Figure 5
Generation of double deletion mutants Δfet3a/Δfet3b, Δfet3c/Δfet3b and Δfet3a/Δfet3c. (A) Schematic representation of wild type and mutant loci after homologous recombination with the disruption fragments of genes fet3b (left) and fet3c (right). The position of the probes used (B and C) and the expected sizes of the restriction fragments are indicated. Dashed lines, sequences not included in the disruption fragment. (B) Southern blot analysis of the wild-type recipient strain (WT) and transformants obtained with the disruption fragments after ten vegetative cycles in selective medium. Genomic DNA (1 μg) was digested with HindIII (transformants for fet3b, left and right) or XbaI (transformants for fet3c, middle) and hybridized with probes B and C, which recognized wild-type and disrupted alleles, but could discriminate between them. The positions and sizes of the GeneRuler DNA ladder mixture (M) (Fermentas) are indicated. The cropped blots are displayed in the main figure, the black lines surrounding blots indicate the cropping lines. The scanned full blots are presented in Supplementary Fig. 3.
