An improved tetO promoter replacement system for regulating the expression of yeast genes

Yeast. 2003 Nov;20(15):1255-62. doi: 10.1002/yea.1037.

Abstract

Regulatable promoters are commonly used to control the expression of, especially, essential genes in a conditional manner. Integration of such promoters upstream of an ORF using one-step PCR-mediated homologous recombination should be particularly efficient. However, integration of the original KanMX4-tetO promoter cassette (Belli et al., 1998a) into the relatively short upstream regions of many yeast genes is often problematic, presumably due to the size (3.9 kb) of the replacement cassette. We have created a new, shorter, KanMX4-tetO cassette by removing the transactivator (tTA) sequence from the original cassette. The transactivator (tTA) has been integrated into the yeast genome to create a new strain for use with the new system, which has a greatly increased efficiency of promoter substitution. With it, we have been able to create strains that could not be made with the original cassette. To increase the throughput of promoter substitutions, we have developed a new assay for testing doxycycline sensitivity, based on liquid culture using microtitre trays. Altogether, the components of this new 'tool kit' greatly increase the efficiency of systematic promoter substitutions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • DNA, Fungal / chemistry
  • DNA, Fungal / genetics
  • DNA-Binding Proteins*
  • Doxycycline / pharmacology
  • Gene Expression Regulation, Fungal
  • Genes, Essential / genetics
  • Genes, Essential / physiology
  • Mutagenesis, Insertional / methods*
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Promoter Regions, Genetic / genetics
  • Promoter Regions, Genetic / physiology
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Securin
  • Trans-Activators / genetics
  • Trans-Activators / metabolism
  • Transformation, Genetic / genetics*

Substances

  • Anti-Bacterial Agents
  • CDC45 protein, S cerevisiae
  • Carrier Proteins
  • Cell Cycle Proteins
  • DNA, Fungal
  • DNA-Binding Proteins
  • MOB1 protein, S cerevisiae
  • Nuclear Proteins
  • PDS1 protein, S cerevisiae
  • Phosphoproteins
  • Saccharomyces cerevisiae Proteins
  • Securin
  • Trans-Activators
  • Doxycycline