Use of Reverse Ligation-PCR to Identify Transcriptional Start Sites in GC-Rich TATA-Less Genes: Application to the Rat IGFBP-2 Gene

摘要

ABSTRACTTATA-less genes are often GC-rich in the region of transcriptional initiation and the corresponding mRNAs are prone to the formation of secondary structure. These properties have made it difficult to determine unambiguously the start sites of transcription for some of these genes by conventional assays such as primer extension and nuclease protection. Using the TATA-less rat IGFBP-2 gene, we demonstrate that tobacco acid pyrophosphatase-reverse ligation polymerase chain reaction (TAP-RLPCR), a novel and sensitive assay, can be used to map the start sites of these genes. First, the validity of TAP-RLPCR was demonstrated by mapping the transcription start site of the rat insulin-like growth factor 1 (IGFBP-1) gene to the correct position (nucleotides −173 relative to ATG, +1). Using total RNA obtained from the rat liver cell line BRL-3A, the transcription start sites of the rat IGFBP-2 gene were mapped to a narrow cluster extending from nucleotides −86 to −90 (ATG, +1), 39 bp downstream of three adjacent GC boxes that are essential to the transcriptional activity of the gene. The assay was also used to map the start sites of a luciferase reporter gene driven by the fragment −1,295 to −32 of the rat IGFBP-2 promoter after transfection in the human embryonic kidney cell line 293. The hybrid gene utilized the same transcription start sites as the rat IGFBP-2 gene, indicating that the elements required for positioning of the transcription initiation complex are contained within the 3′ end terminating at nucleotide −32. These studies demonstrate the usefulness of the TAP-RLPCR assay for verifying the authenticity of ambiguous start sites, and definitively map the start sites of the rat