Figure 4

Detection of the aph VIII gene in transformants and demonstration of its transcription. (A-C) Paromomycin resistant transformants and the parental wild-type strain were analyzed for presence of the aphVIII gene in the genome by genomic PCR. The expected size of the PCR fragment produced in transformants was 324 bp (Figure 3A). The rightmost lane (pPmr3) shows a positive control using pPmr3 plasmid DNA as the template. Lane M refers to the molecular weight marker. (D) RT-PCR analysis of aphVIII expression. RNA from paromomycin-resistant transformants was reverse transcribed and products were amplified by PCR using primers specific for the aphVIII gene construct. Transformants with aphVIII expression were expected to yield a 324-bp cDNA fragment (Figure 3A). The parent wild-type strain was used as a control. Lane M, molecular weight marker. (E) Sequence obtained from the amplified and cloned genomic and cDNA fragments. The 324-bp fragment contains part of the aphVIII gene and part of the 5′ untranslated region from V. carteri rbcS3. The positions of the primers and the start codon (bold) are indicated.