmentioning

confidence: 84%

New selectable host–marker systems for multiple genetic manipulations based on TRP1, MET2 and ADE2 in the methylotrophic yeast Hansenula polymorpha

Cheon

Choo

Ubiyvovk

et al. 2009

Yeast

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Interest has been increasing in the thermotolerant methylotrophic yeast Hansenula polymorpha as a useful system for fundamental research and applied purposes. Only a few genetic marker genes and auxotrophic hosts are yet available for this yeast. Here we isolated and developed H. polymorpha TRP1, MET2 and ADE2 genes as selectable markers for multiple genetic manipulations. The H. polymorpha TRP1 (HpTRP1 ), MET2 (HpMET2 ) and ADE2 (HpADE2 ) genes were sequentially disrupted, using an HpURA3 pop-out cassette in H. polymorpha to generate a series of new multiple auxotrophic strains, including up to a quintuple auxotrophic strain. Unexpectedly, the HpTRP1 deletion mutants required additional tryptophan supplementation for their full growth, even on complex media such as YPD. Despite the clearly increased resistance to 5-fluoroanthranilic acid of the HpTRP1 deletion mutants, the HpTRP1 blaster cassette does not appear to be usable as a counter-selection marker in H. polymorpha. Expression vectors carrying HpADE2, HpTRP1 or HpMET2 with their own promoters and terminators as selectable markers were constructed and used to co-transform the quintuple auxotrophic strain for the targeted expression of a heterologous gene, Aspergillus saitoi MsdS, at the ER, the Golgi and the cell surface, respectively. The nucleotide sequences presented here were submitted to GenBank under Accession Nos AY795576 (HpTRP1 ), FJ226453 (HpMET2 ) and FJ493241 (HpADE2 ), respectively.

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“…This compound, toxic for prototrophic cells, permits in most cases an easy counterselection of ura3 or ura5 genotypes from a randomly mutagenized population. In the same way, recent advances have demonstrated the potential of 5-fluoroanthranilate for counter-selection of trp1, trp3, trp4 or trp5 genotypes in yeast (Toyn et al, 2000;Cheon et al, 2003Cheon et al, , 2009Lebel et al, 2006;Jones et al, 2008).…”

Section: Dominant Selection Markersmentioning

confidence: 88%