Abstract
The molecular action of ricin A chain involves cleavage of the N-glycosidic bond between ribose and the adenine 4324 nucleotides from the 5' end of mammalian 28 S rRNA (Endo, Y., and Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130). In this paper, four ricin- and abrin-resistant Chinese hamster ovary cell mutants that possess ribosomes resistant to this N-glycosidase action are described. Three of the mutant phenotypes, Lec26, Lec27, and Lec28, were recessive in somatic cell hybrids and define at least two new lectin-resistant complementation groups. The most extensively characterized mutant type, LEC17, was dominant in such hybrids. None of the mutants were cross-resistant to modeccin. Post-mitochondrial supernatants from each of the four mutants were resistant to inhibition of cell-free protein synthesis by ricin, ricin A chain, and abrin. In addition, polysomes isolated from mutant cells were resistant to cleavage of the adenine-ribose N-glycosidic bond by ricin A chain or abrin, as assayed by the release of an approximately 470-nucleotide fragment following aniline treatment of ribosomal RNA extracted from toxin-treated polysomes. The unique lectin-resistance properties of the different mutants suggests that the accessibility of adenine 4324 to each toxin differs. It seems likely that the recessive Chinese hamster ovary ribosomal mutants reflect structural changes in different ribosomal proteins while the dominant phenotype may be due to the modification of protein(s) or rRNA involved in toxin-ribosome interaction. Further analysis of these cell lines should provide new insights into the structure/function relationships of eukaryotic ribosomes.
Highlights
The molecular action of ricin A chain involves cleavage of the N-glycosidic bond between ribose and the adenine 4324 nucleotides from the 5’ end of mammalian 28 S rRNA (Endo, Y., and Tsurugi, K. (1987) J
It is apparent that LEC17 extracts were significantly more resistant to the inhibitory action of ricin, abrin, and ricin A chain on protein synthesis compared with parental CHO SlO extracts (22, 9, and 56fold, respectively) yet were as sensitive as the latter to inhibition by modeccin
Four new mutants of CHO cells that are differentially resistant to ricin, abrin, and modeccin have been isolated and partially characterized
Summary
Materials-Sodium [‘251]iodide (100 mCi/ml), L-[4,5-3H]leucine (120-190 Ci/mmol, 1 mCi/ml), human placental ribonuclease inhibitor were purchased from Amersham Corp. The efficacy of the mutagen treatment was estimated by comparing the number of surviving colonies in plates containing 104, 105, and lo cells incubated at 37 “C for 8-10 days in alO%FCS with or without 10 Kg/ml WGA. The amount of cell-associated (cell-surface-bound plus internalized) ‘?ricin was determined as follows: LEC17 and Pro-5 cells (6 x lo cells each) were incubated at 37 “C for 1 h in a final volume of 7 ml of phosphate-buffered saline containing 2% (w/v) BSA Buffer A, containing 0.7 M sucrose, 2.0 M KCl, 0.5 M pmercaptoethanol and absolute ethanol (in the volumes given above), was added to the resuspended pellet and the mixture incubated with stirring in an ice/Hz0 bath for an additional 30 min. XL densitometer to approximate the relative amount of -470 nucleotide fragment obtained compared with an internal standard (5 S rRNA)
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