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.
Highlights
Auxotrophic marker genes such as URA3, LEU2, or HIS3 are ubiquitous in yeast genetics, where they are used to select cells that have been successfully transformed with recombinant DNA
Mutation of the URA3 marker gene results in failure to grow in a medium that lacks uracil, as one would expect given that URA3 encodes a key enzyme in the de novo pathway for uracil biosynthesis [3]
Cells directly suspended in staining buffers exhibited a gradual loss of refractility of the vacuole in solutions containing 0.1% w/v Triton, consistent with the gradual permeabilization of intracellular organelles by the detergent. This finding led to the development of a second formulation of dithizone that would be suitable for staining cell suspensions
Summary
Auxotrophic marker genes such as URA3, LEU2, or HIS3 are ubiquitous in yeast genetics, where they are used to select cells that have been successfully transformed with recombinant DNA (reviewed in [1]). Growth of ura mutants in uracil-deficient medium can be restored, if the cells have been transformed with DNA containing the URA3 gene. This exquisite control over growth by the simple presence or absence of a marker gene has been exploited since the earliest attempts to transform yeast cells with recombinant DNA [4]. Several useful applications of counterselections involving 5-fluoroorotic acid have been developed, including the replacement of one genetic allele with another using an intermediate state containing URA3 between the two alleles [7] and the recycling of the URA3 marker for as many as 20 sequential rounds of transformation [8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
