Southern Analysis of pFCEN in the Parasites.

<p>(A) To determine the copy number of pCon (lane a) and pFCEN (lane b), Southern analyses were performed by using two probe DNAs, the <i>gfp</i> and <i>pfsir2A</i> genes. The <i>gfp</i> and <i>pfsir2A</i> probes recognised the plasmids and the single, endogenous genomic <i>pfsir2A</i> copy, respectively. In this analysis, the signal from the <i>pfsir2A</i> gene was used as the internal control. The copy number of each plasmid was determined by comparing the signal intensities of the plasmids with that of the internal control. The asterisks mark three unexpected signals found in the genomic DNA including pCon. In contrast, corresponding signals were not detected in the genomic DNA including pFCEN. Therefore, these signals likely result from various derivatives, which might be irregularly generated from pCon during the replication, suggesting that <i>pfcen5-1.5</i> ensured the accurate replication of the plasmid. (B) The parasites transfected with the pFCEN and the pCon plasmids were maintained in the presence of the drug for 2 months, and their genomic DNA, including the plasmids, were purified. Southern analysis of both plasmids in the parasites was performed using purified genomic DNA and the gfp gene as a probe DNA. In this analysis, the undigested genomic DNA (lane a, pCon, and lane c, pFCEN) and the <i>Bam</i>HI-digested DNA (lane b, pCon, and lane d, pFCEN) were used. The arrows indicate the <i>Bam</i>HI-digested pFCEN and pCon. The upper and lower triangles indicate the closed circular and the super-coiled forms of pFCEN, respectively. Asterisks indicate concatemers of pCon that were detected in the undigested genomic DNA containing pCon.