Demonstration of co- transformation and transcription of heterologous genes. (A1-A3) Paromomycin-resistant transformants were analyzed for the presence of the co-transformed, non-selectable DNA in the genome. PCRs were conducted using genomic DNA from transformants co-transformed with the pPsaD-GLuc (A1), pHRLucP (A2), or pHsp70A-GLuc (A3) plasmids as template. The parental wild-type strain was analyzed as a control. Primers were specific for the heterologous gluc gene, and a 342-bp PCR fragment (Figure 3B–D) was expected in co-transformants. The rightmost lane shows a positive control using DNA of the co-transformed plasmid as the template. Lane M refers to the molecular weight marker. (A4) Sequence obtained from the amplified and cloned gluc fragments. Primer positions, the stop codon (bold), and a BamHI restriction site (italics) are indicated. (B) Southern blot analysis of HindIII- or BamHI-digested genomic DNA from transformants EuHR-8 and EuHR-12, which were produced using the pPmr3 and pHRLucP plasmids, and from the parental wild-type strain. The blot was probed using a 258-bp fragment of the gluc gene (Figure 3D). (C1-C3) Transcription analysis by RT-PCR. RNA from paromomycin-resistant transformants co-bombarded with the non-selectable plasmids pPsaD-GLuc (C1), pHRLucP (C2), or pHsp70A-GLuc (C3) was reverse transcribed, and products were amplified by PCR using gluc-specific primers. Co-transformants were expected to yield a 342-bp cDNA fragment of the gluc gene (Figure 3B–D). The parental wild-type strain was used as a control. (C4) Sequence obtained from the cloned gluc cDNA fragments. Primer positions, the stop codon (bold), and a BamHI restriction site (italics) are indicated. Lane M refers to the molecular weight marker.