The effect of exon 5 and its downstream sequences on mA3 intron 5 splicing.

<p>(A) Distributions of sequence polymorphisms in the intron 5 of the <i>Apobec3</i> gene locus between the B6 allele [NT_039621] and the Celera database sequence of mixed mouse DNA [NW_001030577.1]. Shorter horizontal lines below the thick one representing intron 5 indicate regions of sequenced BALB/c genome [DDBJ accession No. AB646261-AB646265], which show nucleotide sequences identical to corresponding Celera database sequences. Spans of the analyzed regions are 1–620, 661–1597, 1643–2058, 2759–3443, and 5558–6247 in base numbers starting from the first nucleotide of intron 5. SNP are shown with vertical lines, single-base indels with arrows, and deletions of ≥2 bases with triangles. Indels and deletions above the thick horizontal line that represents intron 5 are deletions in the B6 allele relative to the Celera sequence, while those underneath the horizontal line are deletions in the Celera sequence relative to the B6 allele sequence. (B) Plasmid constructions for the splicing assays. The exon 5–6 plasmids harbored either the B6 or BALB/c genomic fragment encompassing exons 5 and 6, including the entire intron 5 of the corresponding allele. Each of the remaining plasmids possessed a sequentially reduced length of the intron 5 as indicated. The precise size of each PCR-generated 5′ fragment included was as follows: 3177bp and 3185bp for B6 and BALB/c intron 5-Δ3′; 2106bp and 2086bp for B6 and BALB/c 200bp intron 5; 1120bp and 1110bp for B6 and BALB/c 1100bp intron 5; and 634bp and 620bp for B6 and BALB/c 600bp intron 5, respectively. The primers g–h are the same as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002478#ppat-1002478-g004" target="_blank">Figure 4</a>. (C) RT-PCR detection of spliced messages expressed from the B6 and BALB/c exon 5–6 plasmids along with those from the exon 4–7 plasmids as controls. Primers g and h were used. A lack of expression of the spliced message in cells transfected with the B6 exon 5–6 plasmid was evident. (D and F) Splicing assays using intron 5 deletion plasmids. The intron 5 fragment included in each plasmid is shown in (B). RT-PCR assays were performed with primers g and h. A portion of the transfected cells were utilized for luciferase assays to compare transfection efficiencies among the samples as shown in (C). Comparable levels of luciferase activities were observed in all samples in each experiment (data not shown). Quantitative real-time PCR data show averages of three reaction wells and SD. (E) Reciprocal chimeras were produced between B6 and BALB/c exon 5–6 by exchanging the cloned genomic DNA fragment at position 1100 within intron 5. The exact location of the above position 1100 for B6 and BALB/c intron 5 is described in the legend for (B). RT-PCR detection of spliced messages was performed with primers g and h. A portion of the transfected cells were utilized for luciferase assays to compare transfection efficiencies.