Journal ArticleDOI
Applications of high efficiency lithium acetate transformation of intact yeast cells using single-stranded nucleic acids as carrier.
TLDR
Co‐transformation showed that 30–40% of the transformation‐competent cells take up more than one DNA molecule which can be used to enrich for integration and delection events 30‐ to 60‐fold.Abstract:
The highly efficient yeast lithium acetate transformation protocol of Schiestl and Gietz (1989) was tested for its applicability to some of the most important need of current yeast molecular biology. The method allows efficient cloning of genes by direct transformation of gene libraries into yeast. When a random gene pool ligation reaction was transformed into yeast, the LEU2, HIS3, URA3, TRP1 and ARG4 genes were found among the primary transformations at a frequency of approximately 0·1%. The RAD4 gene, which is toxic to Escherichia coli, was also identified among the primary transformants of a ligation library at a frequency of 0·18%. Non-selective transformation using this transformation proctocol was shown to increase the frequency of gene disruption three-fold. Co-transformation showed that 30–40% of the transformation-competent cells take up more than one DNA molecule which can be used to enrich for integration and delection events 30- to 60-fold. Co-transformation was used in the construction of simultaneous double gene disruptions as well as disrupting both copies of one gene in a diploid which occurred at 2–5% the frequency of the single event.read more
Citations
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Journal ArticleDOI
Improved method for high efficiency transformation of intact yeast cells.
Journal ArticleDOI
A New Efficient Gene Disruption Cassette for Repeated Use in Budding Yeast
TL;DR: A loxP-kanMX-loxP gene disruption cassette is developed, which combines the advantages of the heterologous kanr marker with those from the Cre- lox P recombination system, and will be of great advantage for the functional analysis of gene families.
Journal ArticleDOI
Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters.
TL;DR: It is found that overexpression of the gene encoding 14DM either by gene amplification or by gene deregulation was not the major cause of resistance among these clinical isolates of fluconazole-resistant C. albicans, and that resistance could be mediated by energy-requiring efflux pumps such as ATP-binding cassette (ABC) multidrug transporters.
Book ChapterDOI
Yeast Transformation by the LiAc/SS Carrier DNA/PEG Method
R. Daniel Gietz,Robin A. Woods +1 more
TL;DR: The technique for the transformation of Saccharomyces cerevisiae using the LiAc/SS Carrier DNA/PEG method is described and a rapid method, for use when large numbers of transformants are not necessary, is described.
Journal ArticleDOI
The Partial Agonist Activity of Antagonist-Occupied Steroid Receptors Is Controlled by a Novel Hinge Domain-Binding Coactivator L7/SPA and the Corepressors N-CoR or SMRT
Twila A. Jackson,Jennifer K. Richer,David L. Bain,Glenn S. Takimoto,Lin Tung,Kathryn B. Horwitz +5 more
TL;DR: The data suggest that the direction of transcription by antagonist-occupied steroid receptors can be controlled by the ratio of coactivators to corepressors recruited to the transcription complex by promoter-bound receptors.
References
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TL;DR: This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters that can be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography.
Journal ArticleDOI
A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity
TL;DR: A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described, and these "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.
A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity
TL;DR: In this article, a technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described, where DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers.
Journal ArticleDOI
Studies on transformation of Escherichia coli with plasmids
Douglas Hanahan,Douglas Hanahan +1 more
TL;DR: Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmid molecules.
Journal ArticleDOI
A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.
Robert S. Sikorski,Philip Hieter +1 more
TL;DR: A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae to perform most standard DNA manipulations in the same plasmid that is introduced into yeast.
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A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.
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