Genomic structure of the promoter region of human AC133 and alternative splicing within its 5′-UTR. (A) Exon 1A, exon 1B, exons D1, D2, and D3 from cluster 1D, and exon E4 from cluster 1E are alternatively spliced to a common exon 2, according to our RACE data. Translation initiation site is located in exon 2. 5′-RACE revealed the presence of exons 1A, 1B, and exon clusters 1D and 1E, while exon 1C was found as a single EST from skeletal muscle. P1, P2, P4, P5, and possibly P3 are alternative promoters for the AC133 gene. Exons 1A, 1B, 1C, and their corresponding promoters are located within a CpG island. (B) 5′-RACE analysis of AC133 mRNA. mRNAs from brain (B) and kidney (K) were subjected to 5′-RACE analysis. The PCR products (second round of amplification; nested primers) were resolved on 1.5% agarose gel, stained with ethidium bromide. Size marker 1 kb plus is shown on the right (1 kb+). C_B^neg and C_K^neg are negative controls for brain and kidney, respectively. One band from brain and 2 bands from kidney were isolated, subcloned into pCR4-TOPO, and sequenced. The band from brain was found to comprise exon 1B fragments. The lower band from kidney was found to comprise a majority of exon 1A fragments, with a minority of exon 1B fragments. The upper band from kidney was found to comprise exon 1A fragments from an alternate transcription start point, 55 bp longer than those in the lower band. (C) 5′-RACE analysis of AC133 mRNA in testis (T). Multiple bands were observed for testis 5′-RACE products. Since multiple bands were observed, the entire pool of PCR products was subcloned into pCR4-TOPO directly from the PCR product mix. PCR mix was determined to contain 5 different alternative splice variants containing exons from either cluster 1E or cluster 1D.