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. 1989 Feb;9(2):415–420. doi: 10.1128/mcb.9.2.415

Selection and characterization of ricin toxin A-chain mutations in Saccharomyces cerevisiae.

A Frankel 1, D Schlossman 1, P Welsh 1, A Hertler 1, D Withers 1, S Johnston 1
PMCID: PMC362616  PMID: 2469000

Abstract

A DNA sequence encoding the A chain of ricin toxin (RTA) from the castor bean plant, Ricinus communis, was placed under GAL1 promoter control and transformed into Saccharomyces cerevisiae. Induction of expression of RTA was lethal. This lethality was the basis for a selection of mutations in RTA which inactivated the toxin. A number of mutant alleles which encoded cross-reactive material were sequenced. Eight of the first nine mutant RTAs studied showed single-amino-acid changes involving residues located in the proposed active-site cleft.

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Selected References

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

  1. Adams B. G. Induction of galactokinase in Saccharomyces cerevisiae: kinetics of induction and glucose effects. J Bacteriol. 1972 Aug;111(2):308–315. doi: 10.1128/jb.111.2.308-315.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Degnen G. E., Cox E. C. Conditional mutator gene in Escherichia coli: isolation, mapping, and effector studies. J Bacteriol. 1974 Feb;117(2):477–487. doi: 10.1128/jb.117.2.477-487.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Eiklid K., Olsnes S., Pihl A. Entry of lethal doses of abrin, ricin and modeccin into the cytosol of HeLa cells. Exp Cell Res. 1980 Apr;126(2):321–326. doi: 10.1016/0014-4827(80)90270-0. [DOI] [PubMed] [Google Scholar]
  4. Elder R. T., Loh E. Y., Davis R. W. RNA from the yeast transposable element Ty1 has both ends in the direct repeats, a structure similar to retrovirus RNA. Proc Natl Acad Sci U S A. 1983 May;80(9):2432–2436. doi: 10.1073/pnas.80.9.2432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Endo Y., Mitsui K., Motizuki M., Tsurugi K. The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins. J Biol Chem. 1987 Apr 25;262(12):5908–5912. [PubMed] [Google Scholar]
  6. Halling K. C., Halling A. C., Murray E. E., Ladin B. F., Houston L. L., Weaver R. F. Genomic cloning and characterization of a ricin gene from Ricinus communis. Nucleic Acids Res. 1985 Nov 25;13(22):8019–8033. doi: 10.1093/nar/13.22.8019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Hovde C. J., Calderwood S. B., Mekalanos J. J., Collier R. J. Evidence that glutamic acid 167 is an active-site residue of Shiga-like toxin I. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2568–2572. doi: 10.1073/pnas.85.8.2568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Johnston M., Davis R. W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. doi: 10.1128/mcb.4.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Johnston S. A., Zavortink M. J., Debouck C., Hopper J. E. Functional domains of the yeast regulatory protein GAL4. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6553–6557. doi: 10.1073/pnas.83.17.6553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. 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]
  14. Montfort W., Villafranca J. E., Monzingo A. F., Ernst S. R., Katzin B., Rutenber E., Xuong N. H., Hamlin R., Robertus J. D. The three-dimensional structure of ricin at 2.8 A. J Biol Chem. 1987 Apr 15;262(11):5398–5403. [PubMed] [Google Scholar]
  15. O'Hare M., Roberts L. M., Thorpe P. E., Watson G. J., Prior B., Lord J. M. Expression of ricin A chain in Escherichia coli. FEBS Lett. 1987 May 25;216(1):73–78. doi: 10.1016/0014-5793(87)80759-7. [DOI] [PubMed] [Google Scholar]
  16. Olsnes S., Pihl A. Treatment of abrin and ricin with -mercaptoethanol opposite effects on their toxicity in mice and their ability to inhibit protein synthesis in a cell-free system. FEBS Lett. 1972 Nov 15;28(1):48–50. doi: 10.1016/0014-5793(72)80674-4. [DOI] [PubMed] [Google Scholar]
  17. Piatak M., Lane J. A., Laird W., Bjorn M. J., Wang A., Williams M. Expression of soluble and fully functional ricin A chain in Escherichia coli is temperature-sensitive. J Biol Chem. 1988 Apr 5;263(10):4837–4843. [PubMed] [Google Scholar]
  18. Ready M. P., Katzin B. J., Robertus J. D. Ribosome-inhibiting proteins, retroviral reverse transcriptases, and RNase H share common structural elements. Proteins. 1988;3(1):53–59. doi: 10.1002/prot.340030105. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Yocum R. R., Hanley S., West R., Jr, Ptashne M. Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Oct;4(10):1985–1998. doi: 10.1128/mcb.4.10.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Zakian V. A., Scott J. F. Construction, replication, and chromatin structure of TRP1 RI circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA. Mol Cell Biol. 1982 Mar;2(3):221–232. doi: 10.1128/mcb.2.3.221. [DOI] [PMC free article] [PubMed] [Google Scholar]

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