Demonstration of co-transformation of heterologous genes by genomic PCR. PCRs were done using genomic template DNA of paromomycin-resistant transformants co-bombarded with the non-selectable plasmids ptubar4 (A1), pHsp-HA (B1), pPsaD-GLuc (C1), or pHsp70A-GLuc (C2). Primers were specific for the co-bombarded genes. Co-transformants were expected to yield gene fragments of the V. carteri arylsulfatase (ars) (212 bp, A1), the V. carteri hsp70A (479 bp, B1), or the Gaussia luciferase (luc) (343 bp, C1 and C2). Sequences obtained from cloned fragments are shown in A2 (ars, ptubar4 co-transformants), B2 (hsp70A, pHsp-HA co-transformants), and C3 (luc, pPsaD-GLuc and pHsp70A-GLuc co-transformants). (A1, B1, C1, C2) The parent wild type strain was analyzed as a control; lane M, molecular weight marker. (C1, C2) Control, the co-transformed plasmid was used as a template. (A2, B2, C3) Primer positions are indicated; upper case (grey background), exon; lower case and italics (white background), intron; boxed: intron ends. (B2) Upper case, italics, and bold: HA-tag. (C3) Bold, stop codon; italics, artificial BamHI site.