Mutagen specificity and position effects on mutation in T4rII nonsense sites.

In 14 sites in the T4rII region, spontaneous and induced interconversions of ochre (UAA) and opal (UGA) alleles, as well as the reversion of the nonsense sites to r+, were studied. The mutagens employed were 2-aminopurine (2AP), 5-bromouracil (5BU), N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and hydroxylamine (HA). With the test system employed, mutagen specificity (i.e., the preferential induction of A: T leads to G: C or G: C leads to A: T mutation at a given site) can be studied. Simultaneously, the response of similar base pairs at various locations in the same or in different nucleotide triplets throughout the rII region, to a given mutagen, can be compared. 2-Aminopurine can induce transitions of both A: T and G: C base pairs at high rates. This mutagen shows no preference for either direction. Furthermore, there is a correlation between the response to 2AP of an A: T pair occupying a given site, and that of a G: C pair occupying the same site. NTG and HA induce G: C leads to A: T transitions almost exclusively. However, there is a correlation between the low rates of A: T leads to G: C transition induced in each of the various sites by these mutagens and those of G: C leads to A: T. 5-Bromouracil induces transitions from G: C to A: T more readily than from A: T to G: C. With 5BU-induced mutation, there is no correlation between the rates of G: C leads to A: T transitions and those of A: T leads to G: C. In UAA sites, all three adenine:thymidine paris respond to 2AP mutagenesis in a similar pattern, In each position in the triplet, response to 2 AP is correlated with that to 5BU. In UGA sites, there are correlations among the spontaneous as well as the 2AP-, HA- and NTG-induced transition rates. 5BU-induced transition rates are usually not correlated with those induced by other mutagens or with the sponatneous ones.     

Nonsense Mutants in the rII A Cistron of Bacteriophage T4

After in vitro treatment of bacteriophage T4 with hydroxylamine (HA), 54 nonsense mutants in the rII A cistron were isolated. These mutants were characterized by growth on suppressor strains of Escherichia coli, and the mutational sites were mapped in the rII A cistron. Twenty-five (9 sites) were amber (UAG), 20 (6 sites) were opal (UGA), and 9 (6 sites) were ochre (UAA). Mapping experiments further indicated that there were three closely linked pairs of amber and opal mutations, conceivably involving mutations occurring in adjacent nucleotides. Based on the specificity of HA mutagenesis (GC → AT), the amino acid codons in which the mutations occurred have been inferred. It is suggested that the three amber-opal pairs arose in tryptophan codons (UGG) and the six ochre mutants arose in glutamine codons (CAA). The six unpaired ambers and the three unpaired opals have been tentatively assigned to glutamine codons (CAG) and arginine codons (CGA), respectively, in the wild-type phage.     

Intragenic localization of chain-terminating triplets arising by transitions and transversions

Mutational changes involving transitions can convert only one sense codon to ochre, two codons to amber, and two codons to UGA. One codon, UGG for tryptophan, can be converted by transitions to either amber or UGA. By transversion changes 15 other codons can be converted to ochre and/or amber and/or UGA. Ten amino acids can never be replaced by chain termination as a result of transition and transversion mutagenesis of single base-pairs. For two systems (bacteriophage T4 lysozyme and Escherichia coli K12 tryptophan synthetase A protein) in which the poly-peptide gene product has been completely sequenced one can construct predictive intra-genic distribution maps for the location of all possible chain-terminating mutations arising as a result of transitions and transversions.     

Evidence that the supE44 Mutation of Escherichia coli Is an Amber Suppressor Allele of glnX and that It Also Suppresses Ochre and Opal Nonsense Mutations

Translational readthrough of nonsense codons is seen not only in organisms possessing one or more tRNA suppressors but also in strains lacking suppressors. Amber suppressor tRNAs have been reported to suppress only amber nonsense mutations, unlike ochre suppressors, which can suppress both amber and ochre mutations, essentially due to wobble base pairing. In an Escherichia coli strain carrying the lacZU118 episome (an ochre mutation in the lacZ gene) and harboring the supE44 allele, suppression of the ochre mutation was observed after 7 days of incubation. The presence of the supE44 lesion in the relevant strains was confirmed by sequencing, and it was found to be in the duplicate copy of the glnV tRNA gene, glnX. To investigate this further, an in vivo luciferase assay developed by D. W. Schultz and M. Yarus (J. Bacteriol. 172:595-602, 1990) was employed to evaluate the efficiency of suppression of amber (UAG), ochre (UAA), and opal (UGA) mutations by supE44. We have shown here that supE44 suppresses ochre as well as opal nonsense mutations, with comparable efficiencies. The readthrough of nonsense mutations in a wild-type E. coli strain was much lower than that in a supE44 strain when measured by the luciferase assay. Increased suppression of nonsense mutations, especially ochre and opal, by supE44 was found to be growth phase dependent, as this phenomenon was only observed in stationary phase and not in logarithmic phase. These results have implications for the decoding accuracy of the translational machinery, particularly in stationary growth phase.Translation termination is mediated by one of the three stop codons (UAA, UAG, or UGA). When such stop codons arise in coding sequences due to mutations, referred to as nonsense mutations, they lead to abrupt arrest of the translation process. However, the termination efficiency of such nonsense codons is not 100%, as certain tRNAs have the ability to read these nonsense codons. Genetic code ambiguity is seen in several organisms. Stop codons have been shown to have alternate roles apart from translation termination. In organisms from all three domains of life, UGA encodes selenocysteine through a specialized mechanism. In Methanosarcinaceae, UAG encodes pyrrolysine (3). UAA and UAG are read as glutamine codons in some green algae and ciliates such as Tetrahymena and Diplomonads (24), and UAG alone encodes glutamine in Moloney murine leukemia virus (32). UGA encodes cysteine in Euplotes; tryptophan in some ciliates, Mycoplasma species, Spiroplasma citri, Bacillus, and tobacco rattle virus; and an unidentified amino acid in Pseudomicrothorax dubius and Nyctotherus ovalis (30). In certain cases the context of the stop codon in translational readthrough has been shown to play a role; for example, it has been reported that in vitro in tobacco mosaic virus, UAG and UAA are misread by tRNA^Tyr in a highly context-dependent manner (34, 9).Termination suppressors are of three types, i.e., amber, ochre, and opal suppressors, which are named based on their ability to suppress the three stop codons. Amber suppressors can suppress only amber codons, whereas ochre suppressors can suppress ochre codons (by normal base pairing) as well as amber codons (by wobbling) and opal suppressors can read opal and UGG tryptophan codon in certain cases. As described by Sambrook et al. (27), a few amber suppressors can also suppress ochre mutations by wobbling. The suppression efficiency varies among these suppressors, with amber suppressors generally showing increased efficiency over ochre and opal suppressors. supE44, an amber suppressor tRNA, is an allele of and is found in many commonly used strains of Escherichia coli K-12. Earlier studies have shown that supE44 is a weak amber suppressor and that its efficiency varies up to 35-fold depending on the reading context of the stop codon (8).Translational accuracy depends on several factors, which include charging of tRNAs with specific amino acids, mRNA decoding, and the presence of antibiotics such as streptomycin and mutations in ribosomal proteins which modulate the fidelity of the translational machinery. Among these, mRNA decoding errors have been reported to occur at a frequency ranging from about 10⁻³ to 10⁻⁴ per codon. Translational misreading errors also largely depend on the competition between cognate and near-cognate tRNA species. Poor availability of cognate tRNAs increases misreading (18).Several studies with E. coli and Saccharomyces cerevisiae have shown the readthrough of nonsense codons in suppressor-free cells. In a suppressor-free E. coli strain, it has been shown in vitro that glutamine is incorporated at the nonsense codons UAG and UAA (26). It has been reported that overexpression of wild-type tRNA^Gln in yeast suppresses amber as well as ochre mutations (25). In this study, we have confirmed the presence of an amber suppressor mutation in the glnX gene in a supE44 strain by sequence analysis. This was done essentially because we observed that supE44 could also suppress lacZ ochre mutations, albeit inefficiently. On further investigation using an in vivo luciferase reporter assay system for tRNA-mediated nonsense suppression (28), we found that the efficiency of suppression of amber lesion by supE44 is significantly higher than that reported previously in the literature. An increased ability to suppress ochre and opal nonsense mutations was observed in cells bearing supE44 compared to in the wild type. Such an effect was observed only in the stationary phase and was abolished in logarithmic phase.     

Differentiation between Amber and Ochre Mutants of Yeast by Reversion with 4-Nitroquinoline-1-Oxide

4-nitroquinoline-1-oxide (NQO) induces high frequencies of intragenic revertants of amber (UAG) but not ochre (UAA) mutants of yeast. Distinction of the amber and ochre codons was made with well-characterized nonsense mutants of the iso-1-cytochrome c gene (cyc1 mutants) as well as with nonsense mutants having nutritional requirements. Thus the NQO-induced reversion frequencies corroborated the assignments that were based on the pattern of amino acid replacements in intragenic revertants and on the speficity of suppression. It was concluded from these results and from the results of a previous investigation with other cyc1 mutants (Prakash, Stewart and Sherman 1974) that NQO induces transversions of G:C base pairs at many sites and that the specificity is not strongly influenced by neighboring base pairs in at least the strains examined in these studies. NQO was previously shown to induce G:C → A:T transitions at least at one site and this and the previous study established that it does not significantly mutate A:T base pairs at numerous sites. Thus NQO can be used to selectively mutate G:C base pairs and to determine if the pathways of reverse mutations involve G:C base pairs. Suppressors that act on either amber or ochre mutants were induced with NQO, indicating that they can arise by mutations of G:C base pairs.     

Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant suppression of three different termination codons in an mRNA in mammalian cells

We describe the generation of a complete set of orthogonal 21st synthetase-amber, ochre and opal suppressor tRNA pairs including the first report of a 21st synthetase-ochre suppressor tRNA pair. We show that amber, ochre and opal suppressor tRNAs, derived from Escherichia coli glutamine tRNA, suppress UAG, UAA and UGA termination codons, respectively, in a reporter mRNA in mammalian cells. Activity of each suppressor tRNA is dependent upon the expression of E.coli glutaminyl-tRNA synthetase, indicating that none of the suppressor tRNAs are aminoacylated by any of the twenty aminoacyl-tRNA synthetases in the mammalian cytoplasm. Amber, ochre and opal suppressor tRNAs with a wide range of activities in suppression (increases of up to 36, 156 and 200-fold, respectively) have been generated by introducing further mutations into the suppressor tRNA genes. The most active suppressor tRNAs have been used in combination to concomitantly suppress two or three termination codons in an mRNA. We discuss the potential use of these 21st synthetase-suppressor tRNA pairs for the site-specific incorporation of two or, possibly, even three different unnatural amino acids into proteins and for the regulated suppression of amber, ochre and opal termination codons in mammalian cells.     

Genetic studies of the lac repressor. IV. Mutagenic specificity in the lacI gene of Escherichia coli.

An extensive set of amber and ochre sites in the lacI gene has been characterized with respect to the base change required to generate the nonsense codon (Miller et al., 1977; Coulondre &; Miller, 1977). These mutations have been used to analyze the forward mutational spectrum of a series of mutagens in Escherichia coli. The sites induced by N′-methyl-N′-nitro-N-nitrosoguanidine, ethyl methanesulfonate, 4-nitroquinoline-1-oxide, and ultraviolet light, were examined, as well as those which arose spontaneously. Sites induced by the G · C → A · T transition were compared with those generated by 2-aminopurine mutagenesis. All together, more than 4000 independent occurrences of amber and ochre mutations were tabulated in order to define the respective mutagenic specificities. With the exception of the A · T → G · C change, all base substitutions lead to the generation of nonsense codons from wild-type. The A · T → G · C transition was monitored in a reversion system, in which the ochre to amber conversion (UAA → UAG) was scored, as well as the UAA → CAA reversion.Both NG³ and EMS were found to be highly specific for the G · C → A · T transition, less than 1% transversions appearing in either case. At between 1% and 5% the level of the G · C → A · T change, NG can stimulate the A · T → G · C transition. EMS stimulates the A · T → G · C transition at a significantly lower rate. NQO is also highly specific for G · C base-pairs, but approximately 10% of the changes found at these sites are transversions. Mutations found spontaneously or after irradiation with ultraviolet light showed none of the specificities found for EMS, NG or NQO. All transversions were detected in both cases. Moreover, a significant number of tandem double base changes were found to be induced by u.v. irradiation. Some of these have been verified directly by protein sequencing. The frequencies of occurrence of amber and ochre mutations arising from the G · C → A · T transition have been compared for different mutagens, revealing several striking hotspots. The implications of these findings with respect to the mechanism of mutagenesis and the application of different mutagens are discussed.     

Nonsense and Sense Suppression Abilities of Original and Derivative Methanosarcina mazei Pyrrolysyl-tRNA Synthetase-tRNAPyl Pairs in the Escherichia coli BL21(DE3) Cell Strain

Systematic studies of nonsense and sense suppression of the original and three derivative Methanosarcina mazei PylRS-tRNA^Pyl pairs and cross recognition between nonsense codons and various tRNA^Pyl anticodons in the Escherichia coli BL21(DE3) cell strain are reported. is orthogonal in E. coli and able to induce strong amber suppression when it is co-expressed with pyrrolysyl-tRNA synthetase (PylRS) and charged with a PylRS substrate, N^ε-tert-butoxycarbonyl-l-lysine (BocK). Similar to, is also orthogonal in E. coli and can be coupled with PylRS to genetically incorporate BocK at an ochre mutation site. Although is expected to recognize a UAG codon based on the wobble hypothesis, the PylRS- pair does not give rise to amber suppression that surpasses the basal amber suppression level in E. coli. E. coli itself displays a relatively high opal suppression level and tryptophan (Trp) is incorporated at an opal mutation site. Although the PylRS- pair can be used to encode BocK at an opal codon, the pair fails to suppress the incorporation of Trp at the same site. fails to deliver BocK at an AGG codon when co-expressed with PylRS in E. coli.     

A temperature-sensitive mutant of Escherichia coli that shows enhanced misreading of UAG/A and increased efficiency for some tRNA nonsense suppressors

Summary A spontaneous mutant was isolated that harbors a weak suppressing activity towards a UAG mutation, together with an inability to grow at 43° C in rich medium. The mutation is shown to be associated with an increased misreading of UAG at certain codon contexts and UAA. UGA, missense or frameshift mutations do not appear to be misread to a similar extent. The mutation gives an increased efficiency to several amber tRNA suppressors with-out increasing their ambiguity towards UAA. The ochre suppressors SuB and Su5 are stimulated in their reading of both UAG and UAA with preference for UAG. An opal suppressor is not affected. The effect of the mutation on the efficiency of amber and ochre suppressors is dependent on the codon context of the nonsense codon.The mutated gene (uar) has been mapped and found to be recessive both with respect to suppressor-enhancing ability as well as for temperature sensitivity. The phenotype is partly suppressed by the ochre suppressor SuC. It is suggested that uar codes for a protein, which is involved in translational termination at UAG and UAA stop codons.     

Streptomycin-Induced Phenotypic Suppression of Hydroxylamine-Induced Nonsense Mutants in the rII A Cistron of Bacteriophage T4

Twenty-one hydroxylamine-induced rII A cistron nonsense mutants were tested for streptomycin (SM)-induced phenotypic suppression by exposing Escherichia coli SBO (nonpermissive host) to phage in the presence and absence of SM. All nine amber, four of six ochre, and five of six opal mutants were phenotypically suppressible by SM. For suppressible mutants, the ratio of the average burst size in the presence of SM to size in the absence of SM ranged from 12 to 242 for the ambers, 3 to 33 for the ochres, and 4 to 14 for the opals. Increased susceptibility of the amber mutants to SM-induced phenotypic suppression relative to the susceptibility of the opal and ochre mutants may reflect a neighboring base effect, such that a 3′-terminal adenine inhibits misreading of a 5′-terminal uracil.     

Synthesis of an ochre suppressor tRNA gene and expression in mammalian cells.

We have used site-specific mutagenesis to change the anticodon of a Xenopus laevis tyrosine tRNA gene so that it would recognize ochre codons. This tRNA gene is expressed when amplified in monkey cells as part of a SV40 recombinant and efficiently suppresses termination at both the ochre codon separating the adenovirus 2 hexon gene from a 23-kd downstream gene and the ochre codon at the end of the NS1 gene of influenza virus A/Tex/1/68. Termination at an amber codon of a NS1 gene of another influenza virus strain was not suppressed by the (Su+) ochre gene suggesting that in mammalian cells amber codons are not recognized by ochre suppressor tRNAs. Finally, microinjection into mammalian cells of both (Su+) ochre tRNA genes and selectible genes containing ochre nonsense mutations gives rise to colonies under selective conditions. We conclude that it should be possible to isolate a wide assortment of mammalian cell lines with ochre suppressor activity.     

Neighbour and temperature effects on base-analogue-induced mutation in phage T4

Summary The effect of neighbouring base pairs and of temperature on mutation frequencies were measured at nonsense sites in the T4rII region. 2AP-induced ATGC transition frequencies are insensitive to nearest-neighbour effects, while 5BU-induced ones are promoted by GC neighbours on the 5 side. The effect of temperature on 2AP-and 5BU-induced mutation frequencies shows no simple dependence on nearest neighbours. These results are incompatible with a unitary mechanism as explanation for the effects of nearest neighbours and temperature on baseanalogue-induced mutagenesis.     

Context Effects on Nonsense Codon Suppression in ESCHERICHIA COLI

The influence of mRNA context on nonsense codon suppression has been studied by suppression measurements at one site in the Escherichia coli trpE gene and at two sites in the trpA gene. The ratio of suppression efficiencies of amber and ochre codons at each site (homotopic pairs) has been compared using ochre suppressing derivatives of tRNATyr. This ratio is independent of differential effects of the inserted amino acid on enzyme function. We have found that mRNA context can change the ratio of suppression efficiencies of homotopic nonsense codons at the three sites in the trp gene system over a ten-fold range. The causes of such variation, and, in particular the effect of certain adjacent nucleotides on nonsense codon suppression are considered.     

Effects of surrounding sequence on the suppression of nonsense codons

Using a lacI-Z fusion system, we have determined the efficiency of suppression of nonsense codons in the I gene of Escherichia coli by assaying beta-galactosidase activity. We examined the efficiency of four amber suppressors acting on 42 different amber (UAG) codons at known positions in the I gene, and the efficiency of a UAG suppressor at 14 different UGA codons. The largest effects were found with the amber suppressor supE (Su2), which displayed efficiencies that varied over a 35-fold range, and with the UGA suppressor, which displayed a 170-fold variation in efficiency. Certain UGA sites were so poorly suppressed (less than 0.2%) by the UGA suppressor that they were not originally detected as nonsense mutations. Suppression efficiency can be correlated with the sequence on the 3' side of the codon being suppressed, and in many cases with the first base on the 3' side. In general, codons followed by A or G are well suppressed, and codons followed by U or C are poorly suppressed. There are exceptions, however, since codons followed by CUG or CUC are well suppressed. Models explaining the effect of the surrounding sequence on suppression efficiency are considered in the Discussion and in the accompanying paper.     

RNA cleavage linked with ribosomal action

Ribonuclease LS in Escherichia coli is a potential antagonist of bacteriophage T4. When T4 dmd is mutated, this RNase efficiently cleaves T4 mRNAs and leads to the silencing of late genes, thus blocking T4 growth. We previously found that, when two consecutive ochre codons were placed in the open reading frame of T4 soc, RNase LS cleaved soc mRNA at a specific site downstream of the ochre codons. Here, we demonstrate that RNase LS cleaves soc RNA at the same site even when only a single ochre codon is present or is replaced with either an amber or an opal codon. On the other hand, disruption of the Shine-Dalgarno sequence, a ribosome-binding site required for the initiation of translation, eliminates the cleavage. These results strongly suggest that RNase LS cleaves in a manner dependent on translation termination. Consistent with this suggestion, the cleavage dependency on an amber codon was considerably reduced in the presence of amber-codon-suppressing tRNA. Instead, two other cleavages that depend on translation of the region containing the target sites occurred farther downstream. Additional analysis suggests that an interaction of the ribosome with a stop codon might affect the site of cleavage by RNase LS in an mRNA molecule. This effect of the ribosome could reflect remodeling of the high-order structure of the mRNA molecule.     

Nonsense suppression in eukaryotes: the use of the Xenopus oocyte as an in vivo assay system.

Amber, ochre, and opal nonsense suppressor tRNAs isolated from yeast were injected into Xenopus laevis oocytes together with purified mRNAs (globin mRNA from rabbit, tobacco mosaic virus-RNA). Yeast opal suppressor tRNA is able to read the UGA stop codon of the rabbit beta-globin mRNA, thus producing a readthrough protein. A large readthrough product is also obtained upon coinjection of yeast amber or ochre suppressor tRNA with TMV-RNA. The amount of readthrough product is dependent on the amount of injected suppressor tRNA. The suppression of the terminator codon of TMV-RNA is not susceptible to Mg++ concentration or polyamine addition. Therefore, the Xenopus laevis oocyte provides a simple, sensitive, and well buffered in vivo screening system for all three types of eukaryotic nonsense suppressor tRNAs.     

In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal,ochre, and amber suppressor tRNAs

Amber suppressor tRNAs are widely used to incorporate nonnatural amino acids into proteins to serve as probes of structure, environment, and function. The utility of this approach would be greatly enhanced if multiple probes could be simultaneously incorporated at different locations in the same protein without other modifications. Toward this end, we have developed amber, opal, and ochre suppressor tRNAs derived from Escherichia coli, and yeast tRNA^Cys that incorporate a chemically modified cysteine residue with high selectivity at the cognate UAG, UGA, and UAA stop codons in an in vitro translation system. These synthetic tRNAs were aminoacylated in vitro, and the labile aminoacyl bond was stabilized by covalently attaching a fluorescent dye to the cysteine sulfhydryl group. Readthrough efficiency (amber > opal > ochre) was substantially improved by eRF1/eRF3 inhibition with an RNA aptamer, thus overcoming an intrinsic hierarchy in stop codon selection that limits UGA and UAA termination suppression in higher eukaryotic translation systems. This approach now allows concurrent incorporation of two different modified amino acids at amber and opal codons with a combined apparent readthrough efficiency of up to 25% when compared with the parent protein lacking a stop codon. As such, it significantly expands the possibilities for incorporating nonnative amino acids for protein structure/function studies.     

Site-specific mutagenesis of Escherichia coli gltT yields a weak, glutamic acid-inserting ochre suppressor

Of all the Escherichia coli tRNA genes that can give rise to an amber or an ochre suppressor by a single-nucleotide mutation, only the tRNAGlu genes have not been observed to do so. A study of the relationship between the sequences of tRNAs and the codons they translate predicts that the ochre suppressor derived from tRNAGlu would function very poorly on the ribosome. We have used site-specific mutagenesis to create the gene for such a tRNA in order to test this prediction. We cloned the tRNAGlu-Suoc gene into a high copy number plasmid, under control of the lacUV5 promoter. The mutant tRNA suppresses both amber and ochre nonsense mutations. As predicted, it is less efficient than other suppressors expressed under similar conditions.     

Gene selective suppression of nonsense termination using antisense agents

An estimated one third of all inherited genetic disorders and many forms of cancer are caused by premature (nonsense) termination codons. Aminoglycoside antibiotics are candidate drugs for a large number of such genetic diseases; however, aminoglycosides are toxic, lack specificity and show low efficacy in this application. Because translational termination is an active process, we considered that steric hindrance by antisense sequences could trigger the ribosome's "default mode" of readthrough when positioned near nonsense codons. To test this hypothesis, we performed experiments using plasmids containing a luciferase reporter with amber, ochre and opal nonsense mutations within the luxB gene in Escherichia coli. The nonspecific termination inhibitors gentamicin and paromomycin and six antisense peptide nucleic acids (PNA) spanning the termination region were tested for their potential to suppress the luxB mutation. Gentamicin and paromomycin increased luciferase activity up to 2.5- and 10-fold, respectively. Two of the PNAs increased Lux activity up to 2.5-fold over control levels, with no significant effect on cell growth or mRNA levels. Thus, it is possible to significantly suppress nonsense mutations within target genes using antisense PNAs. The mechanism of suppression likely involves enhanced readthrough, but this requires further investigation. Nonsense termination in human cells may also be susceptible to suppression by antisense agents, providing a new approach to address numerous diseases caused by nonsense mutations.     

Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene.

Amber, ochre and opal suppressor tRNA genes have been generated by using oligonucleotide directed site-specific mutagenesis to change one or two nucleotides in a human serine tRNA gene. The amber and ochre suppressor (Su+) tRNA genes are efficiently expressed in CV-1 cells when introduced as part of a SV40 recombinant. The expressed amber and ochre Su+ tRNAs are functional as suppressors as demonstrated by readthrough of the amber codon which terminates the NS1 gene of an influenza virus or the ochre codon which terminates the hexon gene of adenovirus, respectively. Interestingly, several attempts to obtain the equivalent virus stock of an SV40 recombinant containing the opal suppressor tRNA gene yielded virus lacking the opal suppressor tRNA gene. This suggests that expression of an efficient opal suppressor derived from a human serine tRNA gene is highly detrimental to either cellular or viral processes.