ANALYSIS OF THE STRUCTURE AND CELL-SPECIFIC TRANSCRIPTION OF THE BOVINE PARATHYROID HORMONE GENE (MICROCOCCAL NUCLEASE)

Two EcoR I restriction fragments containing PTH gene sequences were isolated from a partial bovine library prepared using EcoR I cleaved high molecular weight bovine DNA and lambda phage charon 30 as a cloning vector. Restriction and sequence analyses of the two fragments indicate that they are identical. This finding supports the conclusion from genomic Southern blot data that there is one copy of the PTH gene per haploid genome. The complete sequence of the bovine PTH gene has been determined and compared to the sequence published for human and rat PTH genes. Remarkable sequence features of the bovine gene include: the high AT content (68%), including very long stretches of alternating AT, the exon-functional domain correspondence, and the presence of two TATA sequences in the 5' flanking region of the gene. Primer extension and S1 mapping of the 5' termini of PTH mRNA indicate that both TATA boxes function in directing transcription in vivo. In addition, both TATA boxes appear to direct transcription in vitro. However, the relative levels of transcriptional initiation from the two TATA boxes were different in vitro as compared to in vivo.;To facilitate the assay of promoter activity of 5' flanking sequences in the PTH gene, several fusion genes were constructed combining varying extents of this region of the PTH gene with a marker gene, the bacterial chloramphenicol acetyltransferase (CAT) gene. Very low levels of CAT expression were detected after transfection of heterologous cells with these fusion genes. Insertion of the SV40 enhancer 2500 bp away from the PTH promoter in fusion-gene plasmids resulted in a dramatic increase in CAT gene expression, demonstrating the transcriptional competence of the PTH promoter. Deletion of PTH sequence from -50 increased expression of the PTH-CAT fusion gene almost two orders of magnitude and increased expression of enhancer-containing fusion-gene plasmids approximately to the level of the intact SV40 promoter. This result suggests that a silencer element is contained within the 5' flanking sequence which functions to repress transcription of the PTH gene in nonparathyroid cells.