Splicing activity and predicted secondary structure of the group I intron of Pf-WMP3.

<p>A. <i>In vivo</i> splicing of the Pf-WMP3 intron. RNA isolated from Pf-WMP3-infected <i>P. foveolarum</i> was analyzed by reverse transcription. PCR was done using intron primers P3f1 and P3r1 on genomic DNA (gDNA), RNA before reverse transcription (RNA), cDNA and a no-template control (NTC). The times of RNA isolation (in minutes) after infection are indicated. An ethidium bromide-stained agarose gel (2% agarose [wt vol⁻¹]) is shown. The sizes (in bases) of prominent bands of the DNA size standard (lane M) are indicated on the left. B. Determination of the splicing point by DNA sequencing of RT-PCR products of <i>in vivo</i> spliced transcripts. T7 primer in pEASY-T1 was used. C. <i>In vitro</i> splicing analysis of the Pf-WMP3 intron. PCR was done using intron primers P3f3 and P3r2 on genomic DNA (gDNA), cDNA (RNA was from <i>in vitro</i> transcription), <i>in vitro</i>-transcribed RNA and a no-template control (NTC). An ethidium bromide-stained agarose gel (2% agarose [wt vol⁻¹]) is shown. The sizes (in bases) of prominent bands of the DNA size standard (lane M) are indicated on the left. D. Predicted secondary structure of the group I intron of Pf-WMP3. Exon and intron sequences are in lower and upper case letters respectively. Filled arrows indicate 5′ and 3′ splice sites. Conserved structural elements P1 through P9 are shown. Boxed regions designate the P10 pairing. Gray boxes indicate the start and stop codons of the I-PfoP3I coding sequence. The line next to AGGAGGU in the stem P6b indicates the putative ribosomal binding site (RBS). The 438-nucleotide sequence of the intron-encoded ORF is indicated.