Aminoglycoside suppression at UAG, UAA and UGA codons inEscherichia coli and human tissue culture cells

Martin, Robert H.; Mogg, Anne E.; Heywood, Louise A.; Nitschke, Lars; Burke, Julian F.

doi:10.1007/bf02464911

Cited by 74 publications

(60 citation statements)

References 40 publications

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“…The efficiency of read-through varies inversely to the efficiency of a stop codon, the ranking order for aminoglycoside read-through efficiency generally being UGA Ͼ UAA Ͼ UAG (28). On the other hand, this ranking varies from one species to another, and is influenced as well by the context within which a stop codon appears, especially with the ''wobble'' of the ϩ4 nucleotide (22,29).…”

Section: Discussionmentioning

confidence: 99%

Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Lai

Chun

Nahas

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

146

102

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Cells were irradiated with 10 Gy and were incubated for 45 min at 37°C before fixation on coverslips. ATM pS1981 IRIFs were not observed in untreated cells; maximum intensity of staining was seen in the nuclei of cells exposed to 75 g͞ml. Nonirradiated cells, untreated or exposed to comparable concentrations of geneticin, showed only occasional foci. A taxia-telangiectasia (A-T) is an autosomal recessive neurodegenerative disorder with onset in early childhood, resulting from mutations in the A-T mutated (ATM) gene (1). The ATM protein is a hierarchical serine-threonine kinase, phosphorylating many substrates involved in repair of doublestranded DNA breaks, control of cell cycle checkpoints, and responses to oxidative stress, as well as in radiosensitivity, cancer susceptibility, immune function, and neurological development (2). ATM protein levels are undetectable in the cells of most A-T patients by conventional testing. However a few patients, often with a milder phenotype, have some detectable ATM protein (3,4). This finding encouraged us to seek compounds, such as aminoglycosides, that have the potential to read through premature termination codons and restore ATM protein function (5, 6).To test these effects, we selected 13 lymphoblastoid cell lines (LCLs) with primary premature termination codon (PTC) mutations from a repository of Ͼ400 lines derived from A-T patients. Herein, we show representative data from 5 of the 13 LCLs. Using protein truncation testing (PTT) driven by plasmid templates containing PTC mutations in the ATM gene (7), we observed in vitro read-through effects of various magnitudes with three of four aminoglycosides tested. Full-length ATM protein was also documented ex vivo by immunoprecipitation. Correction of radioresistant DNA synthesis and radiosensitivity, as well as autophosphorylation of ATM, suggested that this readthrough produces functional ATM protein. Experimental ProceduresCell Lines. Lymphoblastoid cell lines were maintained in RPMI medium 1640 (Invitrogen) with 15% FBS (HyClone) and 1% penicillin͞streptomycin (Invitrogen) at 37°C and 5% CO 2 . In ex vivo experiments, aminoglycosides were added daily into culture media at the indicated doses and times. Because streptomycin is also an aminoglycoside that can perturb proofreading by binding to another ribosomal site, ex vivo experiments were performed with or without streptomycin; no differences were noted.Mutations. We selected only PTC mutations that resulted directly from the mutation, i.e., primary PTC mutations, and not those caused indirectly by upstream mutations or aberrant splicing, because the latter would have no potential therapeutic benefits. The LCLs carried the following mutations (www.benaroyaresearch.org͞bri investigators͞atm.htm): AT185LA, homozygous 3673C 3 T (a UAA G stop codon in PTT fragment 4); TAT51, homozygous 5623C 3 T (a UGA C stop codon in PTT fragment 5); AT187LA, homozygous 5908C 3 T (a UAA G stop codon in PTT fragment 6); AT153LA, homozygous 8977C 3 T (a UGA A stop codon in fragment 8); and AT...

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Section: Discussionmentioning

confidence: 99%

Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Lai

Chun

Nahas

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

146

102

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“…These context effects are observed in the human cell lines 293 and MRC5V1 and in the simian cell line COS-7. We also show that a second human suppressor, tRNA Gln UAG, and misreading induced by the aminoglycoside antibiotic G418 (11,35) both display broadly similar sensitivities to the 3Ј codon context.…”

mentioning

confidence: 64%

“…In an earlier report, an RSVgal vector containing an unsequenced UAG mutation in which ␤-galactosidase was on the order of 0.01% of the wild-type level was described (35). The lacZ UAG mutation was introduced into pRSVgal via the plasmid pMLB100.…”

Section: Construction Of Prsvgal Reporters Containing Uag In Differenmentioning

confidence: 99%

Context Effects on Misreading and Suppression at UAG Codons in Human Cells

Phillips‐Jones

Hill

Atkinson

et al. 1995

Molecular and Cellular Biology

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The effect of the 3 codon context on the efficiency of nonsense suppression in mammalian tissue culture cells has been tested. Measurements were made following the transfection of cells with a pRSVgal reporter vector that contained the classical Escherichia coli lacZ UAG allele YA559. The position of this mutation was mapped by virtue of its fortuitous creation of a CTAG MaeI restriction enzyme site. Determination of the local DNA sequence revealed a C3T mutation at codon 600 of the lacZ gene: CAG3TAG. Site-directed mutagenesis was used to create a series of vectors in which the base 3 to the nonsense codon was either A, C, G, or U. Suppression of the amber-containing reporter was achieved by cotransfection with genes for human tRNA Ser or tRNA Gln UAG nonsense suppressors and by growth in the translational error-promoting aminoglycoside drug G418. Nonsense suppression was studied in the human cell lines 293 and MRC5V1 and the simian line COS-7. Overall, the rank order for the effect of changes to the base 3 to UAG was C < G ‫؍‬ U < A. This study confirms and extends earlier findings that in mammalian cells 3 C supports efficient nonsense suppression while 3 A is unsympathetic for read-through at nonsense codons. The rules for the mammalian codon context effect on nonsense suppression are therefore demonstrably different from those in E. coli.Normally, nonsense or stop codons catalyze the effective cessation of protein synthesis. However, when tRNA species within the cell are able to recognize a nonsense codon, the function of the stop is suppressed. Nonsense suppression has been exploited as a means for the conditional expression of genes containing stop codons in order to study their function (23,44). Nonsense suppression has also been used extensively in Escherichia coli as a tool for monitoring the efficiency of translation at an individual codon (46,47,51). Of particular interest is the facility that nonsense suppression affords for determining the effects of 5Ј and 3Ј contexts on the interaction of translation factors and aminoacyl-tRNAs with their mRNA targets (19,39,45,49,52). Recently, nonsense suppression has been harnessed in order to carry out in vitro protein engineering. In this process, chemically acylated nonsense suppressor tRNAs are used to insert nonnatural amino acid residues at specified locations by targeting a nonsense codon which interrupts the coding sequence (2, 3, 33).There have been comparatively few reports of nonsense suppression in mammalian cells, but the transfection of sensitively detected reporters now allows this technique to be applied to cells in culture (6,12,13,15,43). In a previous report from this laboratory, we described the effects of alterations in the 3Ј mRNA context on the efficiency of a human tRNA Ser UAG nonsense suppressor (40). The function of the suppressor was monitored by determining the efficiency of translation at a UAG codon in a ␤-galactosidase reporter gene which had been cotransfected into human 293 cells with a plasmid vector expressing a human UAG su...

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“…Aminoglycosides such as G418 are reported to induce stop codon readthrough in Escherichia coli, yeast, and human cultured cells (23,24). To confirm the stop codon readthrough of P0 mRNA, we performed in vitro transcription/translation in the presence of G418 using a mammalian in vitro transcription/ translation system.…”

Section: Elimination Of Immunoreactivity For the 36-kda Band By The Smentioning

confidence: 99%

L-MPZ, a Novel Isoform of Myelin P0, Is Produced by Stop Codon Readthrough

Yamaguchi

Hayashi

Campagnoni

et al. 2012

Journal of Biological Chemistry

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Background:The structure and function of myelin P0-related 36-kDa protein are totally unknown. Results: A novel isoform of P0, L-MPZ, contains an extra C terminus derived from 3Ј-UTR of P0 mRNA and is expressed in peripheral myelin. Conclusion: L-MPZ is produced by stop codon readthrough and probably related with peripheral myelinogenesis. Significance: Analyses of L-MPZ are crucial for understanding readthrough mechanism in mammals and myelinogenesis.

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Aminoglycoside suppression at UAG, UAA and UGA codons inEscherichia coli and human tissue culture cells

Cited by 74 publications

References 40 publications

Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Context Effects on Misreading and Suppression at UAG Codons in Human Cells

L-MPZ, a Novel Isoform of Myelin P0, Is Produced by Stop Codon Readthrough

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