Cycloheximide resistance conferred by novel mutations in ribosomal protein L41 of Chlamydomonas reinhardtii
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O R I GI N A L P A P E R
D. R. Stevens á A. Atteia á L.-G. FranzeÂn á S. Purton
Cycloheximide resistance conferred by novel mutations in ribosomal protein L41 of Chlamydomonas reinhardtii
Received: 27 January 2000 / Accepted: 29 August 2000 / Published online: 25 October 2000 Ó Springer-Verlag 2000
Abstract Although most eukaryotic cells are sensitive to the 80S ribosome inhibitor cycloheximide (CYH), naturally occurring CYH resistance is widespread amongst yeast species. The primary determinant of resistance appears to be a single residue within ribosomal protein L41; resistance is acquired by the substitution of a conserved proline (P56) by a glutamate residue. We have isolated the L41 gene (RPL41) from the green alga Chlamydomonas reinhardtii, and investigated the molecular basis of CYH resistance in various mutant strains. In both the wild-type strain and the mutant act-1, a proline is found at the key position in L41. However, analysis of six independently isolated act-2 mutants reveals that all have point mutations that replace the proline with either leucine or serine. Of the two changes, the leucine mutation confers signi®cantly higher levels of CYH resistance. This work identi®es the ACT-2 locus as RPL41 and provides a possible dominant marker for nuclear transformation of C. reinhardtii. Key words Chlamydomonas á Cycloheximide resistance á Ribosomal protein L41 á Intron Communicated by C. A. M. J. J. van den Hondel D. R. Stevens á S. Purton (&) Department of Biology, University College London, Gower Street, London WC1E 6BT, UK E-mail: [email protected] Tel.: +44-20-76792675 Fax: +44-20-76797096 A. Atteia1 á L.-G. FranzeÂn Department of Plant Physiology, Botanical Institute, GoÈteborg University, SE-405 30 GoÈteborg, Sweden L.-G. FranzeÂn School of Business and Engineering, University of Halmstad, P.O. Box 823, SE-301 18 Halmstad, Sweden Present address: Departamento de GeneÂtica Molecular, Instituto de FisiologõÂ a Celular, Universidad Nacional AutoÂnoma de MeÂxico, MeÂxico City, MeÂxico 1
Introduction The glutarimide antibiotic cycloheximide (CYH) is an eective inhibitor of cytosolic protein synthesis in a wide range of eukaryotic organisms. Although its precise mechanism of action is not yet understood, CYH is known to bind to the large subunit of the 80S ribosome with 1:1 stoichiometry and is able to inhibit all three phases of translation (initiation, elongation and termination) depending on the drug concentration (Oleinick 1977). Early studies of yeast species identi®ed many that were naturally resistant to high levels of CYH, and this property has been used in their classi®cation (Barnett et al. 1983). More recent molecular analysis has revealed that CYH resistance (CYHR) or sensitivity (CYHS) in yeasts correlates with a single amino acid change in ribosomal protein L41 (named L44 in higher eukaryotes). If a glutamate residue is present at position 56 in the L41 sequence then the resulting ribosome is CYHR, whereas a proline at this position gives rise to CYHS ribosomes (Kawai et al. 1992). Fu
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