Nonsense mutations in the SUP45 gene lead to omnipotent suppression. A. Plate assays showing the growth of yeast strains bearing nonsense mutations in the SUP45 gene (101-1B-D1606, 102-1B-D1606, 104-1B-D1606, 105-1B-D1606, 107-1B-D1606) in the synthetic medium without histidine (SC-His), lysine (SC-Lys), adenine (SC-Ade) or tryptophan (SC-Trp) at 25°C compared with the parent strain 1B-D1606. The types of nonsense mutations in parent strain are denoted under media indications. SUP45 mutant alleles are indicated. Ten serial dilutions of yeast suspension of the same density were used. Five independent clones were tested, representative results are shown. B. Determination of the level of nonsense suppression in sup45-n mutants. UAA, UAG and UGA suppression levels were quantified by measuring β-galactosidase levels in the strains 1B-D1606 (wild type) as control and in mutants transformed with plasmids pUKC817, pUKC818 or pUKC819 bearing a specific stop codon (TAA, TAG or TGA, respectively) in frame that precedes lacZ gene. The efficiency of nonsense codon readthrough (%) was quantified as a ratio of β-galactosidase activity in cells harboring lacZ with stop codon to that in cells without a stop codon in-frame with the lacZ gene in pUKC815 plasmids. The same strains as in Fig. 2A were used. Results are the means of three separate experiments. C. Western blot showing the synthesis of full-length eRF1 (49 kDa) and truncated eRF1 proteins with predicted molecular mass 43.0 kDa, 35.2 kDa and 31.5 kDa for mutants 105, 107 and 104, respectively. (*) Indicates a non-specific band. A search of yeast proteome with the N-terminal peptide sequence that was used for antibody production, revealed the presence of one protein (methionyl-tRNA synthetase, GenBank accession number CAA24627) containing this sequence that has an expected molecular weight near 85 kDa. Cultures of the same strains as in (A) were grown to mid-log phase in medium selective for plasmid and ribosome fractions were prepared. The same amount of each sample was loaded per lane. Immunoblot analysis was performed using polyclonal antibodies directed against the N-terminal part of eRF1.
Moskalenko et al. BMC Molecular Biology 2003 4:2 doi:10.1186/1471-2199-4-2