title: Multiple Antibiotic Resistance Plasmids Allow Scalable, PCR-Mediated DNA Manipulation and Near-Zero Background Cloning.
abstract: We have constructed two plasmids that can be used for cloning as templates for PCR- -based gene disruption, mutagenesis and the construction of DNA chromosome translocation cassettes. To our knowledge, these plasmids are the first vectors that confer resistance to ampicillin, kanamycin and hygromycin B in bacteria, and to geneticin (G418) and hygromycin B inSaccharomyces cerevisiaesimultaneously. The option of simultaneously using up to three resistance markers provides a highly stringent control of recombinant selection and the almost complete elimination of background resistance, while unique restriction sites allow easy cloning of chosen genetic material. Moreover, we successfully used these new vectors as PCR templates for the induction of chromosome translocation in budding yeast by the bridge-induced translocation system. Cells in which translocation was induced carried chromosomal rearrangements as expected and exhibited resistance to both, G418 and hygromycin B. These features make our constructs very handy tools for many molecular biology applications.
authors.affiliation: Yeast Molecular Genetics Laboratory, ICGEB, AREA Science Park, Padriciano 99, IT-34149 Trieste, Italy.
authors.forename: Remigiusz
authors.initials: R
authors.lastname: Arnak
authors.affiliation: Yeast Molecular Genetics Laboratory, ICGEB, AREA Science Park, Padriciano 99, IT-34149 Trieste, Italy.
authors.forename: Burcin
authors.initials: B
authors.lastname: Altun
authors.affiliation: Yeast Molecular Genetics Laboratory, ICGEB, AREA Science Park, Padriciano 99, IT-34149 Trieste, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Tosato
authors.affiliation: Yeast Molecular Genetics Laboratory, ICGEB, AREA Science Park, Padriciano 99, IT-34149 Trieste, Italy.
authors.forename: Carlo V
authors.initials: CV
authors.lastname: Bruschi
keywords: DNA manipulation; functional analysis; gene knock-out; multi-marker vectors; multiple-antibiotic resistance; yeast