The trp1- delta FA designer deletion for PCR-based gene functional analysis in Saccharomyces cerevisiae.
PCR-based gene deletion and modification are now common techniques for rapid gene manipulation in the yeast Saccharomyces cerevisiae. The techniques work best when the host strain lacks sequence homology to the PCR-amplified selectable markers. One of the most versatile sets of PCR deletion/modification vectors is the pFA system described by Longtine et al.(1998), which is based on both heterologous (kanMX6 and HIS3MX6) and homologous (TRP1) markers. Here we describe the trp1-DeltaFA designer deletion allele that removes precisely from the genome TRP1 sequences carried in the pFA vectors. The trp1-DeltaFA allele can be introduced easily into TRP1 and most trp1 starting strains, and its use increases the frequency of correct integrants when using the pFA system's TRP1-based constructs. Unlike trp1-Delta1, trp1-DeltaFA does not remove neighbouring GAL3 upstream activating sequences and therefore does not interfere with GAL gene induction.
Horecka
J
J
Department of Molecular Biology, NIBH, AIST, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. horecka@nibh.go.jp
Jigami
Y
Y
D019747:Aldose-Ketose Isomerases; D000483:Alleles; D017931:DNA Primers / Q000737:chemistry; D004271:DNA, Fungal / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D017353:Gene Deletion; D005822:Genetic Vectors / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D010957:Plasmids / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000032:analysis
D017931:DNA Primers; D004271:DNA, Fungal; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D001616:beta-Galactosidase; D019747:Aldose-Ketose Isomerases; C490353:TRP1 protein, S cerevisiae