Designer deletion strains derived fromSaccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications

Shuttle vectors allow for an efficient transfer of recombinant DNA into yeast cells and are widely used in fundamental research and biotechnology. While available shuttle vectors are applicable in many experimental settings, their use in quantitative biology is hampered by insufficient copy number control. Moreover, they often have practical constraints, such as limited modularity and few unique restriction sites. We constructed the pRG shuttle vector series, consisting of single-and multi-copy integrative, centromeric and episomal plasmids with marker genes for the selection in all commonly used auxotrophic yeast strains. The vectors feature a modular design and a large number of unique restriction sites, enabling an efficient exchange of every vector part and expansion of the series. Integration into the host genome is achieved using a double-crossover recombination mechanism, resulting in stable single-and multi-copy modifications. As centromeric and episomal plasmids give rise to a heterogeneous cell population, an analysis of their copy number distribution and loss behaviour was performed. Overall, the shuttle vector series supports the efficient cloning of genes and their maintenance in yeast cells with improved copy number control.

Section: Discussionmentioning

confidence: 99%

Section: Single-copy Integrative Plasmidsmentioning

confidence: 99%

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

Gnügge

Liphardt

Rudolf

2016

“…The parent strain of the deletion strains was BY4742 (MATα, his3 1 leu2 0 lys2 0 ura3 0), which was derived from S288C (Brachmann et al, 1998). Otherwise, S. cerevisiae CEN.…”

Section: Strains and Plasmidsmentioning

confidence: 99%

The ORF YNL274c (GOR1) codes for glyoxylate reductase in Saccharomyces cerevisiae

Rintala

Pitkänen

Vehkomäki

et al. 2006

The enzyme glyoxylate reductase reversibly reduces glyoxylate to glycolate, or alternatively hydroxypyruvate to D-glycerate, using either NADPH or NADH as a co-factor. The enzyme has multiple metabolic roles in different organisms. In this paper we show that GOR1 (ORF YNL274c) encodes a glyoxylate reductase and not a hydroxyisocaproate dehydrogenase in Saccharomyces cerevisiae, even though it also has minor activity on α-ketoisocaproate. In addition, we show that deletion of the glyoxylate reductase-encoding gene leads to higher biomass concentration after diauxic shift.

“…The pYCG plasmids, which were used to test for complementation, carried the corresponding genes, including upstream and downstream sequences, inserted into the centromeric plasmid, pRS416 (Sikorski and Hieter, 1989). In a few cases in which pYCG plasmids were not available, the phenotype was further checked in deletants made in another genetic background (BY) (Brachmann et al, 1998). Some strains known to be defective in various steps of the secretory/vacuolar pathways were also used as controls.…”

Section: Yeast Strains and Plasmidsmentioning

confidence: 99%

Mutants defective in secretory/vacuolar pathways in the EUROFAN collection of yeast disruptants

Avaro

Belgareh‐Touzé

Sibella-Argüelles

et al. 2002

We have screened the EUROFAN (European Functional Analysis Network) deletion strain collection for yeast mutants defective in secretory/vacuolar pathways and/or associated biochemical modifications. We used systematic Western immunoblotting to analyse the electrophoretic pattern of several markers of the secretory/vacuolar pathways, the soluble a-factor, the periplasmic glycoprotein invertase, the plasma membrane GPIanchored protein Gas1p, and two vacuolar proteins, the soluble carboxypeptidase Y and the membrane-bound alkaline phosphatase, which are targeted to the vacuole by different pathways. We also used colony immunoblotting to monitor the secretion of carboxypeptidase Y into the medium, to identify disruptants impaired in vacuolar targeting. We identified 25 mutants among the 631 deletion strains. Nine of these mutants were disrupted in genes identified in recent years on the basis of their involvement in trafficking (VPS53, VAC7, VAM6, APM3, SYS1), or glycosylation (ALG12, ALG9, OST4, ROT2). Three of these genes were identified on the basis of trafficking defects by ourselves and others within the EUROFAN project (TLG2, RCY1, MON2). The deletion of ERV29, which encodes a COPII vesicle protein, impaired carboxypeptidase Y trafficking from the endoplasmic reticulum to the Golgi apparatus. We also identified eight unknown ORFs, the deletion of which reduced Golgi glycosylation or impaired the Golgi to vacuole trafficking of carboxypeptidase Y. YJR044c, which we identified as a new VPS gene, encodes a protein with numerous homologues of unknown function in sequence databases.