A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae

Cell integrity in Saccharomyces cerevisiae is ensured by a rigid cell wall whose synthesis is controlled by a highly conserved MAP kinase signal transduction cascade. Stress at the cell surface is detected by a set of sensors and ultimately transmitted through this cascade to the transcription factor Rlm1, which governs expression of many genes encoding enzymes of cell wall biosynthesis. We here report on a number of versatile reporter constructs which link activation of a hybrid, Rlm1-lexA, by the MAP kinase Mpk1/Slt2 to the expression of the bacterial lacZ gene. This system was adapted to automated microwell screening and shown to be activated by a number of compounds inhibiting cell wall biosynthesis or interfering with plasma membrane function. In addition, we tested tea tree oil and two of its purified constituents (α-terpineol, terpinen-4-ol) for their effects on growth and on cell integrity signalling using such reporter strains. Tea tree oil was found to inhibit growth of wild-type and slg1/wsc1 mutant cells at a threshold of approximately 0.1% v/v, with the purified compounds acting already at half these concentrations. A mid2 deletion displayed hyper-resistance. Tea tree oil also induces the signalling pathway in a dose-dependent manner.

Section: Oligonucleotides Pcr Cassettes and Plasmidsmentioning

confidence: 99%

The effect of tea tree oil and antifungal agents on a reporter for yeast cell integrity signalling

Straede

Corran

Bundy

et al. 2007

“…The method involves two steps. First is the integration of a selectable marker gene at the locus of interest by homologous recombination in strains expressing the desired fluorescent DNA binding protein (Baudin et al, 1993). For this, we modified the 3 ends of the long primers used for classical gene-targeting PCR, to allow amplification of pUC18/19 target sequences flanking the selectable marker gene ( Figure 1A, steps 1 and 2; Table 1).…”

Section: Resultsmentioning

confidence: 99%

Modules for cloning‐free chromatin tagging in Saccharomyces cerevisae

Rohner

Gasser

Meister

2008

We describe a straightforward two-step PCR-based method to insert arrays of lac or tet operators (lacO or tetO) at specific loci in the budding yeast genome. The method entails insertion of a marker generated by PCR with classical long primers recognizing the locus of interest, followed by the replacement of this marker by a linearized plasmid bearing an array of lacI-or tetR-binding motifs. Using this technique, loci located either in the yeast genome or on yeast artificial chromosomes can be efficiently tagged. We provide a set of plasmids with different markers for cloning-free integration of lacO or tetO repeats into the yeast genome.

“…Often, the final PCR product was cut from the gel and re-amplified with the outermost primers (L1 and L4) to yield enough DNA for the transformation step. The advantages of using the LHF method instead of the shortflanking homology method, or SFH (Baudin et al, 1993), is that the LFH method increases the frequency of homologous recombinaiton of the disruption cassette. In fact, Southern blot analysis of genomic DNA from all but one of the 32 independent transformants analysed showed that integration had occurred at the correct locus.…”

Section: Resultsmentioning

confidence: 99%

Disruption of six Saccharomyces cerevisiae ORFs on chromosome XII results in three lethal disruptants

Alloush

Edwards

Valle-Lisboa

et al. 2001

Six ORFs of unknown function from the left arm of chromosome XII of Saccharomyces cerevisiae were chosen for a reverse genetic approach to provide materials to assist in assignment of function. A two-step PCR using long-flanking homology was employed to amplify disruption cassettes consisting of a kanMX gene as selectable marker flanked by 250-350 bp long regions homologous to the target gene. The diploid strains FY1679 and CEN.PK2 were transformed with the replacement cassettes and transformants were selected for geneticin (G418) resistance. Correct targeting of the replacement cassettes at the genomic locus was verified by Southern blot analysis with the kanMX gene as a probe. Disruption cassettes were cloned in pUG7 plasmid for systematic gene inactivation in other yeast strains and the cognate genes were cloned in pRS416 plasmid for gene complementation studies. Sporulation and tetrad analysis of heterozygous disruptants showed that three of the six ORFs [YLR141w (RRN5), YLR145w and YLR147c (SMD3)] were essential genes that were complemented by their cognate genes. ylr146cD (spe4) homozygous diploids showed enhanced sporulation efficiency, whereas ylr147cD heterozygous diploids failed to sporulate in the FY1679 but not in the CEN.PK2 genetic background. The other two disruptants [ylr143w and ylr144c (acf2)] gave no phenotype.