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.

/pdf/transformation-of-yeast-by-lithium-acetate-single-stranded-8e5ucuyx03.pdf

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

An improved lithium acetate (LiAc)/single-stranded DNA (SS-DNA)/polyethylene glycol (PEG) protocol which yields >1 × 106 transformants/μg plasmid DNA and the original protocol described by Schiestl and Gietz (1989) were used to investigate aspects of the mechanism of LiAc/SS-DNA/PEG transformation. The highest transformation efficiency was observed when 1 × 108 cells were transformed with 100 ng plasmid DNA in the presence of 50 μg SS carrier DNA. The yield of transformants increased linearly up to 5 μg plasmid per transformation. A 20-min heat shock at 42°C was necessary for maximal yields. PEG was found to deposit both carrier DNA and plasmid DNA onto cells. SS carrier DNA bound more effectively to the cells and caused tighter binding of 32P-labelled plasmid DNA than did double-stranded (DS) carrier. The LiAc/SS-DNA/PEG transformation method did not result in cell fusion. DS carrier DNA competed with DS vector DNA in the transformation reaction. SS plasmid DNA transformed cells poorly in combination with both SS and DS carrier DNA. The LiAc/SS-DNA/PEG method was shown to be more effective than other treatments known to make cells transformable. A model for the mechanism of transformation by the LiAc/SS-DNA/PEG method is discussed.

Studies on the transformation of intact yeast cells by the LiAc/SS‐DNA/PEG procedure

Mammalian Ras interacts directly with the serine/threonine kinase Raf

Foreign gene expression in yeast: a review.

Selective concentration and anchoring of ionotropic receptors at the synapse is essential for neuronal signaling. Little is known about the molecules that mediate receptor clustering in the CNS. With use of the yeast two-hybrid system to screen a rat brain cDNA library and by in vitro binding assays, we have identified an interaction between NMDA receptor subunits 2A and 2B (NR2A and NR2B) and three distinct members of the PSD-95/SAP90 family of membrane-associated putative guanylate kinases. The interaction is mediated by binding of the C terminus of the NMDA receptor subunits to the first two PDZ (also known as GLGF or DHR) domains of PSD-95/SAP90, an abundant synaptic protein associated with the membrane cytoskeleton. PSD-95 is also known to bind and cluster Shaker-type voltage-gated K+ channels. Similarities between the C-termini of NR2 subunits and K+ channels suggest a common C-terminal binding motif for PDZ domains. These data suggest that PDZ domains can function as modules for protein-protein interactions. Members of the PSD-95 family might serve to anchor NMDA receptors to the submembrane cytoskeleton and aid in the assembly of signal transduction complexes at postsynaptic sites.

https://www.jneurosci.org/content/jneuro/16/7/2157.full.pdf

Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases

/pdf/dmso-enhanced-whole-cell-yeast-transformation-4p94ppvz2u.pdf

DMSO-enhanced Whole cell yeast transformation

http://www.cell.com/article/S2211124722013407/pdf

J. Hill

Papers

DMSO-enhanced Whole cell yeast transformation

Structural basis of activation and antagonism of receptor signaling mediated by interleukin-27