Construction of a vector pRSV<i>cat</i><sup>amb38</sup>for the rapid and sensitive assay of amber suppression in human and other mammalian cells

Burke, Julian F.; Mogg, Anne E.

doi:10.1093/nar/13.4.1317

Cited by 8 publications

(4 citation statements)

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“…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).…”

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“…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).…”

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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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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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“…An additional silent base change was made at the third position of residue 19 to link the creation of a BssHII site to the introduction of the amber mutation. The endogenous promoter of the H-2Kb gene was replaced with a sequence from the Rous sarcoma virus (RSV) long terminal repeat (LTR), known to act as a strong promoter in a variety of mammalian cells (13) (19,20 (18). S1 nuclease analysis was carried out as described (15).…”

Section: Resultsmentioning

confidence: 99%

Rapid detection of ultraviolet-induced reversion of an amber mutation in mouse L cells.

Eccles

Vidal²,

Wrighton³

et al. 1989

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

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An amber codon (TAG) was introduced into the N-terminal coding region of the murine H-2Kb gene. The mutant gene was transfected into mouse L cells, and a clone containing a single unrearranged chromosomally integrated copy of the mutant gene was mutagenized with 254-nm UV radiation. Surviving cells were scored for surface expression of H-2Kb protein with in situ immunoperoxidase staining. Revertants were detected at a frequency of 3 x 10-6 at a dose of 40 J/m2 (3-5% survival). Revertant genes, cloned by plasmid rescue, contained the expected thymine-to-cytosine transitions at the amber codon. These data show that revertants can be rapidly detected in mammalian cells without selection and provide a basis for the development of mammalian cell lines that could be used to study mutational phenomena. During this study the steady-state level of mRNA was reduced in L cells carrying the amber mutant H-2Kb gene compared with L cells containing a wild-type or revertant H-2Kb gene. This reduction was shown not to be due to transcriptional differences, suggesting that the amber mutation decreases stability of the H-2Kb mRNA.

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“…Chloramphenicol acetyl transferase assay Enzyme assays for chloramphenicol acetyl transferase (CAT) activity were performed as described previously (21). The assay mix contained 30 ul cell supernatant 70 ml 0.25 M Tris HCl pH 7.8, 50 uil H20, lMCi [14C] chloramphenicol (60 mCi/mmol) and 1 mul 10 mM Acetyl CoA, and was incubated at 37'C for 90 min.…”

Section: Drosophila Cell Transfectionmentioning

confidence: 99%

High efficiency expression of transfected genes in aDrosophila melanogasterbaploid (1182) cell line

Saunders¹,

Rawls

Wardle³

et al. 1989

Nucl Acids Res

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Drosophila tissue culture cells have been important in the study of homologous promoters and more recently in the study of mammalian transcriptional factors such as CTF and SP1 which bind and stimulate transcription from transfected genes.In this paper we show that a Drosophila melanogaster haploid cell line (1182-4), not previously used for transfection studies, is capable of taking up and expressing DNA without the use of a facilitating agent such as calcium phosphate. Furthermore expression from a variety of Drosophila promoters such as coPia, heatshock and rudimentary as well as a mammalian promoter RSV-LTR, show between 20 and over 100 times more activity in 1182-4 cells than in D.hydei DH33 or D.melanogaster S3, or Dl cell lines. This cell line should prove to be particularly useful for the analysis of weak promoters and heterologous transcription factors. efficiency of transcription; and factors effecting mRNA stability, transport, and translation. Thus frequently only expression from strong ubiquitous promoters such as heat shock or retroviral promoters can be analysed in this way.Methods to increase the level of uptake and expression of transfected DNA in ammalian cells have been tried.

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Construction of a vector pRSVcat^amb38for the rapid and sensitive assay of amber suppression in human and other mammalian cells

Cited by 8 publications

References 23 publications

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

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

Rapid detection of ultraviolet-induced reversion of an amber mutation in mouse L cells.

High efficiency expression of transfected genes in aDrosophila melanogasterbaploid (1182) cell line

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Construction of a vector pRSVcatamb38for the rapid and sensitive assay of amber suppression in human and other mammalian cells

Cited by 8 publications

References 23 publications

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

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

Rapid detection of ultraviolet-induced reversion of an amber mutation in mouse L cells.

High efficiency expression of transfected genes in aDrosophila melanogasterbaploid (1182) cell line

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Construction of a vector pRSVcat^amb38for the rapid and sensitive assay of amber suppression in human and other mammalian cells