These promoters could be the origin of the antisense transcript within S that we describe in wild-type B cells in mice. obtain access to transcribed variable region gene sequences in the context of a complex with the ssDNA-binding protein replication protein A (RPA) (24). Again, however, in such studies, AID only targets the nontemplate strand. Likewise, AID expressed in bacteria can generate SHM, but again primarily on the nontemplate DNA strand (20). Various findings also suggest that AID may access S regions via a non-R-loop mechanism that may relate to the mechanism by which it accesses variable regions during SHM (3, 25). Despite findings that AID targets mainly the nontemplate strand in most and bacterial experiments, both the template and nontemplate strands are targeted equally by AID during normal SHM and CSR (11, 26, 27). Despite intense ongoing research, it is still unknown how the highly mutagenic activity of AID gets targeted mainly to Ig loci and not to other highly transcribed genes. Moreover, it is ABT-888 (Veliparib) not known how AID effects are targeted to the Ig variable region exon and switch regions while the Rabbit Polyclonal to FPR1 intervening IgH intronic enhancer (E) and CH exons are spared. Furthermore, a full explanation is still missing as to why and in bacteria AID gets almost exclusively targeted to the nontemplate strand (17, 19, 20, 23), whereas AID appears to act equally on the template and the nontemplate strand (11, 26, 27). As for the latter question, several nonmutually exclusive possibilities have been suggested. In the experiments, there could be additional factor(s) missing that would allow AID to target both the template and nontemplate strands experiments vs. RNA polymerase II in cells undergoing SHM RNA polymerase (28). Another possibility is that differences in chromatin structure or supercoiled DNA could allow AID to access both strands (29). Finally, it is conceivable that antisense transcription through variable regions and S regions could target AID to both strands (30). To further evaluate the potential role of antisense transcription as a mechanism of AID targeting, we have assayed, for the generation of antisense transcripts of IgH variable region exons, the C constant region exons and S regions in various types of B-lineage cells. Results Sense and Antisense Transcription Within IgH Variable Region Exons. We first assayed for the existence of antisense transcripts within the rearranged VHDJH exons, a known target region for SHM. To this end, we used VB1C8 VHDJH knockin mice (31), which carry an assembled IgH variable region exon in their germ line. These mice offer the advantage for these studies that all B cells produce transcripts of an identical sequence, which facilitates transcript detection by RT-PCR. Na?ve (B220+, PNALO) and activated (B220+, PNAHI) B cells were sorted from Peyer’s patches of VB1C8 knockin mice, and RNA was prepared. Reverse transcription was ABT-888 (Veliparib) conducted with random hexamers or with strand-specific primers to help distinguish between sense and antisense transcripts. PCR amplification of the cDNAs was designed in ABT-888 (Veliparib) a way that the forward primer anneals in the leader exon, whereas the reverse primer anneals at JH2 within the knocked-in VHDJH exon (Fig. 1transcription with T7 RNA polymerase results in either sense or antisense transcripts (1). Strand-specific reverse transcription reactions (primers depicted in red) generate either sense- or antisense-specific cDNA (2), and sense (3a)-, and antisense (3b)-specific PCRs produce only ABT-888 (Veliparib) a product when the according cDNA is used as a template. We immunized wild-type 129sv mice with sheep RBCs (SRBCs) and sorted na?ve (B220+, PNALO) and activated (B220+, PNAHI) B cells from spleens. Applying the strand-specific RT-PCR assay described above, we detected sense and antisense transcripts in both na?ve and activated B cells (Fig. 2and and and ?and22(11, 26, 27). However, in the context of transcription experiments, mainly the nontemplate strand is a target for AID (17, 19, 20, 23). Therefore, we also tested S regions for the ABT-888 (Veliparib) expression.