R. Daniel Gietz

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

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.

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

SummaryA 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 yeast transformation using the LiAc/SS carrier DNA/PEG method

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.

BNIP3 Heterodimerizes with Bcl-2/Bcl-XL and Induces Cell Death Independent of a Bcl-2 Homology 3 (BH3) Domain at Both Mitochondrial and Nonmitochondrial Sites

BNIP3 (formerly NIP3) is a pro-apoptotic, mitochondrial protein classified in the Bcl-2 family based on limited sequence homology to the Bcl-2 homology 3 (BH3) domain and COOH-terminal transmembrane (TM) domain. BNIP3 expressed in yeast and mammalian cells interacts with survival promoting proteins Bcl-2, Bcl-XL, and CED-9. Typically, the BH3 domain of pro-apoptotic Bcl-2 homologues mediates Bcl-2/Bcl-XLheterodimerization and confers pro-apoptotic activity. Deletion mapping of BNIP3 excluded its BH3-like domain and identified the NH2 terminus (residues 1–49) and TM domain as critical for Bcl-2 heterodimerization, and either region was sufficient for Bcl-XL interaction. Additionally, the removal of the BH3-like domain in BNIP3 did not diminish its killing activity. The TM domain of BNIP3 is critical for homodimerization, pro-apoptotic function, and mitochondrial targeting. Several TM domain mutants were found to disrupt SDS-resistant BNIP3 homodimerization but did not interfere with its killing activity or mitochondrial localization. Substitution of the BNIP3 TM domain with that of cytochromeb 5 directed protein expression to nonmitochondrial sites and still promoted apoptosis and heterodimerization with Bcl-2 and Bcl-XL. We propose that BNIP3 represents a subfamily of Bcl-2-related proteins that functions without a typical BH3 domain to regulate apoptosis from both mitochondrial and nonmitochondrial sites by selective Bcl-2/Bcl-XL interactions.

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

Transformation is essential to many molecular and genetic investigations in the yeast Saccharomyces cerevisiae. Yeast transformation protocols utilizing the LiAc/ssDNA/PEG method are presented. Protocols for various applications are listed including a method for transformation in 96-well microtiter plate format and another for the production of frozen competent yeast cells that can be used at a moments notice.

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

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.

Transformation of Saccharomyces cerevisiae by the lithium acetate/single-stranded carrier DNA/polyethylene glycol protocol

▼Transformation of bacteria was first suggested by Griffiths in 1928. It was not until fifty years later that a system was reported by Hinnen et al. (Ref. 1) and Beggs (Ref. 2) for the induction of transformation in Saccharomyces cerevisiae. Further development by Ito et al. (Ref. 3) allowed transformation of intact yeast cells following exposure to alkali cations. This procedure was less complicated, but it yielded only 400 transformants/μg of plasmid DNA. Schiestl and Gietz (Ref. 4) increased the efficiency of the alkali cation protocol to 100 000 transformants/μg plasmid DNA by using single-stranded carrier DNA in the transformation mixture. Since this time, we have streamlined and optimized this protocol to give yields as high as 2.2 × 107 transformants/μg DNA (Ref. 5, 6, 7, 8). High efficiency is essential for transformation of cDNA expression libraries for the two-hybrid system (Ref. 9, 10) as well as other similar systems (Ref. 11, 12, 13). Three transformation protocols are listed here. The ‘standard high-efficiency’ version of the lithium acetate (LiAc)/ single-stranded DNA (ss-DNA)/ polyethylene glycol (PEG) protocol is used when a large number of transformants are required. The ‘large-scale high efficiency’ version is used to obtain the millions of transformants needed to screen complex cDNA libraries. The ‘quick and easy’ version can be used when large numbers of transformants are not required.

Identification of proteins that interact with a protein of interest: applications of the yeast two-hybrid system.

The yeast two-hybrid system is a molecular genetic test for protein interaction. Here we describe a step by step procedure to screen for proteins that interact with a protein of interest using the two-hybrid system. This process includes, construction and testing of the bait plasmid, screening a plasmid library for interacting fusion proteins, elimination of false positives and deletion analysis of true positives. This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with your protein of interest.

Large-scale high-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method

Here, we describe a Library screen transformation protocol using the lithium acetate/single-stranded carrier DNA/PEG method of transformation for Saccharomyces cerevisiae. This method is suitable for screening complex plasmid libraries such as those used for yeast two-hybrid analysis. This procedure takes up to 2.5 h to complete once the yeast culture has been grown.

4 Transformation of Yeast by the Lithium Acetate/Single-Stranded Carrier DNA/PEG Method

Publisher Summary This chapter discusses the transformation of yeast by the lithium acetate/single-stranded carrier DNA/PEG (LiAc/SSDNA/PEG) method. High efficiency transformation of yeast is used most frequently for applications such as the two-hybrid screen for protein-protein interactions, for one-hybrid screens that detect protein–DNA interactions, for inverted one-hybrid screens to identify DNA elements that bind a transcription factor, and for the screens that detect protein–protein interactions dependent on phosphorylation. The high efficiency LiAc/SSDNA/PEG transformation procedure used to induce the yeast, Sacchuromyces cerevisiae, to take up exogenous DNA has aided in the development and utilization of sophisticated technologies, such as the one, two, and inverted one-hybrid systems. Also, the procedure is important for the efficient construction of libraries of null mutants for the functional analysis of novel yeast genes. These and other molecular genetic techniques have increased the usefulness of this premier eukaryotic model organism so that it can now be considered as a laboratory work horse, which will find its way into many research programs.