Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method

Gietz, R. Daniel; Woods, Robin A.

doi:10.1016/s0076-6879(02)50957-5

Cited by 2,462 publications

(591 citation statements)

References 16 publications

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“…The diploid isolates were transformed using a lithium acetate procedure (Gietz & Woods, 2002) with a cassette that targeted the terminal region of the highly expressed PGK1 gene (Figure S2) and contained: (1) either mCherry or GFP, and (2) antibiotic resistance through KanMX (Wach, Brachat, Pohlmann, & Philippsen, 1994), NatMX or HphMX (Goldstein & McCusker, 1999). Plasmids pFA6a‐GFP‐KanMX6 (Longtine et al., 1998) and pBS34‐mCherry‐KanMX6 (Hailey, Davis, & Muller, 2002) were digested with NotI and used as template for PCR (Yeast Resource Center, University of Washington).…”

Section: Methodsmentioning

confidence: 99%

Biofilm formation and toxin production provide a fitness advantage in mixed colonies of environmental yeast isolates

Deschaine

Heysel

Lenhart

et al. 2018

Ecology and Evolution

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Microbes can engage in social interactions ranging from cooperation to warfare. Biofilms are structured, cooperative microbial communities. Like all cooperative communities, they are susceptible to invasion by selfish individuals who benefit without contributing. However, biofilms are pervasive and ancient, representing the first fossilized life. One hypothesis for the stability of biofilms is spatial structure: Segregated patches of related cooperative cells are able to outcompete unrelated cells. These dynamics have been explored computationally and in bacteria; however, their relevance to eukaryotic microbes remains an open question. The complexity of eukaryotic cell signaling and communication suggests the possibility of different social dynamics. Using the tractable model yeast, Saccharomyces cerevisiae, which can form biofilms, we investigate the interactions of environmental isolates with different social phenotypes. We find that biofilm strains spatially exclude nonbiofilm strains and that biofilm spatial structure confers a consistent and robust fitness advantage in direct competition. Furthermore, biofilms may protect against killer toxin, a warfare phenotype. During biofilm formation, cells are susceptible to toxin from nearby competitors; however, increased spatial use may provide an escape from toxin producers. Our results suggest that yeast biofilms represent a competitive strategy and that principles elucidated for the evolution and stability of bacterial biofilms may apply to more complex eukaryotes.

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Section: Methodsmentioning

confidence: 99%

Biofilm formation and toxin production provide a fitness advantage in mixed colonies of environmental yeast isolates

Deschaine

Heysel

Lenhart

et al. 2018

Ecology and Evolution

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show abstract

“…Manipulations were performed using a standard PEG/LiAC transformation protocol 4 . The GFP-Mdh1, GFP-Mdh2, GFP-Mdh3 and OE-mCherry-Mdh3 strains were picked from the SWAT N’ GFP or N’ mCherry yeast libraries that were recently prepared in our lab 5 .…”

Section: Methodsmentioning

confidence: 99%

Validation of a yeast malate dehydrogenase 2 (Mdh2) antibody tested for use in western blots

2018

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Malate dehydrogenases (Mdhs) reversibly convert malate to oxaloacetate and serve as important enzymes in several metabolic pathways. In the yeast Saccharomyces cerevisiae there are three Mdh isozymes, localized to different compartments in the cell. In order to identify specifically the Mdh2 isozyme, GenScript USA produced three different antibodies that we further tested by western blot. All three antibodies recognized the S. cerevisiae Mdh2 with different background and specificity properties. One of the antibodies had a relatively low background and high specificity and thus can be used for specific identification of Mdh2 in various experimental settings.

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“…W303-1A ( MAT a, leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 ) was used for luciferase experiments. Yeast strains (Table S2) were constructed by standard genetic crosses or by LiAc transformation [52]. Deletions and chromosomal tags were created using homologous recombination with PCR products created through methods described in Janke et al and Longtine et al [53], [54].…”

Section: Methodsmentioning

confidence: 99%

SPO24 Is a Transcriptionally Dynamic, Small ORF-Encoding Locus Required for Efficient Sporulation in Saccharomyces cerevisiae

2014

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In Saccharomyces cerevisiae, meiosis and sporulation are highly regulated responses that are driven in part by changes in RNA expression. Alternative mRNA forms with extended 5′ UTRs are atypical in S. cerevisiae, and 5′ extensions with upstream open reading frames (uORFs) are even more unusual. Here we characterize the gene YPR036W-A, now renamed SPO24, which encodes a very small (67-amino-acid) protein. This gene gives rise to two mRNA forms: a shorter form throughout meiosis and a longer, 5′-extended form in mid-late meiosis. The latter form includes a uORF for a 14-amino-acid peptide (Spo24u14). Deletion of the downstream ORF (dORF) leads to sporulation defects and the appearance of pseudohyphae-like projections. Experiments with luciferase reporters indicate that the uORF does not downregulate dORF translation. The protein encoded by the dORF (Spo24d67) localizes to the prospore membrane and is differentially phosphorylated during meiosis. Transcription of the 5′-extended mRNA in mid-meiosis depends upon the presence of two middle sporulation elements (MSEs). Removal of the MSEs severely inhibits the mid-meiotic appearance of the 5′-extended mRNA and limits the ability of plasmid-borne SPO24 to rescue the sporulation defect of a spo24Δ mutant, suggesting that the 5′-extended mRNA is functionally important. These results reveal Spo24d67 as a sporulation-related factor that is encoded by a transcriptionally dynamic, uORF-containing locus.

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Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method

Cited by 2,462 publications

References 16 publications

Biofilm formation and toxin production provide a fitness advantage in mixed colonies of environmental yeast isolates

Biofilm formation and toxin production provide a fitness advantage in mixed colonies of environmental yeast isolates

Validation of a yeast malate dehydrogenase 2 (Mdh2) antibody tested for use in western blots

SPO24 Is a Transcriptionally Dynamic, Small ORF-Encoding Locus Required for Efficient Sporulation in Saccharomyces cerevisiae

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