Figure 9.
Pcdh-α/γ chimeric transcripts. (A) Schematic diagram of the mouse Pcdh gene clusters. Note that all three clusters are transcribed in the same direction, and two additional genes illustrated by ovals are transcribed in the opposite direction. (B) RT-PCR analysis identified chimeric transcripts between the Pcdh-γ variable exons A11, A12, B2, B6, and C4 and the Pcdh-α constant exons (αC) from mouse brain RNA. (C) RT-PCR analysis identified chimeric transcripts between the Pcdhα variable exons α4, α6, and α7 and the Pcdh-γ constant exons (γC). (D) The chimeric proteins (α4γC and B2αC) encoded by the chimeric transcripts. (E) Western blot analysis confirmed the expression of an in-frame α4γC protein in transfected cells. The * indicates a background band serving as the loading control. (F) Interallelic trans-splicing assay of α10γC chimeric transcripts (similar to Fig. 8). An SNP (red C on 129S strain α10 exon) and the targeted IRES-LacZ cassette define allelic differences in both α10 variable exon and γ constant exon 3. Sequence analysis of two different types of α10γC cDNAs showed that each originated from its original allele, suggesting that trans-splicing might have occurred on the same chromosome. (G) Trans-splicing is infrequent on the same chromosome within the Pcdh-γ cluster. The CBA/J-B6 transgene was targeted to the end of the Pcdh-γ locus and is transcribed in the same orientation as the endogenous locus. This mimics the configuration of the γ variable exon spliced to the α constant exons (in B). RT-PCR was used to amplify spliced B6γC cDNA from the undifferentiated ES cells using a specific primer for the constant exon. RFLP analysis of the RT-PCR products showed that splicing predominantly occurred from the endogenous 129S-B6 exon to the γ constant exons but not from the CBA/J-B6 exon (lanes 1 and 2, cf. lane 3 of control ES cells).