Identification of the promoter for the gene encoding the bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase.
The gene encoding rat peptidylglycine alpha-amidating monooxygenase (PAM) contains 26 protein-coding exons. We identified two non-overlapping genomic clones encoding the 5' untranslated region (UTR) of the PAM gene. Exon 1 has 69 nucleotides flanked by perfect splice acceptor and donor sites, with a TATA motif 25 nucleotides upstream. Exon 0 lacks TATA or CAAT motifs and is embedded in a G + C-rich 800-nucleotide CpG island. The major products identified by RNase protection initiated in exon 0; only a minority of mRNAs initiated in exon 1. 5'-rapid amplification of cDNA ends (RACE) identified the same major transcriptional start sites in exon 0 in the atrium and neurointermediate pituitary. The 2.0-kb fragment upstream of exon 0 and the 1.3-kb fragment upstream of exon 1 were placed upstream of a luciferase-based reporter gene in both sense and antisense orientations. Expression of luciferase was observed in neuroendocrine and nonneuroendocrine cells with both sense constructs. A 0.2-kb fragment of the exon 0 PAM promoter containing multiple GC box elements supported expression of luciferase activity in all cell types. Expression of reporter genes in cells that do not normally express PAM suggests a need for more upstream or intronic information, a role for methylation, or a need for chromatin scaffolding for tissue-specific expression of the endogenous gene.
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Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004274:DNA, Recombinant; D005091:Exons / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D005796:Genes; D008297:Male; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D017422:Sequence Analysis, DNA; D013737:Testis; D014158:Transcription, Genetic / Q000235:genetics
D018076:DNA, Complementary; D004274:DNA, Recombinant; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase