Fig. 5

TRE5-A expression in agnC ^GA mutants. a Construction of agnC “gene activation” mutants. The agnC locus on chromosome 2 is indicated by nucleotide positions. The gene activation cassette consisted of a hybrid actin6/actin15 promoter (arrows indicate transcription direction). The BamHI arm contained a 1200 bp DNA fragment covering the complete coding sequence of gene DDB_G0271884, including 273 bp of upstream sequence. The HindIII arm contained 1200 bp of agnC coding sequence, including the original translation start site. After double-recombination of the agnC ^GA vector with genomic DNA, the expression of agnC was driven by the act15 promoter, whereas expression of the neighboring gene DDB_G0271884 was unaffected. b Semi-quantitative RT-PCR analysis of RNA from AX2, JH.D, and three independent agnC ^GA mutants demonstrating overexpression of agnC, normal expression of the neighboring gene DDB_G0271884 and gpdA (loading control), and silencing of TRE5-A (ORF1 and ORF2 sequences). NTC: no template control. c Quantitative RT-PCR of TRE5-A (ORF1) expression on RNA from JH.D and three agnC ^GA mutants. Expression levels were compared to AX2 cells and are expressed as fold change of expression, meaning that values <1 represent lower levels of TRE5-A in the mutants relative to wild-type AX2 cells. Data represent means from four independent cultures of the indicated strains ± SD