Nucleotide sequence of the human parathyroid hormone gene.

Abstract

From a A phage gene library we have isolated phage containing the gene encoding human preproparathyroid hormone. The phage were isolated by using both the plaque-hybridization technique and the in vivo recombination-selection technique. The human preproparathyroid hormone gene contains two intervening sequences that separate the gene into a 5' noncoding domain, a "prepro" sequence domain, and a domain containing the parathyroid hormone sequence and the 3' noncoding region. The gene is approximately 4,200 base pairs long. Restriction endonuclease analysis of human leukocyte DNA shows that the haploid human-genome contains one copy of the preproparathyroid hormone gene. A 14-base-pair sequence of alternating purines and pyrimidines that has the potential of adopting the Z-DNA conformation lies 134 base pairs upstream from the presumed site of initiation of transcription.Parathyroid hormone (PTH) is the principal homeostatic regulator of blood calcium, and, in turn, the secretion of PTH is closely regulated by the blood level of calcium. While the regulation of secretion of PTH has been studied extensively, the regulation of the synthesis of PTH has received comparatively little attention. We (1) and others (2, 3) have cloned cDNA encoding bovine PTH. More recently, we have cloned cDNA encoding human PTH as well (4). Here we describe the isolation and DNA sequence analysis of genomic DNA encoding human PTH. The human PTH gene contains two intervening sequences that separate the mRNA sequence into three functional domains. DNA blotting experiments show that the haploid human genome contains only one PTH gene. METHODSScreening a A Phage Library. A human gene library in phage Charon 4A, derived from fetal liver, was provided by T. Maniatis (5). The library was first screened by the procedure of Benton and Davis (6), using plasmid pPTHm113 (4) containing human PTH cDNA as a nick-translated (7) probe. The library was subsequently screened by the method of Seed (8). Plasmid DNA was isolated from Escherichia coli strain W3110r m+(p3)(irVX) by an alkaline miniprep procedure (9), the DNA was electrophoresed-through a 0. 7% agarose gel, and the irVX plasmid was isolated from the gel. By using phage T4 DNA ligase, the Bgl II/Xba I fragment from pPTHm122 (4) was inserted into the corresponding sites on the irVX plasmid. The resultant plasmid, irVX-PTH, containing the PTH cDNA linked to the selectable marker supF, was introduced into E. coli strain W3110r-m+ (p3). One million library phage were then amplified on one plate of the resultant strain W3110r-m+(p3)(QrVX-PTH). Then 5 X 108 of the resultant phage were grown on one plate of strain W3110r-m+ Su-, a strain containing no amber suppressor tRNA gene. Growth of Charon 4A phage on E. coli W3110r-m+ Suselected for phage that had incorporated the supF gene, encoding an amber suppressor tRNA, from the irVX-PTH plasmid. All work with organisms containing recombinant DNA was performed in a PI physical containment facility according to the then current guidelines of the Nat...

The murine gene encoding parathyroid hormone: genomic organization, nucleotide sequence and transcriptional regulation

“…The structural organization of the mouse gene is identical to that reported for the human (Vasicek et al 1983) and rat genes (Heinrich et al 1984), with complete conservation of exonintron structures amongst species. Likewise, the open reading frame of the putative mouse Pth gene is contained in two exons and encodes a protein of 115 amino acids that shares a high degree of sequence identity with other mammalian homologues.…”

“…The DNA was fractionated on a 0·7% agarose gel and transferred onto a nitrocellulose membrane using standard procedures (Sambrook et al 1989). Hybridization with an [ -32 P]dCTP-labeled random-primed human PTH cDNA probe (Vasicek et al 1983) was carried out at 42 C in 50% formamide, 4 saline sodium citrate (SSC; 1 SSC: 300 mM NaCl, 30 mM sodium citrate, pH 7·0), 2 Denhardt's solution, 10% dextran sulfate, 100 µg/ml salmon sperm DNA (final concentration) for 16 h. The blot was washed with 2 SSC/0·1% SDS for 30 min at room temperature and then with 0·2 SSC/0·1% SDS for 30 min at 50 C, before autoradiography was performed.…”

“…Overall, these changes represent conservative amino (Vasicek et al 1983), macaque (unpublished, Q9XT35), rat (Heinrich et al 1984), bovine (Kronenberg et al 1979), porcine (Sauer et al 1974), and canine (Rosol et al 1995) PTH are used. Residues conserved across most species are left unshaded, whereas somewhat conserved and highly variable residues have been highlighted by light and dark shading, respectively, using the BoxShade program (www.ch.embnet.org).…”

Analysis of the c-Ha-ras-1 gene for deletion, mutation, amplification and expression in lymph node metastases of human head and neck carcinomas

“…These probes were obtained from the 6.6 kb c-Ha-ras-l gene of EJ cells. Probes used as markers on chromosome 11 were: D I S12 from clone pADJ762 (Barker et al, 1984); HBBC (yG and 'A of the ,B globin cluster) from clone JW151 (Antonarakis et al, 1982); PTH (human parathyroid hormone) from clone pPTH 2.5 (Vasicek et al, 1983); CALC (calcitonin) from clone phTB3 (Craig et al, 1982); CAT (catalase) from clone pCAT41 (Korneluk et al, 1984).…”

Cell-specific expression of the human parathyroid hormone gene in rat pituitary cells.

“…Both TATA boxes of the human parathyroid hormone gene accurately directed transcription in GH4C1 cells; the parathyroid hormone promoter was inactive in HeLa cells.The genes encoding human, bovine, and rat parathyroid hormone (PTH) have recently been isolated, and the DNA sequences of the genes have been determined (6,22,24). The three genes show substantial evolutionary conservation of sequences upstream from the transcription start site for hundreds of base pairs, suggesting that this region has an important regulatory role.…”