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...

We have cloned in Escherichia coli a DNA copy of mRNA coding for bovine preproparathyroid hormone. Double-stranded DNA was inserted into the Pst I site in pliismid pBR322 by using the poly(dG)poly(dC) homopolymer extension technique to join the DNA molecules. Recombinant plasmids coding for preproparathyroid hormone were identified by the plasmid's ability to arrest specifically the translation of preproparathyroid hormone mRNA. The nucleotide sequence of the largest recombinant was determined by using both chemical and enzymatic techniques. The parathyroid insert contains 470 nucleotides-102 nucleotides from the 5' noncoding region of the mRNA, 345 nucleotides representing the entire coding region, and 23 nucleotides from the 3' noncoding region. The coding sequence clarifies the hormone's amino acid sequence, which has been disputed. Codon usage is discussed.Parathyroid hormone (PTH) regulates the blood level of calcium, and in turn, blood calcium regulates the secretion of PTH. Although modulators of PTH secretion have been extensively delineated (1), the regulation of synthesis of PTH is poorly understood. Pulse-chase studies of parathyroid gland slices (2) and translation of mRNA in cell-free systems (3) have shown that PTH (84 amino acids) is first synthesized as a 115-amino acid precursor, preproparathyroid hormone (PreproPTH), with a 31-amino acid NH2-terminal extension. The first 25 amino acids are rapidly cleaved to yield the 90-amino acid proparathyroid hormone, which is subsequently converted to PTH. As a first step in studying further the regulation of PTH biosynthesis, we have cloned in Escherichia coli a DNA copy of PTH mRNA. Nucleotide sequence analysis of the cloned DNA demonstrates that one clone contains the entire coding region plus portions of both the 5' and 3' noncoding regions of the mRNA.MATERIALS AND METHODS Enzymes. Reverse transcriptase was provided by J. W. Beard (Life Sciences, St. Petersburg, FL). DNA polymerase I and polynucleotide kinase were purchased from Boehringer Mannheim, S1 nuclease and lysozyme were from Sigma, terminal transferase was from Bethesda Research (Cockeysville, MD), and restriction endonucleases and DNA polymerase I (large fragment) were from New England Biolabs.Bacteria and Nucleic Acids. E. coli X1776 (4), a certified EK2 host, was provided by R. Curtiss and E. coli carrying plasmid pBR322 (5) was provided by A. Bothwell. pBR322, a certified EK2 vector, § after amplification with chloramphenicol was purified on two CsCI2 gradients after use of the cleared lysate technique (6). PTH mRNA was partially purified as described (7). Vesicular stomatitis virus RNA was isolated as described (8).Synthesis of Poly(dC)-Tailed Double-Stranded DNA. DNA complementary in sequence to PTH mRNA was synthesized by using minor modifications of the protocols of Friedman and Rosbash (9) and Efstratiadis et al. (10). mRNA (18 ,ug) was incubated in a 2.2-ml reaction mixture containing 169 units of reverse transcriptase (220 ,ul), 50 mM Tris-HCl (pH 8.3), oligo(dT) at 27 ,ug...

2016-11-15T19:40:59

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