Yeast vectors for the integration/expression of any sequence at the TYR1 locus.
We have constructed new yeast vectors for targeted integration and conditional expression of any sequence at the Saccharomyces cerevisiae TYR1 locus which becomes disrupted. We show that vector integration is not neutral, causing prototrophy for tyrosine and auxotrophy for the vector's selectable marker (uracil or leucine, depending on the vector used). This feature allows a double screening of transformed yeast cells, improving the identification of colonies with the desired chromosomal structure. The GAL10 gene promoter has been added to drive conditional expression of cloned sequences. Using these vectors, chromosomal structure verification of recombinant clones is no longer necessary, since the noise of non-homologous recombination, as well as spontaneous reversion of the selected phenotype, can easily be identified. The ability of the vector to conditionally control gene expression has been confirmed using the gene for the green fluorescent protein (GFP) as a reporter.
Mirisola
Mario G
MG
Dipartimento di Biopatologia e Metodologie Biomediche, Università degli Studi di Palermo, Via Divisi 83, 90133 Palermo, Italy. mirisola@unipa.it
Colomba
Leonarda
L
Gallo
Alessia
A
Amodeo
Roberta
R
De Leo
Giacomo
G
D001483:Base Sequence; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D017422:Sequence Analysis, DNA; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics*
D004271:DNA, Fungal; D049452:Green Fluorescent Proteins; D014443:Tyrosine