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Fig. 6. Identification of two alternate upstream PIK3CA exons. 5' RACE analysis was used to determine the presence of upstream exons. (A) A 1:100 dilution of the primary PCR product of the tailed cDNA was amplified with GSP3 and AUAP primers, demonstrating the presence of a single band of approximately 500 bp in size. The diluted PCR product of no tail cDNA amplified with GSP3 and AUAP was the negative control. (B) The presence of putative exon1b was confirmed using a 1:100 dilution of the primary PCR product of the tailed cDNA amplified with GSP3 and GSP5, resulting in a 442 bp band. GSP3 binds to exon2(1), whereas GSP6 and GSP5 bind to exons1a and 1b, respectively. GSP3 and GSP6 were used as positive control (429 bp) to amplify the newly identified exon1a. (C) A diagram of the first three exons of PIK3CA showing the nucleotide positions of the newly identified exons 1a and 1b, thereby demonstrating their nucleotide distance from exon2(1). The first nucleotide of exon1a is marked as 1 and the positions of the first and last nucleotides of the first three exons [1a, 1b and 2(1)] are accordingly labeled. The exons are indicated by the black boxes labeled 1a, 1b and 2(1); the introns are indicated by the horizontal lines connecting them. Exon2(1) [coordinates, chr3:180,399,240-180,399,668 (human genome assembly 17)] contains the translational start site (arrow). Exon1a [coordinates, chr3:180,348,661-180,348,709 (human genome assembly 17)] is in the 5' UTR and is 50,579 bp upstream of exon2(1). Exon1b [coordinates, chr3:180,349,013-180,349,093 (human genome assembly 17)] is also in the 5' UTR and is 50,227 bp upstream of exon2(1). Exon1a and exon1b are highly conserved, and their first nucleotides are 352 bp apart. (D) A diagram showing that there are two alternate PIK3CA transcripts. Exons 1a and 1b splice alternatively to exon2(1). (E) Phylogenetic footprinting and p53-binding-site analysis of promoter-proximal sequences. p53 interacts with DNA through binding to two tandem copies of a well-defined half-site. The PIK3CA promoter region was analyzed with two half-site filters to predict candidate binding sites. The specific profile (lines) requires the presence of two nucleotides at critical positions within the recognition sequence (CnnG). Because empirical observation suggests that some bonafide p53 target sequences diverge from the constrain in one of the two half-sites, the sensitive profile (circles) requires only one perfect match to the core sequences. The locations of the oligonucleotides used in mobility shift assays and the amplified PCR products from the ChIP experiments are indicated.
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