title: Dithizone staining of intracellular zinc: an unexpected and versatile counterscreen for auxotrophic marker genes in Saccharomyces cerevisiae.
abstract: Auxotrophic marker genes such as URA3, LEU2, and HIS3 in Saccharomyces cerevisiae have long been used to select cells that have been successfully transformed with recombinant DNA. A longstanding challenge in working with these genes is that counterselection procedures are often lacking. This paper describes the unexpected discovery of a simple plate assay that imparts a bright red stain to cells experiencing nutritional stress from the lack of a marker gene. The procedure specifically stains a zinc-rich vesicular compartment analogous to the zinc-rich secretory vesicles found in insulin-secreting pancreatic islet cells and glutamate-secreting neurons. Staining was greatly diminished in zap1 mutants, which lack a homeostatic activator of zinc uptake, and in cot1 zrc1 double mutants, which lack the two yeast homologs of mammalian vesicle-specific zinc export proteins. Only one of 93 strains with temperature-sensitive alleles of essential genes exhibited an increase in dithizone staining at its non-permissive temperature, indicating that staining is not simply a sign of growth-arrested or dying cells. Remarkably, the procedure works with most commonly used marker genes, highlights subtle defects, uses no reporter constructs or expensive reagents, requires only a few hours of incubation, yields visually striking results without any instrumentation, and is not toxic to the cells. Many potential applications exist for dithizone staining, both as a versatile counterscreen for auxotrophic marker genes and as a powerful new tool for the genetic analysis of a biomedically important vesicular organelle.
authors: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America. dyuan@jhmi.edu
authors: Daniel S
authors: DS
authors: Yuan
mesh_terms: D002470:Cell Survival; D002999:Clone Cells / Q000166:cytology; D003116:Color; D004230:Dithizone / Q000378:metabolism*; D005800:Genes, Fungal / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics*; D042541:Intracellular Space / Q000378:metabolism*; D010539:Permeability; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics; D022142:Secretory Vesicles / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D015032:Zinc / Q000378:metabolism*
chemical_list: D005819:Genetic Markers; D004230:Dithizone; D015032:Zinc