Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1984 Mar;3(3):617–621. doi: 10.1002/j.1460-2075.1984.tb01857.x

Biosynthesis of thyrotropin releasing hormone in the skin of Xenopus laevis: partial sequence of the precursor deduced from cloned cDNA.

K Richter, E Kawashima, R Egger, G Kreil
PMCID: PMC557397  PMID: 6425056

Abstract

Skin of Xenopus laevis contains relatively large quantities of thyrotropin releasing hormone (TRH). Total mRNA isolated from skin was cloned in the plasmid pUC8. Among 1400 cDNA clones, one was found with an insert of 478 nucleotides coding for the amino-terminal part of prepro-TRH. This clone was detected using a mixture of two synthetic undecanucleotides for colony hybridization. The single open reading frame starts with a methionine residue and a stretch of hydrophobic amino acids, as is typical for signal peptides, and terminates at the poly(C) tail without a stop codon. The deduced polypeptide of 123 amino acids contains three copies of the sequences Lys-Arg-Gln-His-Pro-Gly-Lys Arg-Arg and a fourth incomplete copy at the carboxyl end. Typical pro-hormone processing at this sequence would yield pGlu-His-Pro.NH2,i.e. TRH. It is concluded that the cloned part of the mRNA codes for prepro-TRH and that the TRH precursor from skin of X. laevis is a polyprotein containing at least four copies of the end product in its amino acid sequence.

Full text

PDF
617

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ackermann T., Gramlich V., Klump H., Knäble T., Schmid E. D., Seliger H., Stulz J. Demonstration of G . U wobble base pairs by Raman and IR spectroscopy. Biophys Chem. 1979 Nov;10(3-4):231–238. doi: 10.1016/0301-4622(79)85011-5. [DOI] [PubMed] [Google Scholar]
  2. Bauer K., Lipmann F. Attempts toward biosynthesis of the thyrotropin-releasing hormone and studies on its breakdown in hypothalamic tissue preparations. Endocrinology. 1976 Jul;99(1):230–242. doi: 10.1210/endo-99-1-230. [DOI] [PubMed] [Google Scholar]
  3. Bennett G. W., Balls M., Clothier R. H., Marsden C. A., Robinson G., Wemyss-Holden G. D. Location and release of TRH and 5-HT from amphibian skin. Cell Biol Int Rep. 1981 Feb;5(2):151–158. doi: 10.1016/0309-1651(81)90023-0. [DOI] [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boel E., Vuust J., Norris F., Norris K., Wind A., Rehfeld J. F., Marcker K. A. Molecular cloning of human gastrin cDNA: evidence for evolution of gastrin by gene duplication. Proc Natl Acad Sci U S A. 1983 May;80(10):2866–2869. doi: 10.1073/pnas.80.10.2866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  7. Bradbury A. F., Finnie M. D., Smyth D. G. Mechanism of C-terminal amide formation by pituitary enzymes. Nature. 1982 Aug 12;298(5875):686–688. doi: 10.1038/298686a0. [DOI] [PubMed] [Google Scholar]
  8. Cooke N. E., Coit D., Shine J., Baxter J. D., Martial J. A. Human prolactin. cDNA structural analysis and evolutionary comparisons. J Biol Chem. 1981 Apr 25;256(8):4007–4016. [PubMed] [Google Scholar]
  9. Dagert M., Ehrlich S. D. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene. 1979 May;6(1):23–28. doi: 10.1016/0378-1119(79)90082-9. [DOI] [PubMed] [Google Scholar]
  10. Docherty K., Steiner D. F. Post-translational proteolysis in polypeptide hormone biosynthesis. Annu Rev Physiol. 1982;44:625–638. doi: 10.1146/annurev.ph.44.030182.003205. [DOI] [PubMed] [Google Scholar]
  11. Eipper B. A., Mains R. E., Glembotski C. C. Identification in pituitary tissue of a peptide alpha-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper, and ascorbic acid. Proc Natl Acad Sci U S A. 1983 Aug;80(16):5144–5148. doi: 10.1073/pnas.80.16.5144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Erspamer V. Biogenic amines and active polypeptides of the amphibian skin. Annu Rev Pharmacol. 1971;11:327–350. doi: 10.1146/annurev.pa.11.040171.001551. [DOI] [PubMed] [Google Scholar]
  13. Erspamer V., Melchiorri P., Broccardo M., Erspamer G. F., Falaschi P., Improota G., Negri L., Renda T. The brain-gut-skin triangle: new peptides. Peptides. 1981;2 (Suppl 2):7–16. doi: 10.1016/0196-9781(81)90003-6. [DOI] [PubMed] [Google Scholar]
  14. Furutani Y., Morimoto Y., Shibahara S., Noda M., Takahashi H., Hirose T., Asai M., Inayama S., Hayashida H., Miyata T. Cloning and sequence analysis of cDNA for ovine corticotropin-releasing factor precursor. Nature. 1983 Feb 10;301(5900):537–540. doi: 10.1038/301537a0. [DOI] [PubMed] [Google Scholar]
  15. Gubler U., Monahan J. J., Lomedico P. T., Bhatt R. S., Collier K. J., Hoffman B. J., Böhlen P., Esch F., Ling N., Zeytin F. Cloning and sequence analysis of cDNA for the precursor of human growth hormone-releasing factor, somatocrinin. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4311–4314. doi: 10.1073/pnas.80.14.4311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Guillemin R. Peptides in the brain: the new endocrinology of the neuron. Science. 1978 Oct 27;202(4366):390–402. doi: 10.1126/science.212832. [DOI] [PubMed] [Google Scholar]
  17. Hoffmann W., Richter K., Kreil G. A novel peptide designated PYLa and its precursor as predicted from cloned mRNA of Xenopus laevis skin. EMBO J. 1983;2(5):711–714. doi: 10.1002/j.1460-2075.1983.tb01489.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  19. Husain I., Tate S. S. Formation of the COOH-terminal amide group of thyrotropin-releasing-factor. FEBS Lett. 1983 Feb 21;152(2):277–281. doi: 10.1016/0014-5793(83)80395-0. [DOI] [PubMed] [Google Scholar]
  20. Jackson I. M., Reichlin S. Thyrotropin-releasing hormone: abundance in the skin of the frog, Rana pipiens. Science. 1977 Oct 28;198(4315):414–415. doi: 10.1126/science.410104. [DOI] [PubMed] [Google Scholar]
  21. Jay E., Bambara R., Padmanabhan R., Wu R. DNA sequence analysis: a general, simple and rapid method for sequencing large oligodeoxyribonucleotide fragments by mapping. Nucleic Acids Res. 1974 Mar;1(3):331–353. doi: 10.1093/nar/1.3.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kakidani H., Furutani Y., Takahashi H., Noda M., Morimoto Y., Hirose T., Asai M., Inayama S., Nakanishi S., Numa S. Cloning and sequence analysis of cDNA for porcine beta-neo-endorphin/dynorphin precursor. Nature. 1982 Jul 15;298(5871):245–249. doi: 10.1038/298245a0. [DOI] [PubMed] [Google Scholar]
  23. Kurjan J., Herskowitz I. Structure of a yeast pheromone gene (MF alpha): a putative alpha-factor precursor contains four tandem copies of mature alpha-factor. Cell. 1982 Oct;30(3):933–943. doi: 10.1016/0092-8674(82)90298-7. [DOI] [PubMed] [Google Scholar]
  24. Land H., Grez M., Ruppert S., Schmale H., Rehbein M., Richter D., Schütz G. Deduced amino acid sequence from the bovine oxytocin-neurophysin I precursor cDNA. Nature. 1983 Mar 24;302(5906):342–344. doi: 10.1038/302342a0. [DOI] [PubMed] [Google Scholar]
  25. Land H., Schütz G., Schmale H., Richter D. Nucleotide sequence of cloned cDNA encoding bovine arginine vasopressin-neurophysin II precursor. Nature. 1982 Jan 28;295(5847):299–303. doi: 10.1038/295299a0. [DOI] [PubMed] [Google Scholar]
  26. Lehrach H., Frischauf A. M., Hanahan D., Wozney J., Fuller F., Boedtker H. Construction and characterization of pro alpha 1 collagen complementary deoxyribonucleic acid clones. Biochemistry. 1979 Jul 10;18(14):3146–3152. doi: 10.2196/47873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lund P. K., Goodman R. H., Dee P. C., Habener J. F. Pancreatic preproglucagon cDNA contains two glucagon-related coding sequences arranged in tandem. Proc Natl Acad Sci U S A. 1982 Jan;79(2):345–349. doi: 10.1073/pnas.79.2.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  29. Mayo K. E., Vale W., Rivier J., Rosenfeld M. G., Evans R. M. Expression-cloning and sequence of a cDNA encoding human growth hormone-releasing factor. Nature. 1983 Nov 3;306(5938):86–88. doi: 10.1038/306086a0. [DOI] [PubMed] [Google Scholar]
  30. Mitnick M., Reichlin S. Enzymatic synthesis of thyrotropin releasing hormone (TRH) by hypothalamic "TRH synthetase". Endocrinology. 1972 Nov;91(5):1145–1153. doi: 10.1210/endo-91-5-1145. [DOI] [PubMed] [Google Scholar]
  31. Nawa H., Hirose T., Takashima H., Inayama S., Nakanishi S. Nucleotide sequences of cloned cDNAs for two types of bovine brain substance P precursor. Nature. 1983 Nov 3;306(5938):32–36. doi: 10.1038/306032a0. [DOI] [PubMed] [Google Scholar]
  32. Nelson T., Brutlag D. Addition of homopolymers to the 3'-ends of duplex DNA with terminal transferase. Methods Enzymol. 1979;68:41–50. doi: 10.1016/0076-6879(79)68005-9. [DOI] [PubMed] [Google Scholar]
  33. Noda M., Furutani Y., Takahashi H., Toyosato M., Hirose T., Inayama S., Nakanishi S., Numa S. Cloning and sequence analysis of cDNA for bovine adrenal preproenkephalin. Nature. 1982 Jan 21;295(5846):202–206. doi: 10.1038/295202a0. [DOI] [PubMed] [Google Scholar]
  34. Noyes B. E., Mevarech M., Stein R., Agarwal K. L. Detection and partial sequence analysis of gastrin mRNA by using an oligodeoxynucleotide probe. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1770–1774. doi: 10.1073/pnas.76.4.1770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Roninson I. B., Ingram V. M. cDNA sequence of a new chicken embryonic rho-globin. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4782–4785. doi: 10.1073/pnas.78.8.4782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rupnow J. H., Hinkle P. M., Dixon J. E. A macromolecule which gives rise to thyrotropin releasing-hormone. Biochem Biophys Res Commun. 1979 Jul 27;89(2):721–728. doi: 10.1016/0006-291x(79)90689-2. [DOI] [PubMed] [Google Scholar]
  37. Schally A. V. Aspects of hypothalamic regulation of the pituitary gland. Science. 1978 Oct 6;202(4363):18–28. doi: 10.1126/science.99816. [DOI] [PubMed] [Google Scholar]
  38. Scott J., Urdea M., Quiroga M., Sanchez-Pescador R., Fong N., Selby M., Rutter W. J., Bell G. I. Structure of a mouse submaxillary messenger RNA encoding epidermal growth factor and seven related proteins. Science. 1983 Jul 15;221(4607):236–240. doi: 10.1126/science.6602382. [DOI] [PubMed] [Google Scholar]
  39. Singer-Sam J., Simmer R. L., Keith D. H., Shively L., Teplitz M., Itakura K., Gartler S. M., Riggs A. D. Isolation of a cDNA clone for human X-linked 3-phosphoglycerate kinase by use of a mixture of synthetic oligodeoxyribonucleotides as a detection probe. Proc Natl Acad Sci U S A. 1983 Feb;80(3):802–806. doi: 10.1073/pnas.80.3.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Suchanek G., Kreil G. Translation of melittin messenger RNA in vitro yields a product terminating with glutaminylglycine rather than with glutaminamide. Proc Natl Acad Sci U S A. 1977 Mar;74(3):975–978. doi: 10.1073/pnas.74.3.975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  42. Volckaert G., Tavernier J., Derynck R., Devos R., Fiers W. Molecular mechanisms of nucleotide-sequence rearrangements in cDNA clones of human fibroblast interferon mRNA. Gene. 1981 Nov;15(2-3):215–223. doi: 10.1016/0378-1119(81)90131-1. [DOI] [PubMed] [Google Scholar]
  43. Wallace R. B., Johnson M. J., Hirose T., Miyake T., Kawashima E. H., Itakura K. The use of synthetic oligonucleotides as hybridization probes. II. Hybridization of oligonucleotides of mixed sequence to rabbit beta-globin DNA. Nucleic Acids Res. 1981 Feb 25;9(4):879–894. doi: 10.1093/nar/9.4.879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zain S., Sambrook J., Roberts R. J., Keller W., Fried M., Dunn A. R. Nucleotide sequence analysis of the leader segments in a cloned copy of adenovirus 2 fiber mRNA. Cell. 1979 Apr;16(4):851–861. doi: 10.1016/0092-8674(79)90100-4. [DOI] [PubMed] [Google Scholar]
  45. von Heijne G. Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem. 1983 Jun 1;133(1):17–21. doi: 10.1111/j.1432-1033.1983.tb07424.x. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES