Functional characterization of the human prion protein promoter in neuronal and endothelial cells.
Human prion diseases such as Creutzfeld-Jakob disease and kuru are of major medical and biological importance because of their fatal course, epidemic potential, and unique pathophysiology. Endogenous expression of the normal cellular prion protein (PrP(C)) is necessary for infection and prion replication. However, knowledge of human PrP(C) gene regulation is rudimentary. We therefore cloned1543 bp of the 5' untranslated and promoter region of the PrP gene. Using transient transfection assays, the full-length promoter and serial deletion mutants subcloned in a luciferase reporter vector were analyzed in neuronal (KELLY) and endothelial (EA.hy926) cell lines, which both express PrP(C) as shown by RT/PCR. Analysis of promoter constructs in KELLY cells indicated two activating regions at -131/-284 and -1303/-1543, relative to the 3'-terminal end of exon 1, and also two repressing elements at -254/-567 and -567/-909 in neuronal cells. In EA.hy926 cells, activating elements were identified at -131/-284 and -284/-567, and one repressing region was localized at -567/-909. In addition, transcriptional start sites were determined by 5'-RACE reaction and RNase protection assay, revealing one major transcriptional start site located at -47 (in KELLY cells), -53 (in human thalamus) and at about -55 (in EA.hy926 cells).
Funke-Kaiser
H
H
Institute of Clinical Pharmacology and Toxicology, Department of Clinical Pharmacology, Benjamin Franklin Medical Center, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany.
Theis
S
S
Behrouzi
T
T
Thomas
A
A
Scheuch
K
K
Zollmann
F S
FS
Paterka
M
M
Paul
M
M
Orzechowski
H D
HD
D020121:5' Untranslated Regions; D002460:Cell Line; D003001:Cloning, Molecular; D004727:Endothelium / Q000166:cytology* / Q000378:metabolism; D005091:Exons; D017353:Gene Deletion; D017930:Genes, Reporter; D006801:Humans; D016296:Mutagenesis; D009474:Neurons / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D018621:PrPC Proteins / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D020121:5' Untranslated Regions; D018621:PrPC Proteins; D012260:Ribonucleases