Pamp Am

Figure 1. Genomic organization of human AM gene. Dotted area within the exons are translated into the peptide, proadrenomedullin. The heavily hatched area corresponds to mature AM and the proadrenomedullin N-terminal 20 peptide (PAMP).

Southern blot analysis of the DNA from 20 human-hamster somatic cell lines carrying certain human chromosomes was performed using the cloned genomic DNA fragment encoding the human AM gene as a probe (Ishimitsu et al., 1994b). The labeled probe DNA was hybridized only with the DNA from cell lines containing human chromosome 11 (Ishimitsu et al., 1994b). Therefore, it was determined that the human AM gene is localized on chromosome 11. Further chromosomal sublocalization of the human AM gene was performed with the fluorescence in situ hybridization (FISH) technique (Ishimitsu et al., 2001). The cultured human lymphocytes treated with BrdU for synchronization were fixed on slides and hybridized with a fluorescence-labeled genomic DNA fragment encoding the human AM gene in full length. The fluorescent signal from the probe was detected on the distal end of the short arm of chromosome 11. Thus, the human AM gene was located at chromosome 11 (Ishimitsu et al, 2001), region pl5.1-3, as depicted in Figure 1. Near the location of human AM gene, There exist several other known genes, such as sphingomyelinase (pl5.1-4), parathyroid hormone (pl5.1-2) and lactate dehydrogenase (pl4-15.1) (da Veiga Pereira et al, 1991; Arnold et al, 1989; Li et al, 1988).

2. PROMOTER ACTIVITY OF 5'-FLANKING REGION OF HUMAN AM GENE

Besides the adrenal gland, the mRNA of AM is widely expressed in cardiovascular tissues. The cultured cells such as fibroblasts, vascular endothelial cells and smooth muscle cells have been shown to express AM mRNA prominently (Sugo et al, 1994a; Sugo et al, 1994b; Tomoda et al, 2001). We examined the promoter activity of the human AM gene in cultured human aortic endothelial cells (HAEQ (Ishimitsu et al, 1998). First, the transcription start site was determined by the primer extension method using total RNA extracted from HAEC as the template. As framed in Figure 2, two transcription start sites were identified, both at the cytosine nucleotides. These are 21 and 25 bases downstream from the TATA box and 19 and 23 bases upstream from the 5'-end of the human AM cDNA sequence determined by Kitamura et al. (Kitamura et al, 1993b). Hereafter, the upstream transcription start site at nucleotide C is assigned the nucleotide number 1.

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