Alternative transcripts of the mouse Gli1 gene. (A) Detection of alternative transcription start sites (TSS). RACE analysis was performed on NIH3T3 cells using reverse primers within exon 4 of the Gli1 gene followed by agarose gel electrophoresis. White, black and gray triangles indicate transcripts, which are transcribed from known (TSS-S) or novel (TSS-M and TSS-L) TSSs, respectively. Note that the RACE product indicated by the black triangle also includes a minor variant that is transcribed from TSS-L and skips exon 1B. The MspI digested pBR322 molecular weight markers are also shown. (B) Genomic structure of the mouse Gli1 5' UTR region. Exons are represented by black boxes with the splicing pattern indicated. The white box represents the newly identified exon 1 region, which is transcribed from the novel TSSs (black and gray arrows). The exon 1B skipping event is highlighted by a bold line. The initiator methionine codon (ATG) in exon 2 is also shown. (C) Analysis of skipping events of Gli1 5' exons in NIH3T3 cells, Ptch1-/- MEFs and mouse embryos. Cells were treated either with methanol (MeOH) or the S mo ag onist, SAG. Alternative splicing was evaluated by RT-PCR analysis using primer sets within exons 1 and 4. The individual PCR products detected on agarose gel electrophoresis were sequence-verified. The MspI digested pBR322 molecular weight markers are also shown.