The mouse proline-rich protein MP6 promoter binds isoprenaline-inducible parotid nuclear proteins via a highly conserved NFkB/rel-like site.
Proline-rich protein (PRP) gene MP6 was isolated from a mouse BALB/c genomic DNA library in lambda EMBL3, characterised by hybridisation and restriction mapping and the promoter region, from -162 to +72 around the PRP consensus cap-site, was sequenced. In gel shift assays this region formed complexes C1 and C2 with parotid nuclear proteins which were induced by the beta-adrenergic agonist isoprenaline. DNA competition studies and direct binding assays of promoter subfragments showed that it was the sequence from -157 to -91 that was forming the isoprenaline-dependent complexes. All PRP genes conserve a 23bp. sequence, termed PRP Box1, with ets and NFkB/rel binding site-like elements, upstream of their promoters. In the MP6 promoter, PRP Box1 was within the region forming the complexes. Further gel shift assays using PRP Box1 oligonucleotides as competitors and targets indicated that the NFkB/rel binding site-like element was important in formation of the isoprenaline-inducible complexes. HeLa nuclear extracts also formed complexes with PRP Box1 similar to C1 and C2 but nuclear extracts from spleen, submandibular gland and liver did not. These complexes are thus candidate regulators for the isoprenaline-dependent and tissue-specific transcription of PRP genes.
Roberts
S G
SG
Krebs Institute for Biomolecular Research, Department of Molecular Biology, University of Sheffield, Western Bank, South Yorkshire, UK.
Layfield
R
R
McDonald
C J
CJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification; D055232:Proline-Rich Protein Domains; D011401:Promoter Regions, Genetic / Q000235:genetics*
D016328:NF-kappa B; D009687:Nuclear Proteins; D009841:Oligonucleotides; D010455:Peptides; D007545:Isoproterenol