Exploring redundancy in the yeast genome: an improved strategy for use of the cre–loxP system

Delneri, Daniela; Tomlin, Gregory C.; Wixon, Jo; Hutter, Anton; Sefton, Mark; Louis, Edward J.; Oliver, Stephen G.

doi:10.1016/s0378-1119(00)00217-1

Cited by 112 publications

(96 citation statements)

References 27 publications

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“…The 5' loxP site in UBC9 was introduced by homologous recombination of a loxP-KANMX-loxP cassette generated from pUG6 (DELNERI et al 2000) using oligonucleotides UBC9lox5F and UBC9lox5R in the diploid strain UCC8600.…”

Section: Construction Of Loxp Target Genesmentioning

confidence: 99%

The Mother Enrichment Program: A Genetic System for Facile Replicative Life Span Analysis in Saccharomyces cerevisiae

2009

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The replicative life span (RLS) of Saccharomyces cerevisiae has been established as a model for the genetic regulation of longevity despite the inherent difficulty of the RLS assay, which requires separation of mother and daughter cells by micromanipulation after every division. Here we present the mother enrichment program (MEP), an inducible genetic system in which mother cells maintain a normal RLS-a median of 36 generations in the diploid MEP strain-while the proliferative potential of daughter cells is eliminated. Thus, the viability of a population over time becomes a function of RLS, and it displays features of a survival curve such as changes in hazard rate with age. We show that viability of mother cells in liquid culture is regulated by SIR2 and FOB1, two opposing regulators of RLS in yeast. We demonstrate that viability curves of these short-and long-lived strains can be easily distinguished from wild type, using a colony formation assay. This provides a simplified screening method for identifying genetic or environmental factors that regulate RLS. Additionally, the MEP can provide a cohort of cells at any stage of their life span for the analysis of ageassociated phenotypes. These capabilities effectively remove the hurdles presented by RLS analysis that have hindered S. cerevisiae aging studies since their inception 50 years ago.

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Section: Construction Of Loxp Target Genesmentioning

confidence: 99%

The Mother Enrichment Program: A Genetic System for Facile Replicative Life Span Analysis in Saccharomyces cerevisiae

2009

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“…YHE1254 containing URE2 from S. paradoxus terminating with a TAA codon was created using URE2 tagged with a 39-located HIS3 gene flanked by loxP sites as a marker. HIS3 was removed by expression of CRE from plasmid YEp351-cre-cyh (Delneri et al 2000).…”

Section: Mediamentioning

confidence: 99%

Prion Variants and Species Barriers Among Saccharomyces Ure2 Proteins

et al. 2009

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As hamster scrapie cannot infect mice, due to sequence differences in their PrP proteins, we find ''species barriers'' to transmission of the [URE3] prion in Saccharomyces cerevisiae among Ure2 proteins of S. cerevisiae, paradoxus, bayanus, cariocanus, and mikatae on the basis of differences among their Ure2p prion domain sequences. The rapid variation of the N-terminal Ure2p prion domains results in protection against the detrimental effects of infection by a prion, just as the PrP residue 129 Met/Val polymorphism may have arisen to protect humans from the effects of cannibalism. Just as spread of bovine spongiform encephalopathy prion variant is less impaired by species barriers than is sheep scrapie, we find that some [URE3] prion variants are infectious to another yeast species while other variants (with the identical amino acid sequence) are not. The species barrier is thus prion variant dependent as in mammals.[URE3] prion variant characteristics are maintained even on passage through the Ure2p of another species. Ure2p of Saccharomyces castelli has an N-terminal Q/N-rich ''prion domain'' but does not form prions (in S. cerevisiae) and is not infected with [URE3] from Ure2p of other Saccharomyces. This implies that conservation of its prion domain is not for the purpose of forming prions. Indeed the Ure2p prion domain has been shown to be important, though not essential, for the nitrogen catabolism regulatory role of the protein.

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“…The LYS2 gene was removed from pDP6 on a SalI-PvuII fragment (coordinates 1-4631 of GenBank M36287; SalI is immediately before the XbaI site at position 1 of this sequence) and ligated into the SalI-EcoRV sites of pFA6 to generate pFA6-LYS2. A pUC19-LYS2 plasmid has been previously described by Delneri et al (2000).…”

Section: Construction Of the Pfa6 Vectorsmentioning

confidence: 99%

A new family of yeast vectors and S288C‐derived strains for the systematic analysis of gene function

Tomlin

Wixon

Bolotin‐Fukuhara

et al. 2001

Yeast

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The yeast genome has been shown to contain a significant number of gene families with more than three members. In order to study these families it is often necessary to generate strains carrying deletions of all members of the family, which can require a wide range of auxotrophic markers. To facilitate such studies, we have generated yeast strains containing deletions of a selection of nutritional marker genes (ade2, ade4, ade8, met3 and met14). We have also cloned the corresponding cognate genes, allowing their use in PCR-based gene disruptions. Two new pRS family Saccharomyces cerevisiae-Escherichia coli shuttle vectors containing ADE8 (one low-copy, pRS4110, and one high-copy, pRS4210) have been produced for use in conjunction with the new strains. A system for easier synthetic lethal screening using one of these new markers is also presented. The ADE8 and HIS3 genes have been cloned together on a high-copy vector (pRS4213), providing a plasmid for red-white colour screening in the ade2D0 ade8D0 strains we have generated. In contrast to some conventional systems, this plasmid allows for screening using gene libraries constructed in URA3 plasmids.

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Exploring redundancy in the yeast genome: an improved strategy for use of the cre–loxP system

Cited by 112 publications

References 27 publications

The Mother Enrichment Program: A Genetic System for Facile Replicative Life Span Analysis in Saccharomyces cerevisiae

The Mother Enrichment Program: A Genetic System for Facile Replicative Life Span Analysis in Saccharomyces cerevisiae

Prion Variants and Species Barriers Among Saccharomyces Ure2 Proteins

A new family of yeast vectors and S288C‐derived strains for the systematic analysis of gene function

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