Nucleotide sequence of cloned cDNAs encoding human preproparathyroid hormone.

Abstract

We have cloned cDNA copies of human preproparathyroid hormone in Escherichia coli after insertion of doublestranded DNA into the Pst I site of plasmid pBR322 using the poly(dG).poly(dC) homopolymer extension technique. Recombinant plasmids coding for preproparathyroid hormone were identified by filter hybridization assay using as a probe 32P-labeled bovine preproparathyroid cDNA. Nucleotide sequence analysis of five recombinant plasmids permitted the assignment of 74 nucleotides of the 5' noncoding region, the entire coding region of 345 nucleotides, and the entire 3' noncoding region of 348 nucleotides of the mRNA. The coding sequence predicts the previously unknown "pre" amino acid sequence and clarifies the hormone's amino acid sequence, which has been disputed. The 5' noncoding region contains an AUG codon followed by a UGA stop codon before the authentic initiator codon. The 3' noncoding region is 120 nucleotides longer than in bovine preproparathyroid mRNA and contains two A-A-U-A-A-A sequences, potential signals for polyadenlylation.Parathyroid hormone (PTH), an 84-amino acid polypeptide, is a major regulator of the level of blood calcium. It is biosynthesized as a 115-amino acid precursor, preproparathyroid hormone (preproPTH). Kronenberg et aL (1) and Kemper et aL (2) have described the cloning and sequence analysis of DNA copies of bovine preproPTH mRNA. In order to study further the regulation ofPTH biosynthesis in man, we have synthesized and amplified, by cloning in Escherichia coli, DNA copies ofhuman preproPTH mRNA. Nucleotide sequence analysis ofthe cloned cDNAs has revealed almost the entire primary structure of the mRNA.MATERIALS AND METHODS Enzymes. Reverse transcriptase was provided byJ. W. Beard (Life Sciences, St. Petersburg, FL). T4 polynucleotide kinase was purchased from Boehringer Mannheim and New England Nuclear. DNA polymerase I (large fragment) and restriction endonucleases were from New England BioLabs. S1 nuclease and lysozyme were from Sigma; terminal transferase and restriction endonucleases were from Bethesda Research Laboratories (Rockville, MD).Bacteria and Nucleic Acids. E. coli xy1776 and pBR322 were obtained as described (1). Human parathyroid mRNA was prepared as follows. Human parathyroid adenomas and hyperplastic tissue obtained at surgery performed to cure hyperparathyroidism were frozen in liquid nitrogen. Polyadenylylated RNA was prepared from 5 g of this tissue by phenoVchloroform/isoamyl alcohol extraction followed by oligo(dT)-cellulose chromatography as described (3).Synthesis of Poly(dC)-Tailed Double-Stranded DNA. DNA complementary in sequence to PTH mRNA was synthesized and made double-stranded as described (1). Poly(dC) homopolymer extensions were added by using terminal transferase as described (4). Similarly, poly(dG) homopolymer extensions were added to Pst I-cut pBR322 DNA.Transfection of E. colixVX776. Poly(dG)-tailed pBR322 (300 ng) and 20 ng of poly(dC)-tailed parathyroid DNA were annealed in 0.1 M NaCl/l0mM Tris-HCl, pH 7.6/0.1mM EDTA for 2 mi...

Isolation and characterization of the bioactive circulating human parathyroid hormone, hPTH‐1–37

“…These vesicles fuse with the plasma membrane and PTH is released in response to decreased serum calcium levels. Under physiological conditions, secretion occurs predominantly as the intact hPTH-1-84 molecule with a molecular weight of 9425 Da [2], and a minor fraction of hPTH was shown to be metabolized prior to secretion. For human pathological parathyroid tissue, a Ca 2+ -dependent release of quantities of N-terminal PTH fragments about 3-6 times greater than those of intact hPTH-1-84 is described [3].…”

Parathyroid hormone: past and present

“…Nucleotide sequence analysis of cDNA for human and bovine PTH was used to confirm the amino acid sequences that had originally been determined by conventional peptide sequence analysis (Kronenberg et al 1979, Hendy et al 1981. The amino acid sequences of rat, chicken and dog PTH were determined exclusively by molecular cloning techniques without isolation of the protein (Heinrich et al 1984, Khosla et al 1988, Russell & Sherwood 1989, Jüppner et al 2000.…”

Termination-reinitiation occurs in the translation of mammalian cell mRNAs.

“…Many naturally occurring cellular (14,44,55,58) and viral mRNAs (5,7,10,11,17,19,30,31,53,57) are now known to initiate translation at internal AUGs. Moreover, internal initiation has been demonstrated in molecules constructed artificially by recombinant DNA methods (29,38,50,52).…”