Isolation and Identification of Yeasts from Natural Habitats -- Maintenance and Culture of Yeasts -- Mutagenesis in Yeast -- Rare-Mating and Cytoduction in Saccharomyces cerevisiae -- Protoplast Fusion in Saccharomyces cerevisiae -- Meiotic Analysis -- Ascus Dissection -- Karyotyping of Yeast Strains by Pulsed-Field Gel Electrophoresis -- Chromosomal Localization of Genes Through Pulsed-Field Gel Electrophoresis Techniques -- Isolation, Purification, and Analysis of Nuclear DNA in Yeast Taxonomy -- Isolation of Yeast DNA -- Isolation of Mitochondrial DNA -- Isolation of Yeast Plasma Membranes -- The Extraction and Analysis of Sterols from Yeast -- Peroxisome Isolation -- Transformation of Lithium-Treated Yeast Cells and the Selection of Auxotrophic and Dominant Markers -- Biolistic Transformation of Yeasts -- Genomic Yeast DNA Clone Banks -- Yeast Colony Hybridization -- Determination of Plasmid Copy Number in Yeast -- Determination of Chromosome Ploidy in Yeast -- Chromosome Engineering in Yeast with a Site-Specific Recombination System from a Heterologous Yeast Plasmid -- Insertional Mutagenesis by Ty Elements in Saccharomyces cerevisiae -- Reporter Gene Systems for Assaying Gene Expression in Yeast -- Preparation and Use of Yeast Cell-Free Translation Lysate -- Electrophoretic Analysis of Yeast Proteins -- Preparation of Total RNA -- mRNA Abundance and Half-Life Measurements -- Polysome Analysis -- Induction of Heat Shock Proteins and Thermotolerance -- Rhodamine B Assay for Estimating Activity of Killer Toxins Permeabilizing Cytoplasmic Membranes -- Nystatin-Rhodamine B Assay for Estimating Activity of Killer Toxin from Kluyveromyces lactis -- Application of Killer Toxins in Stepwise Selection of Hybrids and Cybrids Obtained by Induced Protoplast Fusion -- Killer Plaque Technique for Selecting Hybrids and Cybrids Obtained by Induced Protoplast Fusion -- Genotoxicity Testing in Schizosaccharomyces pombe -- Purification and Quantification of Saccharomyces cerevisiae Cytochrome P450 -- Calorimetry of Whole Yeast Cells -- Light Microscopy Methods -- Fluorescence Microscopy Methods -- Immunoelectron Microscopy.

Yeast Protocols intends to offer a selection of well-proven proto­ cols in cell and molecular biology, applicable to yeasts including, but certainly not exclusively aimed at, Saccharomyces cerevisiae. Saccharomyces cerevisiae and its very distant cousin, Schizosacc- romyces pombe, are of course now foremost model eukaryotic organisms, and the focus of wide-ranging experimental studies, especially those using molecular genetic techniques. Many of the lat­ ter, such as DNA sequencing and in vitro mutagenesis, are general DNA techniques, well covered in other volumes of Humana's Meth­ ods in Molecular Biology series, and elsewhere. The inclusion of a number of non-DNA techniques in this book is meant to reflect the resurgent interest in yeast cell biology sparked by the development of gene manipulation methods-for example, cellular localization of cloned gene products using microscopical techniques. The presentation of protocols follows the successful Methods in Molecular Biology series format, with a clear sequence of steps and extensive troubleshooting notes. It is our hope that these protocols will be useful not only to established members of the full-time research community, but also to the less experienced-first degree level and masters students undertaking project work, as well as PhD students starting their experimental programs; I am well aware that these young apprentice scientists are not always able to receive the supervision time that they, and indeed their supervisors, would like.