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Dithizone Staining of Intracellular Zinc: An Unexpected and Versatile Counterscreen for Auxotrophic Marker Genes in Saccharomyces cerevisiae

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 d...

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Autor principal: Yuan, Daniel S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3187812/
https://www.ncbi.nlm.nih.gov/pubmed/21998704
http://dx.doi.org/10.1371/journal.pone.0025830
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author Yuan, Daniel S.
author_facet Yuan, Daniel S.
author_sort Yuan, Daniel S.
collection PubMed
description 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.
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spelling pubmed-31878122011-10-13 Dithizone Staining of Intracellular Zinc: An Unexpected and Versatile Counterscreen for Auxotrophic Marker Genes in Saccharomyces cerevisiae Yuan, Daniel S. PLoS One Research Article 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. Public Library of Science 2011-10-05 /pmc/articles/PMC3187812/ /pubmed/21998704 http://dx.doi.org/10.1371/journal.pone.0025830 Text en Daniel S. Yuan. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Yuan, Daniel S.
Dithizone Staining of Intracellular Zinc: An Unexpected and Versatile Counterscreen for Auxotrophic Marker Genes in Saccharomyces cerevisiae
title Dithizone Staining of Intracellular Zinc: An Unexpected and Versatile Counterscreen for Auxotrophic Marker Genes in Saccharomyces cerevisiae
title_full Dithizone Staining of Intracellular Zinc: An Unexpected and Versatile Counterscreen for Auxotrophic Marker Genes in Saccharomyces cerevisiae
title_fullStr Dithizone Staining of Intracellular Zinc: An Unexpected and Versatile Counterscreen for Auxotrophic Marker Genes in Saccharomyces cerevisiae
title_full_unstemmed Dithizone Staining of Intracellular Zinc: An Unexpected and Versatile Counterscreen for Auxotrophic Marker Genes in Saccharomyces cerevisiae
title_short Dithizone Staining of Intracellular Zinc: An Unexpected and Versatile Counterscreen for Auxotrophic Marker Genes in Saccharomyces cerevisiae
title_sort dithizone staining of intracellular zinc: an unexpected and versatile counterscreen for auxotrophic marker genes in saccharomyces cerevisiae
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3187812/
https://www.ncbi.nlm.nih.gov/pubmed/21998704
http://dx.doi.org/10.1371/journal.pone.0025830
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