title: Functional mapping of the bovine Doppel gene promoter region.
abstract: The PRND gene encodes Doppel (Dpl), a protein that is strongly expressed in testis and at much lower levels in other tissues. Despite the recent discovery of Dpl involvement in spermiogenesis and in apoptotic death of cerebellar neurons, respectively in wild type and transgenic mice, the physiological role of this prion-like protein remains unknown. To better understand which factors may contribute to the modulation of PRND activity, a study of the bovine promoter region was performed. First, the transcription start site of PRND mRNA was identified using an innovative fluorescently labelled oligonucleotide extension (FLOE) method. The initiation site mapped 129 nt upstream of the protein coding sequence and represents a refinement of a previous assignment based on RACE. Second, deletion mutants of the 4530 nt encompassing 2704 nt 5' of the bovine PRND, exon 1, intron 1, and the first 6 nt of exon 2, have been investigated with CAT-reporter assays in order to identify critical elements for the activation of the gene. The results showed that the region -323/+32 (+1 is the transcription start site mapped by FLOE) represents the promoter region and contains positive cis-acting elements (CCAAT and E box) confirming previous reports with the mouse gene. Additional regulatory elements, including binding sites for repressor molecules, have been identified upstream of that region and in the first portion of intron 1, suggesting a complex tissue-specific regulation of Doppel gene expression.
authors.affiliation: Università degli Studi di Pavia, Dipartimento di Genetica e Microbiologia, Pavia, Italy. igordv@ipvgen.unipv.it
authors.forename: Igor
authors.initials: I
authors.lastname: Del Vecchio
authors.forename: Alberto
authors.initials: A
authors.lastname: Azzalin
authors.forename: Elena
authors.initials: E
authors.lastname: Guidi
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Amati
authors.forename: Tiziana
authors.initials: T
authors.lastname: Caramori
authors.forename: Cristina
authors.initials: C
authors.lastname: Uboldi
authors.forename: Sergio
authors.initials: S
authors.lastname: Comincini
authors.forename: Luca
authors.initials: L
authors.lastname: Ferretti
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002417:Cattle / Q000235:genetics*; D045744:Cell Line, Tumor; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D019295:Computational Biology / Q000379:methods; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011506:Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D020218:Response Elements / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods; D013737:Testis / Q000378:metabolism; D024363:Transcription Initiation Site; D014162:Transfection
chemical_list: D011506:Proteins; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase