title: A MultiSite Gateway™ vector set for the functional analysis of genes in the model Saccharomyces cerevisiae.
abstract: BACKGROUND
  Recombinatorial cloning using the Gateway™ technology has been the method of choice for high-throughput omics projects, resulting in the availability of entire ORFeomes in Gateway™ compatible vectors. The MultiSite Gateway™ system allows combining multiple genetic fragments such as promoter, ORF and epitope tag in one single reaction. To date, this technology has not been accessible in the yeast Saccharomyces cerevisiae, one of the most widely used experimental systems in molecular biology, due to the lack of appropriate destination vectors.
  
  
  RESULTS
  Here, we present a set of three-fragment MultiSite Gateway™ destination vectors that have been developed for gene expression in S. cerevisiae and that allow the assembly of any promoter, open reading frame, epitope tag arrangement in combination with any of four auxotrophic markers and three distinct replication mechanisms. As an example of its applicability, we used yeast three-hybrid to provide evidence for the assembly of a ternary complex of plant proteins involved in jasmonate signalling and consisting of the JAZ, NINJA and TOPLESS proteins.
  
  
  CONCLUSIONS
  Our vectors make MultiSite Gateway™ cloning accessible in S. cerevisiae and implement a fast and versatile cloning method for the high-throughput functional analysis of (heterologous) proteins in one of the most widely used model organisms for molecular biology research.
authors.affiliation: Department of Plant Systems Biology, VIB, Technologiepark 927, B-9052, Gent, Belgium.
authors.forename: Astrid
authors.initials: A
authors.lastname: Nagels Durand
authors.forename: Tessa
authors.initials: T
authors.lastname: Moses
authors.forename: Rebecca
authors.initials: R
authors.lastname: De Clercq
authors.forename: Alain
authors.initials: A
authors.lastname: Goossens
authors.forename: Laurens
authors.initials: L
authors.lastname: Pauwels
mesh_terms: D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D008954:Models, Biological; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D011993:Recombinant Fusion Proteins