Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1988 Jul 25;16(14A):6361–6372. doi: 10.1093/nar/16.14.6361

Total synthesis and expression of a gene for the alpha-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin).

T P Sakmar 1, H G Khorana 1
PMCID: PMC338301  PMID: 3165194

Abstract

To facilitate structure-function studies by site-specific mutagenesis, we have synthesized a gene for the alpha-subunit of the bovine rod outer segment (ROS) guanine nucleotide-binding protein (transducin). The gene codes for the native amino acid sequence and contains, by design, 38 unique restriction sites which are uniformly spaced. This enables mutagenesis in any part of the gene by restriction fragment replacement. The gene is 1076 base pairs in length. It was constructed from 44 synthetic oligonucleotides which were joined enzymatically in vitro into four fragments which were cloned. The synthetic transducin gene and cDNA encoding transducin were expressed to similar levels in monkey kidney cells (COS-1) using a vector in which transcription was under the control of the adenovirus major late promoter.

Full text

PDF
6361

Images in this article

6364Image
on p.6364
6366Image
on p.6366
6369Image
on p.6369

Selected References

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

  1. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  3. Ferretti L., Karnik S. S., Khorana H. G., Nassal M., Oprian D. D. Total synthesis of a gene for bovine rhodopsin. Proc Natl Acad Sci U S A. 1986 Feb;83(3):599–603. doi: 10.1073/pnas.83.3.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Franke R. R., Sakmar T. P., Oprian D. D., Khorana H. G. A single amino acid substitution in rhodopsin (lysine 248----leucine) prevents activation of transducin. J Biol Chem. 1988 Feb 15;263(5):2119–2122. [PubMed] [Google Scholar]
  5. HUNTER W. M., GREENWOOD F. C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962 May 5;194:495–496. doi: 10.1038/194495a0. [DOI] [PubMed] [Google Scholar]
  6. Kaufman R. J. Identification of the components necessary for adenovirus translational control and their utilization in cDNA expression vectors. Proc Natl Acad Sci U S A. 1985 Feb;82(3):689–693. doi: 10.1073/pnas.82.3.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Khorana H. G. Total synthesis of a gene. Science. 1979 Feb 16;203(4381):614–625. doi: 10.1126/science.366749. [DOI] [PubMed] [Google Scholar]
  8. Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 1984 Jan 25;12(2):857–872. doi: 10.1093/nar/12.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Lo K. M., Jones S. S., Hackett N. R., Khorana H. G. Specific amino acid substitutions in bacterioopsin: Replacement of a restriction fragment in the structural gene by synthetic DNA fragments containing altered codons. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2285–2289. doi: 10.1073/pnas.81.8.2285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Medynski D. C., Sullivan K., Smith D., Van Dop C., Chang F. H., Fung B. K., Seeburg P. H., Bourne H. R. Amino acid sequence of the alpha subunit of transducin deduced from the cDNA sequence. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4311–4315. doi: 10.1073/pnas.82.13.4311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mogi T., Stern L. J., Hackett N. R., Khorana H. G. Bacteriorhodopsin mutants containing single tyrosine to phenylalanine substitutions are all active in proton translocation. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5595–5599. doi: 10.1073/pnas.84.16.5595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mumby S. M., Kahn R. A., Manning D. R., Gilman A. G. Antisera of designed specificity for subunits of guanine nucleotide-binding regulatory proteins. Proc Natl Acad Sci U S A. 1986 Jan;83(2):265–269. doi: 10.1073/pnas.83.2.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Nassal M., Mogi T., Karnik S. S., Khorana H. G. Structure-function studies on bacteriorhodopsin. III. Total synthesis of a gene for bacterio-opsin and its expression in Escherichia coli. J Biol Chem. 1987 Jul 5;262(19):9264–9270. [PubMed] [Google Scholar]
  15. Oprian D. D., Molday R. S., Kaufman R. J., Khorana H. G. Expression of a synthetic bovine rhodopsin gene in monkey kidney cells. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8874–8878. doi: 10.1073/pnas.84.24.8874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Sekiya T., Besmer P., Takeya T., Khorana H. G. Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 7. Enzymatic joining of the chemically synthesized segments to form a DNA duplex corresponding to the nucleotide sequence 1-26. J Biol Chem. 1976 Feb 10;251(3):634–641. [PubMed] [Google Scholar]
  18. Sompayrac L. M., Danna K. J. Efficient infection of monkey cells with DNA of simian virus 40. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7575–7578. doi: 10.1073/pnas.78.12.7575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Stryer L., Bourne H. R. G proteins: a family of signal transducers. Annu Rev Cell Biol. 1986;2:391–419. doi: 10.1146/annurev.cb.02.110186.002135. [DOI] [PubMed] [Google Scholar]
  20. Venter J. C., di Porzio U., Robinson D. A., Shreeve S. M., Lai J., Kerlavage A. R., Fracek S. P., Jr, Lentes K. U., Fraser C. M. Evolution of neurotransmitter receptor systems. Prog Neurobiol. 1988;30(2-3):105–169. doi: 10.1016/0301-0082(88)90004-4. [DOI] [PubMed] [Google Scholar]
  21. Yatsunami K., Khorana H. G. GTPase of bovine rod outer segments: the amino acid sequence of the alpha subunit as derived from the cDNA sequence. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4316–4320. doi: 10.1073/pnas.82.13.4316. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES