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P C Dee

Publications and source records attributed to P C Dee.

11 recordsLinked to original sources
PubMed1982

Somatostatin-28 encoded in a cloned cDNA obtained from a rat medullary thyroid carcinoma.

We have constructed and cloned in bacteria complementary DNAs derived from a transplantable rat medullary thyroid carcinoma. Using a hybridization probe encoding an anglerfish islet pre-prosomatostatin, a precursor of the tetradecapeptide somatostatin, we have identified and isolated a clone containing a 400-base pair complementary DNA encoding most of the rat carcinoma pre-prosomatostatin. The amino acid sequence of the tetradecapeptide somatostatin and of the amino-terminally extended form, somatostatin-28 was deduced from the nucleotide sequence of the complementary DNA. Somatostatin-28 was found at the COOH terminus of a polypeptide of at least 80 amino acids indicating that somatostatin-28 arises by cleavage from a large precursor. The sequences of somatostatin-28 and somatostatin-14 are strictly conserved between the rat and other mammals. Such conservation of these sequences indicates strong selective pressures during evolution to maintain the sequence and suggests that somatostatin-28 may serve some essential biologic functions apart from, or in addition to, the important regulatory actions of somatostatin-14. Additionally, we found a high degree of homology in the amino acid sequences of the NH2-terminal extension peptides in the anglerfish islet and the rat carcinoma pre-prosomatostatins pointing further to a possible biologic function of these extension peptides.

Amino Acid Sequence↗
PubMed1982

Pancreatic preproglucagon cDNA contains two glucagon-related coding sequences arranged in tandem.

We have constructed and cloned in bacteria recombinant plasmids containing DNA complementary to the mRNA encoding a pancreatic preproglucagon (Mr 14,500), a product of cell-free translation of angler fish islet mRNAs shown previously by immunoprecipitation analyses to be a precursor of glucagon. cDNAs of 630, 180, and 120 base pairs were isolated and correspond to most of the mRNA for the preproglucagon (650 bases). The cDNAs contain a protein coding sequence of 372 nucleotides and 5'- and 3'-untranslated regions of 58 and 206 nucleotides, respectively. From the coding sequence of the cDNAs, we find that the sequence of glucagon, identical to mammalian glucagon in 20 of 29 positions, resides in the preproglucagon of 124 amino acids flanked by NH2- and COOH-peptide extensions of 52 and 43 amino acids, respectively. The peptide extensions are linked to the glucagon by Lys-Arg sequences characteristic of the sites that are cleaved during the posttranslational processing of prohormones. Notable is the finding that, following the initial Lys-Arg sequence in the COOH-peptide extension is a pentapeptide. Ser-Gly-Val-Ala-Glu, followed by another Lys-Arg and a sequence of 34 residues that shows striking homology with glucagon and the other peptides of the glucagon family--gastric inhibitory peptide, vasoactive intestinal peptide, and secretin. Thus, the preproglucagon mRNA contains two glucagon-related coding sequences arranged in tandem. The finding of Lys-Arg sequences flanking the glucagon and glucagon-related sequences suggests that these two peptides and a pentapeptide are formed in vivo by posttranslational cleavages of a common precursor.

Amino Acid Sequence↗
PubMed1981

Calcitonin messenger RNA encodes multiple polypeptides in a single precursor.

Recombinant DNA techniques were used to analyze the structure of the messenger RNA encoding a precursor of calcitonin, a small calcium-regulating hormone of 32 amino acids. Analyses of the nucleotide sequences of cloned complementary DNA's comprising the entire coding sequence of the messenger RNA revealed that calcitonin is flanked at both its amino and carboxyl termini by peptide extensions linked to the hormone by short sequences of basic amino acids. The location of glycine next to the carboxyl terminal prolinamide of calcitonin is consistent with indications that glycine is required for the enzymatic amidation of proline to the prolinamide. During cellular biosynthesis, calcitonin arises from a large precursor protein by cleavages at both amino and carboxyl terminal residues of the hormone. These findings raise questions concerning the regulation of these cleavages and the potential biological functions of the precursor extensions derived from these cleavages.

Amino Acid Sequence↗
PubMed1981

Nucleotide sequence of the mRNA encoding the pre-alpha-subunit of mouse thyrotropin.

We have constructed and cloned in bacteria recombinant DNA molecules containing DNA sequences coding for the precursor of the alpha subunit of thyrotropin (pre-TSH-alpha). Double-stranded DNA complementary to total poly(A)+RNA derived from a mouse pituitary thyrotropic tumor was prepared enzymatically, inserted into the Pst I site of the plasmid pBR322 by using poly(dC).poly(dG) homopolymeric extensions, and cloned in Escherichia coli chi 1776. Cloned cDNAs encoding pre-TSH-alpha were identified by their hybridization to pre-TSH-alpha mRNA as determined by cell-free translations of hybrid-selected and hybrid-arrested RNA. The nucleotide sequences of two cDNAs (510 and 480 base pairs) were determined with chemical methods and corresponded to much of the region coding for the alpha subunit and the 3' untranslated region of pre-TSH-alpha mRNA. The sequence of the 5' end of the mRNA was determined from cDNA synthesized by using total mRNA as template and a restriction enzyme DNA fragment as primer. Together these sequences represented greater than 90% of the coding and noncoding regions of full-length pre-TSH-alpha mRNA, which was determined to be 800 bases long. The amino acid sequence of the pre-TSH-alpha deduced from the nucleotide sequence showed a NH2-terminal leader sequence of 24 amino acids followed by the 96-amino-acid sequence of the apoprotein of TSH-alpha. There is greater than 90% homology in the amino acid sequences among the murine, ruminant, and porcine alpha subunits and 75-80% homology among the murine, equine, and human alpha subunits. Several regions of the sequence remain absolutely conserved among all species, suggesting that these particular regions are essential for the biological function of the subunit. The successful cloning of the alpha subunit of TSH will permit further studies of the organization of the genes coding for the glycoprotein hormone subunits and the regulation of their expression.

Animals↗
PubMed1980

Nucleotide sequence of a cloned structural gene coding for a precursor of pancreatic somatostatin.

We have constructed and cloned, in bacteria, recombinant plasmids containing DNA complementary to mRNA coding for a pancreatic pre-prosomatostatin, a product of the cell-free translation of pancreatic islet mRNAs shown previously by immunoprecipitation to be a precursor of somatostatin. A clone containing a nearly full-length cDNA insert of 550 base pairs was identified and appeared to contain the entire coding sequence for the somatostatin precursor in addition to portions of the 5' and 3' untranslated regions. mRNA coding for the pre-prosomatostatin is 600-630 bases long as determined by agarose gel electrophoresis and hybridization with labeled cDNA. Analyses of the nucleotide sequence of the cDNA revealed a protein of 119 amino acid beginning with methionine followed by a typical leader sequence containing 18 hydrophobic amino acids. The tetradecapeptide somatostatin, identical in sequence to mammalian hypothalamic somatostatin, is located at the carboxy terminus followed immediately by a stop codon. An ARg-Lys sequence immediately preceding the sequence of somatostatin is typical of a prohormone cleavage site. A sequence Ala-Pro-Arg-Glu preceding the Arg-Lys cleavage site is identical to that found in porcine prosomatostatin. The evolutionary conservation of the identical amino acid sequence of the somatostatin tetradecapeptide from fish to mammals is remarkable. In addition, similar conservation, in fish and mammals, of the sequence Ala-Pro-Arg-Glu-Arg-Lys preceding the coding region for somatostatin suggets that this particular sequence may have biologic importance in cellular processing of the somatostatin precursor.

Amino Acid Sequence↗
PubMed1980

Early events in the cellular formation of proparathyroid hormone.

Early events in the cellular synthesis and subsequent transfer into membrane-limited compartments of pre-proparathyroid hormone (pre-proPTH) and proparathyroid hormone (proPTH) were investigated by electrophoretic analyses of newly synthesized proteins in subcellular fractions of parthyroid gland slices pulse-labeled for 0.5-5 min with [(35)S] methionine. During these short times of incubation, both pre-proPTH and proPTH were confined to the microsomal fraction. Labeled pre-proPTH and proPTH were detected in a 30-s interval between 0.5 and 1.0 min of incubation. The radioactivity in proPTH became relatively constant between 3 and 5 min, whereas the radioactivity in ProPTH increased markedly over this period. When corrected for the known content of methionine in the prohormone and the prohormone, we found four times as much radiolabeled prohormone as prehormone between 0.5 and 1.0 min of synthesis. Sequestration of labeled prohomrone into endoplasmic reticulum compartments was shown by treatment of the microsomal fraction with chymotrypsin and trypsin, which resulted in the degradation of the prehormone but not of the prohormones. Approximately 50 percent of pre-prohormone and 25 percent of prohormone were released from the microsomes by their extraction with 1.0 M KCl, whereas 80-90 percent of both was released by treatment with Triton X-100. These results in intact cells support the signal hypothesis proposed by Blobel and his co-workers in studies utilizing cell-free systems, inasmuch as the results indicate transfer of prohormone into the cisternal space of the rough endoplasmic reticulum concomitant with the growth of the nascent polypeptide chain. Appearance of membrane-sequestered proPTH takes place without entry of pre-proPTH into the cisternal space, suggesting that proteolytic removal of the leader peptide occurs during transfer of the polypeptide through the lipid bilayer. Further evidence in support of this process is that pre-proPTH is only partly extracted from the microsomes by treatment with 1.0 M KCl, suggesting that a substantial fraction of the nascent pre-proPTH is integrally inserted into the membranes before it is cleaved to form proPTH.

Animals↗
PubMed1979

Cellular processing of pre-proparathyroid hormone involves rapid hydrolysis of the leader sequence.

The cellular synthesis of parathyroid hormone (PTH) involves two consecutive cleavages of NH2-terminal peptide sequences from a larger precursor, pre-proparathyroid hormone (Pre-ProPTH). The initial cleavage consists of the removal of an NH2-terminal leader sequence either during or shortly after biosynthesis of the polypeptide chain is complete. To determine the fate of the cleaved leader sequence, we prepared, by chemical synthesis, a peptide based on the known structure of the leader sequence of pre-proparathyroid hormone and used this peptide labeled with 125iodine as a marker to monitor the recovery of the putative cellular leader peptide during extraction and electrophoresis of [35S]methionine-labeled proteins from pulse-labeled parathyroid gland slices. Under conditions in which the recovery of the synthetic leader peptide was 50 to 70%, we found no detectable 35S-labeled product in the region of sodium dodecyl sulfate gels where the synthetic peptide migrates. In view of the known methionine content of pre-proparathyroid hormone and proparathyroid hormone (ProPTH), it would have been possible to detect endogenously labeled leader peptide if present in amounts equal to 0.05% of the amount of labeled ProPTH present in the tissues. These observations indicate that the cellular conversion of Pre-ProPTH to ProPTH involves a rapid hydrolysis of the leader peptide either during or immediately after its removal from the precursor.

Animals↗
PubMed1976

Metabolism of amino- and carboxyl-sequence immunoreactive parathyroid hormone in the bovine: evidence for peripheral cleavage of hormone.

Radioimmunoassays that detect specifically peptide sequences within either the biologically active amino region (N-assay) or inactive carboxyl region (C-assay) of parathyroid hormone (PTH) were used to evaluate the metabolism of PTH during and after infusion and injection of homogeneous (containing less than 0.1% hormonal fragments) intact bovine PTH (bPTH) into calves. During continuous infusions of hormone, when constant blood levels of immunoreactive PTH were reached, a dissociation between the concentrations of amino versus carboxyl immunoreactivity was observed; concentrations of hormone measured by the C-assay rose to a level of approximately three times higher than that measured by the N-assay. Analysis by gel filtration of immunoreactive PTH in plasma samples from calves after injection of hormone showed the rapid disappearance of intact hormone (N- and C-assays) and the appearance of a large fragment detected by the C-assay but not by the N-assay. The hormonal fragment lacked antigenic determinants within the amino peptide sequence required for biologic activity. No additional fragments of PTH were detected by gel filtration using the N- and C-assays. No detectable conversion of intact PTH to hormonal fragments occurred during incubation in vitro in bovine serum. The results are consistent with the concept that PTH is metabolized after entry into the circulation at peripheral sites located outside the vascular space, resulting in the rapid disappearance from blood of intact hormone and the appearance of a biologically inactive hormonal fragment(s). These studies done in calves agree with earlier studies done in dogs and man and point to the existence in mammals of common pathways for the peripheral metabolism of PTH.

Amino Acid Sequence↗