Guy A. Caldwell

Total synthesis of the lipopeptide a-mating factor ofSaccharomyces cerevisiae

The a-mating factor of Saccharomyces cerevisiae was synthesized using both solution phase and solid phase strategies. Structure of the final peptide was confirmed using amino acid analysis, fast atom bombardment mass spectroscopy and 400 MHz proton NMR. The synthetic farnesylated dodecapeptide, YIIKGVFWDPAC (S-farnesyl) OCH3, exhibited chromatographic and spectroscopic properties identical to the natural pheromone and had significant biological activity at nanomolar concentrations.

Total in vitro maturation of the Saccharomyces cerevisiaea-factor lipopeptide mating pheromone

The a-factor mating pheromone, produced by Saccharomyces cerevisiae a haploid cells, is post-translationally modified in a manner analogous to that of the ras proto-oncogene product. A consensus C-terminal amino acid sequence, -CAAX (C is cysteine, A is aliphatic amino acid, and X is any amino acid), is the target of these modifications, which include isoprenylation (essential for Ras function), proteolysis of the -AAX sequence, and carboxy methyl esterification. Recently, the RAM/DPR1 gene product was shown to be a component of the activity responsible for isoprenylation of both Ras and a-factor. In this report, we present an in vitro assay which not only detects a-factor isoprenylation, but also proteolysis and carboxy methyl esterification, and directly demonstrates, biochemically, the order of these processing events. This a-factor maturation assay may prove useful for screening agents which block any of the steps involved in the post-translational modification of the a-factor and Ras -CAAX sequences. Such agents would be potential anti-Ras-related cancer therapeutic drugs.

Exercise 15 – Determination of β-Galactosidase in Permeabilized Yeast Cells

In recent years, a number of permeabilization methods for yeast—such as the use of detergents, organic solvents, and desiccation—have been developed. Cell-permeabilization techniques are often useful for many applications relating to enzyme technology. As permeabilization methods are usually rapid and do not destroy cellular enzymes, the quantity of an enzyme associated with a cell can be assayed after permeabilization. This chapter illustrates the procedure in which Saccharomyces cerevisiae ( S. cerevisiae ) cells are treated with a combination of an organic solvent (toluene) and a detergent (sarkosyl or sodium lauroyl sulfate), which effectively dissolves the permeability barriers (membrane lipids), allowing free access of added substrates to intracellular proteins. Most proteins remain entrapped and attached with the cell because the cell wall barrier is not broken down by this treatment. While calculating the final results, the variables like assay time, volume of cells used in the assay, cell density at the start of the assay, combination of absorbance by o-nitrophenol and light scattering by cells, and the light scattering by cells are primarily taken into account.

Exercise 6 – Large-Scale Isolation of Plasmid DNA by Column Chromatography

This chapter illustrates that the advances in the development of novel DNA-affinity matrices now allow for the purification of milligram quantities of plasmid DNA by safe and effective column chromatography methods. The chapter demonstrates the use of a solid-phase anion-exchange resin that selectively enables the separation of nucleic acids from other cellular contaminants by a gravity-based chromatography procedure. The fact that the resin used has a large pore size and is composed of a hydrophilic surface coated with a high density of charged diethylaminoethyl (DEAE) groups allows for a broad separation range that enhances the isolation of plasmid DNA from complex mixtures also containing cellular proteins, RNA, and chromosomal DNA. The biggest advantage of this procedure is that the entire procedure can be performed rapidly (in a few hours) and the purity of the plasmid obtained in this procedure is equivalent to or greater than that resulting from the use of gradient centrifugation with CsCl. Following lysis, the purification of plasmid DNA away from host proteins and the remaining chromosomal DNA contaminants can be accomplished with the help of traditional procedures for large-scale plasmid purification that involves CsCl-gradient centrifugation.

Exercise 7 – Amplification of a lacZ Gene Fragment by the Polymerase Chain Reaction

This chapter focuses on the polymerase chain reaction (PCR), an extremely useful technique that has revolutionized the manner in which investigators can amplify, detect, manipulate, and clone DNA fragments from a wide variety of sources. Biotechnological applications of PCR methodology include everything from improvements in basic molecular biology methods to clinical uses in the diagnostic detection of genetic mutations, viral infections, or molecular “fingerprinting” as applied to forensic medicine. Several factors influence the fidelity and efficiency of the PCR process. The concentration of various components constituting a typical PCR is a primary determinant of successful amplification. These include the concentration of Thermus aquaticus ( Taq ) polymerase, deoxynucleotide triphosphates, magnesium ions, template DNA, and primers in the reaction. The temperature and times of each step within each cycle and total number of cycles can dramatically affect product yield and specificity. The differential effects of temperature and salt concentration in nucleic-acid hybridization is called “stringency” and this is important in discerning appropriate conditions for PCR. The availability of a purified heat-stable DNA polymerase from the thermophilic bacterium Taq polymerase greatly facilitates the enzymatic catalysis of DNA amplification.

Exercise 2 – Preparation of Culture Media

The cultivation of bacteria and yeast requires certain basic nutritional requirements when reared in any medium. The primary nutritional requirements are a carbon source serving as an energy source, water, nitrogen source, a phosphorus source, a sulfur source, and various mineral nutrients, such as iron and magnesium. Bacteria that are capable of growing in a medium consisting of a single carbon source, such as carbohydrate glucose, and a simple nitrogen source—such as ammonium chloride or ammonium sulfate—are Escherichia coli ( E. coli ) and Saccharomyces cerevisiae ( S. cerevisiae ). S. cerevisia also requires some additional vitamins for growth. As the exact chemical composition of this kind of medium is known, it is termed as “defined or synthetic.” In complex media, the basic nutrients are provided by plant and animal extracts. The basic ingredients of the Luria–Bertani (LB) are yeast extracts and peptones broth commonly used for E. coli and the yeast extract-peptone-dextrose (YEPD or YPD) broth commonly used to rear S. cerevisiae . An ideal solidifying agent for a microbiological medium is “agar.” Sterilization is the procedure that eliminates all viable microorganisms.

Exercise 5 – Purification, Concentration, and Quantitation of DNA

Purification of DNA from a complex mixture of cellular molecules is most readily accomplished by the removal of proteins and other molecules into an organic solvent. This extraction procedure takes advantage of the properties of phenol and phenol chloroform that lead to the denaturation of proteins. DNA and RNA are not soluble in organic solvents and therefore remain associated with the aqueous phases of mixtures that contain solvents for protein extraction. DNA precipitated with ethanol can be recovered by centrifugation and redissolved in a small amount of buffer. In this manner, DNA solutions of desired concentration can be obtained from very small amounts of DNA. Precipitation with ethanol also removes traces of phenol and chloroform that are used in the extraction procedure, which, if present, would inhibit restriction enzymes and other enzymes used in molecular cloning. Nucleic acids absorb ultraviolet (UV) light at 260 and 280 nm and bind the fluorescent dye ethidium bromide (EtBr) forms, the base of some of the most convenient and common methods used to quantitate the amount of DNA. To assess the purity of DNA, UV-light absorption is widely used.

Exercise 17 – β-Galactosidase Purification

Protein purification is a prerequisite for the detailed study of its structures and function. A purification scheme is usually devised to separate molecular species on the basis of selective solubility, chromatographic behavior, and molecular weight. At every stage of purification, it is important to know the “specific activity” of an enzyme—the enzymatic activity per milligram of protein—which indicates the degree of purity of the enzyme. Other purification steps that are often employed include ultrafiltration and chromatography, such as ion exchange, affinity, and high performance liquid chromatography. When using gel-filtration chromatography, preparation and calibration of the gel-filtration column is the first and most important step. Gel-filtration chromatography, also called size-exclusion chromatography, is based on the principle that the flow of molecules is impeded because of the differences in their hydrodynamic volumes. The choice of an appropriate gel-filtration matrix depends on the molecular size and chemical properties of the substances to be separated. Gel-filtration yields are usually excellent, as there is little or no retention of substances on column material. Gel filtration can also be used to study chemical equilibria and separate cells and particles.

Exercise 12 – Isolation of Plasmid from Yeast and Escherichia coli Transformation

Although an excellent host for the expression and analysis of genes, the yeast Saccharomyces cerevisiae ( S. cerevisiae ) has some technical drawbacks associated with it. One of these drawbacks is its propensity for recombination, which can lead to gene rearrangements in plasmid constructs that are designed for heterologous expression. While in Escherichia coli ( E. coli ), the majority of chromosomal DNA can be easily separated from plasmid DNA by the virtue of its association with the bacterial cell membrane, pure yeast plasmid mini-preps cannot be prepared that easily owing to its tendency of contamination with mitochondrial and genomic DNA. The most common method of bacterial transformation utilizes high levels of CaCI 2 to facilitate the entry of plasmid DNA vectors into E. coli in conjunction with the structural alteration of the bacterial cell wall. Most E. coli strains exhibit transformation efficiencies and the factors influencing this efficiency are often related to the conditions that render cells competent. The final step in the entire procedure is the alkaline lysis procedure for mini-prep isolation of plasmid DNA from the E. coli transformants, which can also be considered as an alternative to the “boiling” mini-prep procedure.