Improved method for high efficiency transformation of intact yeast cells.

In this chapter we have provided instructions for transforming yeast by a number of variations of the LiAc/SS-DNA/PEG method for a number of different applications. The rapid transformation protocol is used when small numbers of transformants are required. The high efficiency transformation protocol is used to generate large numbers of transformants or to deliver DNA constructs or oligonucleotides into the yeast cell. The large-scale transformation protocol is primarily applicable to the analysis of complex plasmid DNA libraries, such as those required for the yeast two-hybrid system. The microtiter plate versions of the rapid and high efficiency transformation protocols can be applied to high-throughput screening technologies.

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

A method, using LiAc to yield competent cells, is described that increased the efficiency of genetic transformation of intact cells of Saccharomyces cerevisiae to more than 1 × 105 transformants per microgram of vector DNA and to 1.5% transformants per viable cell. The use of single stranded, or heat denaturated double stranded, nucleic acids as carrier resulted in about a 100 fold higher frequency of transformation with plasmids containing the 2μm origin of replication. Single stranded DNA seems to be responsible for the effect since M13 single stranded DNA, as well as RNA, was effective. Boiled carrier DNA did not yield any increased transformation efficiency using spheroplast formation to induce DNA uptake, indicating a difference in the mechanism of transformation with the two methods.

High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier.

Here we describe a high-efficiency version of the lithium acetate/single-stranded carrier DNA/PEG method of transformation of Saccharomyces cerevisiae. This method currently gives the highest efficiency and yield of transformants, although a faster protocol is available for small number of transformations. The procedure takes up to 1.5 h, depending on the length of heat shock, once the yeast culture has been grown. This method is useful for most transformation requirements.

High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method

We evolved muscarinic receptors in yeast to generate a family of G protein-coupled receptors (GPCRs) that are activated solely by a pharmacologically inert drug-like and bioavailable compound (clozapine-N-oxide) Subsequent screening in human cell lines facilitated the creation of a family of muscarinic acetylcholine GPCRs suitable for in vitro and in situ studies We subsequently created lines of telomerase-immortalized human pulmonary artery smooth muscle cells stably expressing all five family members and found that each one faithfully recapitulated the signaling phenotype of the parent receptor We also expressed a Gi-coupled designer receptor in hippocampal neurons (hM4D) and demonstrated its ability to induce membrane hyperpolarization and neuronal silencing We have thus devised a facile approach for designing families of GPCRs with engineered ligand specificities Such reverse-engineered GPCRs will prove to be powerful tools for selectively modulating signal-transduction pathways in vitro and in vivo

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Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand

Efficient transformation of yeast has been the corner stone of molecular studies in yeast for numerous years. It is essential for techniques such as the yeast two-hybrid system as well as genome modification. Here I present protocols for a number of different transformation applications. A quick and easy protocol is for situations in which large numbers of transformants are not required. The high-efficiency protocol generates large numbers of transformants from small amounts of DNA that can be scaled up for the library transformation protocol. A transformation protocol for a microtiter plate format is also included. Finally, a protocol for the production of frozen competent yeast cells is included, which allows cells to be taken from the freezer at a moment's notice and transformed with high efficiency.

Yeast Transformation by the LiAc/SS Carrier DNA/PEG Method

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.

Applications of high efficiency lithium acetate transformation of intact yeast cells using single-stranded nucleic acids as carrier.

Here, we describe a quick and easy version of the lithium acetate/single-stranded carrier DNA/PEG method of transformation for Saccharomyces cerevisiae. This method can be performed when only a few transformants are needed. The procedure can take less than an hour, depending on the duration of the heat shock. It can be used to transform yeast cells from various stages of growth and storage. Cells can be transformed from freshly grown cells as well as cells stored on a plate at room temperature or in a refrigerator.

R. Daniel Gietz

Papers

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

High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method

Yeast Transformation by the LiAc/SS Carrier DNA/PEG Method

Applications of high efficiency lithium acetate transformation of intact yeast cells using single-stranded nucleic acids as carrier.

Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method