Introduction of UAG, UAA, and UGA nonsense mutations at a specific site in the Escherichia coli chloramphenicol acetyltransferase gene: use in measurement of amber, ochre, and opal suppression in mammalian cells.

Abstract

We have used oligonucleotide-directed site-specific mutagenesis to convert serine codon 27 of the Escherichia coli chloramphenicol acetyltransferase (cat) gene to UAG, UAA, and UGA nonsense codons. The mutant cat genes, under transcriptional control of the Rous sarcoma virus long terminal repeat, were then introduced into mammalian cells by DNA transfection along with UAG, UAA, and UGA suppressor tRNA genes derived from a human serine tRNA. Assay for CAT enzymatic activity in extracts from such cells allowed us to detect and quantitate nonsense suppression in monkey CV-1 cells and mouse N1113T3 cells. Using such an assay, we provide the first direct evidence that an opal suppressor tRNA gene is functional in mammalian cells. The pattern of suppression of the three cat nonsense mutations in bacteria suggests that the serine at position 27 of CAT can be replaced by a wide variety of amino acids without loss of enzymatic activity. Thus, these mutant cat genes should be generally useful for the quantitation of suppressor activity of suppressor tRNA genes introduced into cells and possibly for the detection of naturally occurring nonsense suppressors.Nonsense mutations, which cause premature termination of protein synthesis, can be suppressed by tRNAs which recognize termination codons. The availability of a wide variety of nonsense suppressor tRNA genes has played a crucial role in the isolation and study of nonsense mutations in procaryotes and lower eucaryotes (7). In mammalian cells, it has so far not been possible to isolate cell lines carrying nonsense suppressors by using classic genetic selections; consequently, nonsense mutations have been identified in only a few nonviral genes (8,21).Recently, we demonstrated that both a Xenopus laevis tRNATYr gene and a human tRNAser gene could be altered by oligonucleotide-directed site-specific mutagenesis to recognize and suppress amber (UAG) and ochre (UAA) codons in vivo (5,19,20). An opal (UGA) suppressor tRNA gene derived from a human tRNAser gene was also constructed (5). However, since the opal suppressor tRNA gene could not be propagated in mammalian cells as part of a replicating simian virus 40 (SV40) recombinant, it was not possible to demonstrate its functional expression. In addition, the X.laevis tRNATYr amber and ochre suppressors were also used to establish permanent Su' mammalian cell lines (16,19), although the level of nonsense suppression in these cell lines was low (36). It is likely that many other suppressor tRNA genes which are active in mammalian cells will be constructed and used to isolate mammalian cell lines containing functional suppressors. A knowledge of the levels of suppression in these various cell lines will aid significantly in their eventual use for the isolation and characterization of nonsense mutations in cellular and viral genes. It is therefore important to have a rapid, quantitative, and sensitive method to assay for the * Corresponding author. level of suppression of nonsense mutations in different cell types. This paper repo...

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

“…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).…”

“…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).…”

Rescue of wild-type E-cadherin expression from nonsense-mutated cancer cells by a suppressor-tRNA

“…Suppression of nonsense mutations by sup-tRNAs has been demonstrated in Xenopus oocytes 38 and in mammalian cells, 20,22 and was studied later as putative therapeutic tools for diseases like b-thalassaemia, 23 xeroderma pigmentosum 24 and DMD. 15 Nevertheless, formal proof of functional recovery was rarely reported.…”

“…[17][18][19] In contrast, suppressor-tRNAs (sup-tRNAs) are able to induce stop codon readthrough by specifically introducing the cognate amino acid at the premature stop codon (PTC) site. 20 As each sup-tRNA can recognize and interact with one of the three stop codons amber (UAG), opal (UGA) and ochre (UAA), due to an alteration in its anticodon, 15,20 these agents have high stop codon specificity and also show higher nonsense suppression efficiency than other readthrough strategies. 21 Thus, sup-tRNAs emerge as interesting candidates to be used as therapeutic agents to recover the expression of full-length functional proteins.…”

“…Several studies demonstrated efficient nonsense suppression in mammalian models, although the recovery of functional proteins was rarely shown. 15,20,[22][23][24][25] In this work we wish to: (1) evaluate the potential impact of using nonsense suppression in HDGC; (2) determine the number of tRNAs necessary to treat nonsense mutation-carrying HDGC families; and (3) assess the possibility of recovering expression of a functional protein from an allele carrying a nonsense mutation described in HDGC using a sup-tRNA. To our knowledge, this is the first demonstration of functional recovery of a causative gene in cancer by cognate amino acid replacement with sup-tRNAs.…”