Nonsense mutations in the essential gene SUP35 of Saccharomyces cerevisiae are non-lethal
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In the present work we have characterized for the first time non-lethal nonsense mutations in the essential gene SUP35, which codes for the translation termination factor eRF3 in Saccharomyces cerevisiae. The screen used was based on selection for simultaneous suppression of two auxotrophic nonsense mutations. Among 48 mutants obtained, sixteen were distinguished by the production of a reduced amount of eRF3, suggesting the appearance of nonsense mutations. Fifteen of the total mutants were sequenced, and the presence of nonsense mutations was confirmed for nine of them. Thus a substantial fraction of the sup35 mutations recovered are nonsense mutations located in different regions of SUP35, and such mutants are easily identified by the fact that they express reduced amounts of eRF3. Nonsense mutations in the SUP35 gene do not lead to a decrease in levels of SUP35 mRNA and do not influence the steady-state level of eRF1. The ability of these mutations to complement SUP35 gene disruption mutations in different genetic backgrounds and in the absence of any tRNA suppressor mutation was demonstrated. The missense mutations studied, unlike nonsense mutations, do not decrease steady-state amounts of eRF3.
KeywordsYeast Translation termination Release factor eRF3 Readthrough
The work was supported by grants from CRDF ST-012, CRDF N RB1-2336-ST-02, RFBR 03-04-48886 and to S.C. from NATO, ESF 2002/EG02, the Ministry of Education of the Russian Federation A03-2.12-2. We are very grateful to K. Gull for anti-tubulin antibodies, to K. Volkov for plasmids and strains and to H.B. Osborne for critical reading of the manuscript.
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