ABSTRACT
The murine parotid secretory protein (PSP) gene is expressed selectively at high levels in parotid and sublingual salivary glands. Previously, the transcriptional activity of a PSP mini-gene, called Lama, was shown to be dependent on a 1.5 kb region located 3 kb upstream of the transcription start site. Here, functional studies in transgenic mice demonstrate that this proximal regulatory region has properties of a parotid and sublingual gland specific enhancer. Protein-binding experiments identify multiple sequence-specific binding complexes spanning the entire 1.5 kb enhancer region. Several sequence elements bound specifically by parotid and/or sublingual gland nuclear extracts, including consensus binding elements for previously described transcription factors as well as novel binding elements are located in the proximal enhancer region. A deletion analysis of the enhancer region in transgenic mice identified a core sequence of 700 bp. This region contains five elements bound specifically by nuclear proteins isolated from the PSP-expressing parotid and sublingual glands. Two of these elements, denoted parotid gland element I (PGE I) and sublingual gland element I (SLE I), are novel salivary gland specific binding elements, bound uniquely by parotid and sublingual gland nuclear extracts, respectively.
Subject(s)
Enhancer Elements, Genetic , Parotid Gland/metabolism , Salivary Proteins and Peptides/genetics , Sublingual Gland/metabolism , Amino Acid Sequence , Animals , Bacteriophage lambda , Base Sequence , Female , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Transgenic , Molecular Sequence Data , Regulatory Sequences, Nucleic AcidABSTRACT
The murine PSP gene is expressed at a high-level in the parotid glands. To extend the knowledge of parotid gland expression and develop tools for expression of heterologous proteins in this tissue, the regulation of the PSP gene was studied using transgenic mice. High-level parotid gland expression of the PSP gene was indicated to depend on a novel regulatory region situated between -8.0 and -6.5 kb. Together with previous results this indicates that the main regulatory elements in the PSP gene are situated between -8.0 to -3.1 kb. This region was shown to activate a heterologus SV40 early promoter in the parotid glands of transgenic mice, suggesting that the PSP gene is controlled by enhancer sequences. A novel Psp derived 9.7 kb parotid gland expression cassette, Lama IV, carrying all known regulatory regions in the PSP gene was expressed at high-levels in the parotid glands and should prove highly useful for expression of heterologous proteins in the saliva of transgenic mice.
Subject(s)
Enhancer Elements, Genetic/genetics , Gene Expression Regulation , Parotid Gland/metabolism , Salivary Proteins and Peptides/genetics , Transgenes/genetics , Animals , Blotting, Northern , Blotting, Southern , Lac Operon , Luciferases/metabolism , Mice , Mice, Transgenic , Molecular Sequence Data , Promoter Regions, Genetic/geneticsABSTRACT
Histidine-rich glycoprotein (HRG) was purified from bovine plasma and the disulphide bridge arrangement established. Disulphide-bridged peptides were obtained from peptic and tryptic degradation of native bovine HRG. Twelve half-cystine residues were found in bovine HRG (compared to sixteen cysteines in human HRG), all involved in the formation of six disulphide bridges connecting Cys-1 to Cys-12, Cys-2 to Cys-3, Cys-4 to Cys-5, Cys-6 to Cys-11, Cys-7 to Cys-8, and Cys-9 to Cys-10. Additional sequence analysis of 14C-carboxymethylated chymotryptic and Staphylococcus aureus V8 protease generated peptides and CNBr-fragments of bovine HRG yielded a partial amino acid sequence of bovine HRG constituting 78% of the sequence when compared to the human cDNA sequence.
