Sequence features of the human TMP21 gene promoter and identification of the transcription start site. (A) The genomic organization of human TMP21 gene on Chromosome 14q24.3. E represents exon. ATG is the translation start codon and TAA the stop codon. (B) RLM-RACE experiment was performed to map the TMP21 transcription start site. Neuronal RNA was extracted by TRI-Reagent from SH-SY5Y cells. Total RNA was treated with Calf Intestine Alkaline Phosphatase to remove free 5'-phosphates and the RNA was then treated with Tobacco Acid Pyrophosphatase (TAP) to remove the cap structure from full-length mRNA, leaving a 5'-monophosphate. A 45 base RNA Adapter oligonucleotide was ligated to the RNA population using T4 RNA ligase. A random-primed reverse transcription reaction and nested PCR then amplified the 5' end of a specific transcript. The product was analyzed on a 1.5% agarose gel. (C) The PCR product was cloned into pcDNA4-mycHis at BamHI and XhoI sites. DNA sequencing was performed to identify the insert sequence. The first base pair after the adapter was the transcription start site (TSS). The arrow indicates the TSS. (D) The nucleotide sequence of the human TMP21 gene promoter. A 3333-bp fragment of the 5' flanking region of the human TMP21 gene was isolated from a human BAC genomic clone and sequenced by the primer walking strategy. The cytosine +1 represents the TSS mapped by RLM-RACE. The putative transcription factor binding sites are underlined in bold face. Amino acid codons were boxed in boldface. Genbank™accession number is JF694939.