Robin A. Woods

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.

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.

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.

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.

The effects of amidantel (BAY d 8815) and its deacylated derivative (BAY d 9216) on Caenorhabditis elegans

The paralyzing effects of the anthelmintic drugs amidantel (BAY d 8815) and its deacylated derivative (BAY d 9216) on whole and cut C. elegans were investigated. The minimum effective concentrations with whole worms were 350 and 180 microM, respectively, compared to only 4 microM for another anthelmintic drug, levamisole. After rendering the worms permeable by cutting them at their approximate midsections, the minimum effective concentrations were: amidantel 0.30 microM, deacylated amidantel 0.07 microM and levamisole 0.15 microM. Comparison of the effects produced by amidantel and deacylated amidantel with those produced by levamisole, a known cholinergic agonist, suggested a common mode of action for all three drugs. The drugs were moderately potent inhibitors of both E. electricus and C. elegans acetylcholinesterase but at concentrations too high to account for their abilities to contract cut worms. Their primary mode of action appears to be as agonists at the level of the acetylcholine receptor, a view supported by the observation that their effects may be blocked by the nicotinic antagonists d-tubocurarine and gallamine.

Hypoxanthine: Guanine phosphoribosyltransferase mutants in Saccharomyces cerevisiae

SummaryYeast mutants lacking activity of the enzyme hypoxanthine: guanine phosphoribosyltransferase (H:GPRT) have been isolated by selecting for resistance to 8-azaguanine in a strain carrying the wild type allele, ade4+ of the gene coding for amidophosphoribosyltransferase (PRPPAT), the first enzyme of de novo purine synthesis. The mutants excrete purines and are cross-resistant to 8-azaadenine. They are recessive and represent a single complementation group, designated hpt1. Ade4-su, a prototrophic allele of ade4 with reduced activity of PRPPAT, is epistatic to hpt1, suppressing purine excretion and resistance to azaadenine but not resistance to azaguanine. The genotype ade2 hpt1 does not respond to hypoxanthine. Hpt1 complements and is not closely linked to the purine excreting mutants pur1 to pur5. Hpt1 and pur6, a regulatory mutant of PRPPAT, are also unlinked but do not complement, suggesting a protein-protein interaction between H:G-PRT and PRPPAT. Mycophenolic acid (MPA), an inhibitor of de novo guanine nucleotide synthesis, inhibits the growth of hpt1 and hpt1+. Xanthine allows both genotypes to grow in the presence of MPA whereas guanine only allows growth of hpt1+. Activity of A-PRT, X-PRT and H:G-PRT is present in hpt+. Hpt1 lacks activity of H:G-PRT but has normal A-PRT and X-PRT.

Adenine phosphoribosyltransferase mutants in Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae deficient in adenine phosphoribosyltransferase (A-PRT, EC 2,4,2,7) have been isolated following selection for resistance to 8-azaadenine in a prototrophic strain carrying the ade4-su allele of the gene coding for amidophosphoribosyltransferase (EC 2,4,2,14). The mutants were recessive and defined a single gene, apt1. They did not excrete purine when combined with ade4+. The mutants appeared to retain some A-PRT activity in crude extracts, and strains of the genotype ade2 apt1 responded to both adenine and hypoxanthine. Mutants deficient in adenine aminohydrolase (EC 3,5,4,2) activity, aah1, and hypoxanthine:guanine phosphoribosyltransferase (EC 2,4,2,8) activity, hpt1, were used to synthesize the genotypes apt1 hpt1 aah+ and apt1 hpt+ aah1. The absence of A-PRT activity in strains with these genotypes confirmed the hypothesis that the residual A-PRT activity of apt1 mutants was due to adenine aminohydrolase and hypoxanthine:guanine phosphoribosyltransferase acting in concert.

A complex genetic locus controlling purine nucleotide biosynthesis in yeast.

SummaryMutants of the genes pur1 to pur6 excrete purine when in combination with the allele su-pur+ and are resistant to growth inhibition by 8-azaadenine (8-AzAd) and 8-azaguanine (8-AzGu). In combination with su-pur, which suppresses purine excretion, pur1 and pur2 are analogue sensitive; pur3 is slightly resistant to 8-AzAd; pur4 is slightly resistant to both analogues and pur5 is completely resistant to 8-AzGu. Crosses of the pur mutants to dap, which causes sensitivity to 2,6-diaminopurine (2,6-DAP), guanine and 6-mercaptopurine (6-MP), show that dap also suppresses purine excretion and is closely linked to pur6. In combination with dap, pur1 and pur3 are analogue sensitive; pur4 is hypersensitive to guanine but resistant to 6-MP; pur5 is resistant to 2,6-DAP and guanine whilst pur2 is hypersensitive to all three compounds.The gene slw, which, like pur2, potentiates the effects of dap, also suppresses purine excretion but is not linked to any of the pur genes. The diploid slw/pur3 excretes purine.Tests for functional allelism were carried out on the closely linked genotypes su-pur+, su-pur, dap, pur6, PUR6 and ade4. The results of these tests indicate that all six genotypes are functionally allelic. It is suggested that a molecular complex of the products of pur1, pur3, pur4, pur6 and slw is involved in the control of purine nucleotide biosynthesis in yeast.

A Hot Aldehyde-Peroxide Fixation Method for Electron Microscopy of the Free-Living Nematode Caenorhabditis Elegans

An improved method for the fixation of the third and fourth larval stages and adults of Caenorhabditis elegans has been developed. Worms are placed in a mixture of 1.5% paraformaldehyde and 1.0% glutaraldehyde at pH 7.0 and 70 C and the suspension promptly cooled in a water bath at 20 C for 1 hr. The fixed worms are then immersed in a mixture of 5% glutaraldehyde and hydrogen peroxide at 4 C for 1 hr, stained en bloc in uranyl acetate, and embedded in resin for electron microscopy. The procedure results in superior fixation, particularly of microfilaments and microtubules. The high temperature of the initial fixation straightens the worms and thus facilitates serial sectioning.