The impact of TCCT repeat numbers on exon 5 inclusion in mA3 mRNA splicing.

<p>(A) Schematic representation of the genomic DNA structure of the <i>Apobec3</i> gene locus. Boxes indicate exons. LTR, the endogenous retroviral LTR inserted into intron 2 in some strains of mice <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002478#ppat.1002478-Sanville1" target="_blank">[37]</a>. (B) Insert structures of the plasmids used for the splicing assays. BALB/c exon 4–7 and BALB/c ΔTCCT plasmids harbored the same DNA fragment amplified from BALB/c genomic DNA encompassing exons 4 and 7, except that the BALB/c ΔTCCT contains only a single TCCT quadruplet. B6 exon 4–7 plasmid harbored the B6 genomic DNA fragment encompassing exons 4 and 7. C741T and T741C indicate a C to T or reciprocal nucleotide substitution, respectively, within intron 4 at 741-bp downstream from the first nucleotide of exon 4 on the backbone of the BALB/c or B6 exon 4–7 insert. An additional repeat of the TCCT quadruplet was added to the B6 exon 4–6 construct to generate B6 +TCCT. The positions of primers g, h, i, and j used for RT-PCR assays are indicated with the arrows. (C) The plasmid harboring each insert depicted in (B) was transfected into BALB/3T3 cells, total RNA was extracted, and RT-PCR reactions were performed with primer pairs g–h and i–h. A portion of the transfected cells were utilized for luciferase assays to compare transfection efficiencies among the samples. Comparable levels of luciferase activities were observed in all samples in each experiment (data not shown). The predicted splicing products are schematically indicated on the right side of the panel. Quantitative real-time PCR assays were performed with primers g and j, and data are shown here by averages of three reaction wells and SD.