Figure 6

Demonstration of transcription of co-transformed, heterologous genes by RT-PCR. RNA from paromomycin-resistant transformants that were co-transformed with the non-selectable plasmids ptubar4 (A, T-J39), pHsp-HA (B, T-H3), pPsaD-GLuc (C, T-PL2, T-PL3), or pHsp70A-GLuc (C, T-HL2) was reverse transcribed and products were amplified by PCR using primers specific for the heterologous genes. Co-transformants were expected to yield cDNA fragments of the V. carteri arylsulfatase (ars) (265 bp, A), the V. carteri hsp70A (374 bp, B), or the Gaussia luciferase (luc) (343 bp, C). Sequences obtained from cloned fragments are shown in A (ars, co-transformant T-J39), B (hsp70A, co-transformant T-H3), and C (luc, co-transformants T-PL2, T-PL3, and T-HL2). (A-C) The parent wild type strain was analyzed as a control; lane M, molecular weight marker. The positions of the primers and the former positions of introns (two connected arrowheads) are indicated in the sequences. (B) Italics and bold, HA-tag sequence. (C) Bold, stop codon; italics, artificial BamHI site.