Figure 4

Proof of genomic integration of both aph VIII and g-luc genes in transformants. (A) Paromomycin resistant transformants and the parent 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 (gPCR1, Figure 2A and D). (B) Genomic PCR analysis of paromomycin resistant transformants and the parent wild-type strain for the presence of the g-luc gene in the genome. The expected size of the PCR fragment produced in transformants was 119 bp (gPCR2, Figure 2C and D). (C) As a control, a 208 bp fragment of an unaffected Volvox gene (tbpA) was amplified both from all transformants and from wild-type strains just as expected. (A-C) M-lanes refer to the molecular weight marker. PCR fragments were cloned and sequenced. The obtained sequences are given right of each gel image. The positions of the primers and the start codon (underlined, bold) are indicated. This PCR assay was carried out more than 250 generations after transformation of the strains.