Abstract
We have chemically synthesized and expressed in yeast a gene coding for human epidermal growth factor (urogastrone), a 53-amino-acid polypeptide that has been shown to promote epithelial cell proliferation and to inhibit gastric acid secretion. The synthetic gene, consisting of 170 base pairs, was designed with yeast-preferred codons and assembled by enzymatic ligation of synthetic fragments produced by phosphoramidite chemistry. The DNA synthesis protocol used allows for facile synthesis of oligonucleotides larger than 50 bases. Yeast cells were transformed with plasmids containing the synthetic gene under control of a yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter and were shown to synthesize a biologically active human epidermal growth factor.
Full text
PDF




Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Alvarado-Urbina G., Sathe G. M., Liu W. C., Gillen M. F., Duck P. D., Bender R., Ogilvie K. K. Automated synthesis of gene fragments. Science. 1981 Oct 16;214(4518):270–274. doi: 10.1126/science.6169150. [DOI] [PubMed] [Google Scholar]
- Beggs J. D. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978 Sep 14;275(5676):104–109. doi: 10.1038/275104a0. [DOI] [PubMed] [Google Scholar]
- Bennetzen J. L., Hall B. D. Codon selection in yeast. J Biol Chem. 1982 Mar 25;257(6):3026–3031. [PubMed] [Google Scholar]
- Bolivar F. Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules. Gene. 1978 Oct;4(2):121–136. doi: 10.1016/0378-1119(78)90025-2. [DOI] [PubMed] [Google Scholar]
- Brown E. L., Belagaje R., Ryan M. J., Khorana H. G. Chemical synthesis and cloning of a tyrosine tRNA gene. Methods Enzymol. 1979;68:109–151. doi: 10.1016/0076-6879(79)68010-2. [DOI] [PubMed] [Google Scholar]
- Brown N. L., Smith M. Cleavage specificity of the restriction endonuclease isolated from Haemophilus gallinarum (Hga I). Proc Natl Acad Sci U S A. 1977 Aug;74(8):3213–3216. doi: 10.1073/pnas.74.8.3213. [DOI] [PMC free article] [PubMed] [Google Scholar]
- COHEN S. Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the new-born animal. J Biol Chem. 1962 May;237:1555–1562. [PubMed] [Google Scholar]
- Carpenter G., Lembach K. J., Morrison M. M., Cohen S. Characterization of the binding of 125-I-labeled epidermal growth factor to human fibroblasts. J Biol Chem. 1975 Jun 10;250(11):4297–4304. [PubMed] [Google Scholar]
- Carpenter G. The regulation of cell proliferation: advances in the biology and mechanism of action of epidermal growth factor. J Invest Dermatol. 1978 Nov;71(5):283–288. doi: 10.1111/1523-1747.ep12529177. [DOI] [PubMed] [Google Scholar]
- Chow F., Kempe T., Palm G. Synthesis of oligodeoxyribonucleotides on silica gel support. Nucleic Acids Res. 1981 Jun 25;9(12):2807–2817. doi: 10.1093/nar/9.12.2807. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cohen S., Carpenter G. Human epidermal growth factor: isolation and chemical and biological properties. Proc Natl Acad Sci U S A. 1975 Apr;72(4):1317–1321. doi: 10.1073/pnas.72.4.1317. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Crea R., Horn T. Synthesis of oligonucleotides on cellulose by a phosphotriester method. Nucleic Acids Res. 1980 May 24;8(10):2331–2348. doi: 10.1093/nar/8.10.2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Elder J. B., Ganguli P. C., Gillespie I. E., Gerring E. L., Gregory H. Effect of urogastrone on gastric secretion and plasma gastrin levels in normal subjects. Gut. 1975 Nov;16(11):887–893. doi: 10.1136/gut.16.11.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Finnan J. L., Varshney A., Letsinger R. L. Development in the phosphite-triester method of synthesis of oligonucleotides. Nucleic Acids Symp Ser. 1980;(7):133–145. [PubMed] [Google Scholar]
- Gregory H. Isolation and structure of urogastrone and its relationship to epidermal growth factor. Nature. 1975 Sep 25;257(5524):325–327. doi: 10.1038/257325a0. [DOI] [PubMed] [Google Scholar]
- Gregory H., Preston B. M. The primary structure of human urogastrone. Int J Pept Protein Res. 1977;9(2):107–118. doi: 10.1111/j.1399-3011.1977.tb03470.x. [DOI] [PubMed] [Google Scholar]
- Gregory H., Willshire I. R. The isolation of the urogastrones - inhibitors of gastric acid secretion - from human urine. Hoppe Seylers Z Physiol Chem. 1975 Nov;356(11):1765–1774. doi: 10.1515/bchm2.1975.356.2.1765. [DOI] [PubMed] [Google Scholar]
- Heitz P. U., Kasper M., van Noorden S., Polak J. M., Gregory H., Pearse A. G. Immunohistochemical localisation of urogastrone to human duodenal and submandibular glands. Gut. 1978 May;19(5):408–413. doi: 10.1136/gut.19.5.408. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Holland J. P., Holland M. J. Structural comparison of two nontandemly repeated yeast glyceraldehyde-3-phosphate dehydrogenase genes. J Biol Chem. 1980 Mar 25;255(6):2596–2605. [PubMed] [Google Scholar]
- Letsinger R. L., Lunsford W. B. Synthesis of thymidine oligonucleotides by phosphite triester intermediates. J Am Chem Soc. 1976 Jun 9;98(12):3655–3661. doi: 10.1021/ja00428a045. [DOI] [PubMed] [Google Scholar]
- 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]
- Narang S. A., Brousseau R., Hsiung H. M., Michniewicz J. J. Chemical synthesis of deoxyoligonucleotides by the modified triester method. Methods Enzymol. 1980;65(1):610–620. doi: 10.1016/s0076-6879(80)65063-0. [DOI] [PubMed] [Google Scholar]
- O'Keefe E., Hollenberg M. D., Cuatrecasas P. Epidermal growth factor. Characteristics of specific binding in membranes from liver, placenta, and other target tissues. Arch Biochem Biophys. 1974 Oct;164(2):518–526. doi: 10.1016/0003-9861(74)90062-9. [DOI] [PubMed] [Google Scholar]
- Rheinwald J. G., Green H. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature. 1977 Feb 3;265(5593):421–424. doi: 10.1038/265421a0. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Savage C. R., Jr, Inagami T., Cohen S. The primary structure of epidermal growth factor. J Biol Chem. 1972 Dec 10;247(23):7612–7621. [PubMed] [Google Scholar]
- Smith J., Cook E., Fotheringham I., Pheby S., Derbyshire R., Eaton M. A., Doel M., Lilley D. M., Pardon J. F., Patel T. Chemical synthesis and cloning of a gene for human beta-urogastrone. Nucleic Acids Res. 1982 Aug 11;10(15):4467–4482. doi: 10.1093/nar/10.15.4467. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sun T. T., Green H. Cultured epithelial cells of cornea, conjunctiva and skin: absence of marked intrinsic divergence of their differentiated states. Nature. 1977 Oct 6;269(5628):489–493. doi: 10.1038/269489a0. [DOI] [PubMed] [Google Scholar]
- Tanaka T., Letsinger R. L. Syringe method for stepwise chemical synthesis of oligonucleotides. Nucleic Acids Res. 1982 May 25;10(10):3249–3260. doi: 10.1093/nar/10.10.3249. [DOI] [PMC free article] [PubMed] [Google Scholar]