Figure 5
Analysis of T. atroviride co-transformants for the presence of the tga3Q207Lgene. (A) Schematic drawing of the endogenous tga3 gene locus and of the transforming plasmid pKAtga3 harboring the plasmid pKAtga3 with tga3Q207Lexpression cassette gene including 1030-bp of the 5′ and 783-bp of the 3′ non-coding regions. The point mutation resulting in the Q207L amino acid exchange is indicated by a bold type arrow. Grey arrows indicate primers used for the PCR-based approaches. (B) Southern blot of genomic DNA digested with Sac I and hybridized with a probe containing ~ 1118-bp of the tga3 gene. The two bands of 1703-bp and 2272-bp present in all lanes including the wild-type (−) correspond to the endogenous tga3 gene copy. The presence of additional bands in co-transformants 3/3 (lane 7) and 4/5 (lane 11) indicate ectopic integration of the tga3Q207L gene. (C) The tga3 gene locus was amplified by PCR as given in the Methods section and the resulting 3377-bp amplicon digested with Bpu10 I. As an additional Bpu10 I recognition site was generated in tga3Q207L by introducing the A to T mutation, Bpu10 I digestion resulted in the two indicative fragments (1004-bp, 1392-bp) in co-transformants with homologous integration of tga3Q207L whereas the 2396-bp fragment remained undigested in the wild-type and ectopic co-transformants 3/3 (lane 12) and 4/5 (lane 13). WT; wildtype; M, DNA ladder.