Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1988 Jul;82(1):270–275. doi: 10.1172/JCI113582

Human growth hormone gene and the highly homologous growth hormone variant gene display different splicing patterns.

N E Cooke 1, J Ray 1, M A Watson 1, P A Estes 1, B A Kuo 1, S A Liebhaber 1
PMCID: PMC303504  PMID: 3392209

Abstract

Stably transfected cell lines containing the normal human growth hormone (hGH-N) and human growth hormone-variant (hGH-V) genes have been established in order to study the expression of these two highly homologous genes. Each gene was inserted into a bovine papillomavirus shuttle vector under the transcriptional control of the mouse metallothionein gene promoter and the resultant recombinants were transfected into mouse C127 cells. The transfected cells containing the hGH-N gene secrete two hGH proteins, 91% migrating at 22 kD and 9% migrating at 20 kD, the same relative proportions synthesized in vivo by the human pituitary. S1 nuclease analysis of mRNA from these cells confirms that 20 kD hGH is encoded by an alternatively spliced product of the primary hGH-N gene transcript in which the normal exon 3 splice-acceptor site is bypassed for a secondary site 15 codons within exon 3. Although the hGH-V gene is identical to the hGH-N gene for at least 15 nucleotides on either side of the normal and alternative exon 3 AG splice-acceptor sites, hGH-V synthesizes only a 22-kD protein. Reciprocal exchange of exon 3 and its flanking intron sequences between the hGH-N gene and the hGH-V gene, eliminates the synthesis of the 20-kD protein in both resultant chimeric genes. These results directly demonstrate that both the major 22-kD and the minor 20-kD forms of pituitary hGH are encoded by the alternative splicing products of a single hGH-N gene transcript. This alternative splicing is neither species nor tissue-specific and appears to be regulated by at least two separate regions remote from the AG splice-acceptor site.

Full text

PDF
270

Images in this article

271Image
on p.271
271Image
on p.271
271Image
on p.271
271Image
on p.271
271Image
on p.271
271Image
on p.271
272Image
on p.272
273Image
on p.273
273Image
on p.273

Selected References

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

  1. Barsh G. S., Seeburg P. H., Gelinas R. E. The human growth hormone gene family: structure and evolution of the chromosomal locus. Nucleic Acids Res. 1983 Jun 25;11(12):3939–3958. doi: 10.1093/nar/11.12.3939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chapman G. E., Rogers K. M., Brittain T., Bradshaw R. A., Bates O. J., Turner C., Cary P. D., Crane-Robinson C. The 20,000 molecular weight variant of human growth hormone. Preparation and some physical and chemical properties. J Biol Chem. 1981 Mar 10;256(5):2395–2401. [PubMed] [Google Scholar]
  3. Chen E. Y., Howley P. M., Levinson A. D., Seeburg P. H. The primary structure and genetic organization of the bovine papillomavirus type 1 genome. Nature. 1982 Oct 7;299(5883):529–534. doi: 10.1038/299529a0. [DOI] [PubMed] [Google Scholar]
  4. Cooke N. E., Ray J., Emery J. G., Liebhaber S. A. Two distinct species of human growth hormone-variant mRNA in the human placenta predict the expression of novel growth hormone proteins. J Biol Chem. 1988 Jun 25;263(18):9001–9006. [PubMed] [Google Scholar]
  5. DeNoto F. M., Moore D. D., Goodman H. M. Human growth hormone DNA sequence and mRNA structure: possible alternative splicing. Nucleic Acids Res. 1981 Aug 11;9(15):3719–3730. doi: 10.1093/nar/9.15.3719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dvoretzky I., Shober R., Chattopadhyay S. K., Lowy D. R. A quantitative in vitro focus assay for bovine papilloma virus. Virology. 1980 Jun;103(2):369–375. doi: 10.1016/0042-6822(80)90195-6. [DOI] [PubMed] [Google Scholar]
  7. Engvall E. Enzyme immunoassay ELISA and EMIT. Methods Enzymol. 1980;70(A):419–439. doi: 10.1016/s0076-6879(80)70067-8. [DOI] [PubMed] [Google Scholar]
  8. Hamer D. H., Walling M. Regulation in vivo of a cloned mammalian gene: cadmium induces the transcription of a mouse metallothionein gene in SV40 vectors. J Mol Appl Genet. 1982;1(4):273–288. [PubMed] [Google Scholar]
  9. Howley P. M., Sarver N., Law M. F. Eukaryotic cloning vectors derived from bovine papillomavirus DNA. Methods Enzymol. 1983;101:387–402. doi: 10.1016/0076-6879(83)01029-0. [DOI] [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. Lewis U. J., Bonewald L. F., Lewis L. J. The 20,000-dalton variant of human growth hormone: location of the amino acid deletions. Biochem Biophys Res Commun. 1980 Jan 29;92(2):511–516. doi: 10.1016/0006-291x(80)90363-0. [DOI] [PubMed] [Google Scholar]
  12. Lewis U. J., Dunn J. T., Bonewald L. F., Seavey B. K., Vanderlaan W. P. A naturally occurring structural variant of human growth hormone. J Biol Chem. 1978 Apr 25;253(8):2679–2687. [PubMed] [Google Scholar]
  13. Liebhaber S. A., Ray J., Cooke N. E. Synthesis of growth hormone-prolactin chimeric proteins and processing mutants by the exchange and deletion of genomic exons. J Biol Chem. 1986 Oct 25;261(30):14301–14306. [PubMed] [Google Scholar]
  14. Lusky M., Botchan M. Inhibition of SV40 replication in simian cells by specific pBR322 DNA sequences. Nature. 1981 Sep 3;293(5827):79–81. doi: 10.1038/293079a0. [DOI] [PubMed] [Google Scholar]
  15. Martial J. A., Hallewell R. A., Baxter J. D., Goodman H. M. Human growth hormone: complementary DNA cloning and expression in bacteria. Science. 1979 Aug 10;205(4406):602–607. doi: 10.1126/science.377496. [DOI] [PubMed] [Google Scholar]
  16. Mount S. M. A catalogue of splice junction sequences. Nucleic Acids Res. 1982 Jan 22;10(2):459–472. doi: 10.1093/nar/10.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Padgett R. A., Grabowski P. J., Konarska M. M., Seiler S., Sharp P. A. Splicing of messenger RNA precursors. Annu Rev Biochem. 1986;55:1119–1150. doi: 10.1146/annurev.bi.55.070186.005351. [DOI] [PubMed] [Google Scholar]
  18. Pavlakis G. N., Hizuka N., Gorden P., Seeburg P., Hamer D. H. Expression of two human growth hormone genes in monkey cells infected by simian virus 40 recombinants. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7398–7402. doi: 10.1073/pnas.78.12.7398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ruskin B., Krainer A. R., Maniatis T., Green M. R. Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell. 1984 Aug;38(1):317–331. doi: 10.1016/0092-8674(84)90553-1. [DOI] [PubMed] [Google Scholar]
  20. Seeburg P. H. The human growth hormone gene family: nucleotide sequences show recent divergence and predict a new polypeptide hormone. DNA. 1982;1(3):239–249. doi: 10.1089/dna.1.1982.1.239. [DOI] [PubMed] [Google Scholar]
  21. Sharp P. A. Splicing of messenger RNA precursors. Science. 1987 Feb 13;235(4790):766–771. doi: 10.1126/science.3544217. [DOI] [PubMed] [Google Scholar]
  22. Singh R. N., Seavey B. K., Lewis U. J. Heterogeneity of human growth hormone. Endocr Res Commun. 1974;1(5-6):449–464. doi: 10.3109/07435807409089000. [DOI] [PubMed] [Google Scholar]
  23. Solnick D. Alternative splicing caused by RNA secondary structure. Cell. 1985 Dec;43(3 Pt 2):667–676. doi: 10.1016/0092-8674(85)90239-9. [DOI] [PubMed] [Google Scholar]
  24. Strohman R. C., Moss P. S., Micou-Eastwood J., Spector D., Przybyla A., Paterson B. Messenger RNA for myosin polypeptides: isolation from single myogenic cell cultures. Cell. 1977 Feb;10(2):265–273. doi: 10.1016/0092-8674(77)90220-3. [DOI] [PubMed] [Google Scholar]
  25. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  26. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Vogelstein B., Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A. 1979 Feb;76(2):615–619. doi: 10.1073/pnas.76.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES