An inducible mammalian amber suppressor: propagation of a poliovirus mutant

We describe a general protocol for controlled gene amplification, which allows conditional expression of high levels of amber suppressor activity in monkey kidney cells, and we demonstrate its use in the genetic analysis of animal viruses by the generation and propagation of the first nonsense mutant of poliovirus. A human amber suppressor tRNASer gene linked to the SV40 origin of replication and a second DNA carrying a temperature-sensitive SV40 large T antigen gene were cotransfected into monkey cells. Cell lines having stably integrated the DNAs were isolated. Shifting the cells from the nonpermissive temperature to a lower permissive temperature caused the amplification of the suppressor tRNA gene, which resulted in suppression efficiencies at amber codons of 50%-70%, as measured by suppression of an amber codon in the E. coli chloramphenicol acetyltransferase gene. A mutant of poliovirus, in which a serine codon in the replicase gene was converted to an amber codon, was efficiently propagated on the suppressor-positive cell lines. The mutant virus reverted to wild-type by a single base change to a serine codon at a frequency of approximately 2.5 x 10(-6), surprisingly low for a RNA genome.     

Nonsense suppression in Dictyostelium discoideum

We describe the generation of Dictyostelium discoideum cell lines that carry different suppressor tRNA genes. These genes were constructed by primer-directed mutagenesis changing a tRNA(Trp)(CCA) gene from D. discoideum to a tRNA(Trp)(amber) gene and changing a tRNA(Glu)(UUC) gene from D. discoideum to a tRNA(Glu)(ochre) as well as a tRNA(Glu)(amber) gene. These genes were stably integrated into the D. discoideum genome together with a reporter gene. An actin 6::lacZ gene fusion carrying corresponding translational stop signals served as a reported. Active beta-galactosidase is expressed only in D. discoideum strains that contain, in addition to the reporter, a functional suppressor tRNA. Both amber suppressors are active in D. discoideum without interfering significantly with cell growth and development. We failed, however, to establish cell lines containing a functional tRNA(Glu)(ochre) suppressor. This may be due to the fact that nearly every message from D. discoideum known so far terminates with UAA. Therefore a tRNA capable of reading this termination codon may not be compatible with cell growth.     

Retroviral gag gene amber codon suppression is caused by an intrinsic cis-acting component of the viral mRNA.

In some type C retroviruses, translation of the pol gene appears to require translational suppression of the proximal gag amber codon. To identify the region of the viral nucleic acid responsible for synthesis of the pol gene products, a 300-base-pair DNA fragment containing the stop codon from a type C murine virus (AK virus) was inserted into the Escherichia coli lacZ gene such that the translational reading frame was maintained. Introduction of the resulting fusion gene into cells resulted in the suppression of the viral stop codon. As measured by beta-galactosidase production, suppression occurred at a frequency of approximately 10%. Suppression could occur in at least several vertebrate cell types and was not augmented by virus replication or the expression of viral gene products. This indicates that gag amber codon suppression does not require augmented levels of suppressor tRNA species.     

Differential response of human cells to deletions and stop codons in the gamma(1)34.5 gene of herpes simplex virus.

Earlier studies have shown that herpes simplex virus mutants lacking the gamma(1)34.5 gene are totally avirulent on intracerebral inoculation of the virus into mice and induce premature shutoff of protein synthesis in human neuroblastoma (SK-N-SH) cells but not in Vero cells. We report the following. (i) Whereas deletion mutant R3616, lacking 1,000 bp of the gamma(1)34.5 gene, caused premature shutoff of protein synthesis in both SK-N-SH and human foreskin fibroblasts (HFF), mutants R4009 and R930 (mutant F), carrying stop codons in all six frames, 27 and 210 codons from the initiation codon of the gamma(1)34.5 genes, respectively, induced shutoff of protein synthesis in SK-N-SH cells but not in HFF. The differences in behavior between the R3616 deletion and R4009 stop codon mutants cannot be attributed to differences in the rate of induction of premature shutoff of protein synthesis and the multiplicity of infection. HFF do not produce detectable truncated gamma(1)34.5 protein or truncated mRNA. (ii) Some clonal lines of SK-N-SH cells carrying a gamma(1)34.5 gene driven by a metallothionein promoter express the gamma(1)34.5 gene constitutively and do not require induction by cadmium to complement the gamma(1)34.5- virus. One clonal cell line complements the gamma(1)34.5- virus only after induction by cadmium. These results are consistent with previous conclusions that the phenotype of premature shutoff of protein synthesis is associated with absence of the gamma(1)34.5 protein and indicate that the amounts of gamma(1)34.5 protein necessary to complement the gamma(1)34.5- viruses are small. We conclude that human cells differ in the manner in which they respond to the presence of stop codons. Shutoff of protein synthesis in HFF infected with the stop codon mutants could have been precluded by small amounts of gamma(1)34.5 protein produced by splicing out of an intron containing the stop codon, downstream initiation of translation, or tRNA suppression of the stop codon.     

Nonsense mutation in open reading frame E2 of bovine papillomavirus DNA.

Oligonucleotide-directed mutagenesis was used to construct a nonsense mutation in open reading frame (ORF) E2 of bovine papillomavirus DNA. A single base substitution mutation was constructed which converted a TAC codon into a TAG amber stop codon at a position in the ORF that did not overlap with any other viral ORFs. Full-length viral DNA containing the mutation induced only approximately 2% of the transformed foci of mouse C127 cells that were induced by wild-type DNA. In a different transformation assay, approximately one-half of the C127 cells which had acquired the mutant DNA gave rise to colonies containing at least some cells with transformed morphology. The constructed mutation was maintained in cell lines derived from cells which had acquired the mutant viral DNA, but the viral DNA appeared to be integrated into the host cell genome. Genetic mapping experiments proved that the constructed amber mutation caused the decrease in focus-forming activity and the integration of the mutant viral DNA. These results suggest that ORF E2 encodes a protein which is involved either directly or indirectly in some aspects of oncogenic transformation by bovine papillomavirus and in maintaining the viral DNA as a plasmid in transformed cells.     

The amber codon in the gene encoding the monomethylamine methyltransferase isolated from Methanosarcina barkeri is translated as a sense codon.

Each of the genes encoding the methyltransferases initiating methanogenesis from trimethylamine, dimethylamine, or monomethylamine by various Methanosarcina species possesses one naturally occurring in-frame amber codon that does not appear to act as a translation stop during synthesis of the biochemically characterized methyltransferase. To investigate the means by which suppression of the amber codon within these genes occurs, MtmB, a methyltransferase initiating metabolism of monomethylamine, was examined. The C-terminal sequence of MtmB indicated that synthesis of this mtmB1 gene product did not cease at the internal amber codon, but at the following ochre codon. Antibody raised against MtmB revealed that Escherichia coli transformed with mtmB1 produced the amber termination product. The same antibody detected primarily a 50-kDa protein in Methanosarcina barkeri, which is the mass predicted for the amber readthrough product of the mtmB1 gene. Sequencing of peptide fragments from MtmB by Edman degradation and mass spectrometry revealed no change in the reading frame during mtmB1 expression. The amber codon position corresponded to a lysyl residue using either sequencing technique. The amber codon is thus read through during translation at apparently high efficiency and corresponds to lysine in tryptic fragments of MtmB even though canonical lysine codon usage is encountered in other Methanosarcina genes.     

Mischarging mutants of Su+2 glutamine tRNA in E. coli. II. Amino acid specificities of the mutant tRNAs

Among the mischarging mutants isolated from strains with Su+2 glutamine tRNA, two double-mutants, A37A29 and A37C38, have been suggested to insert tryptophan at the UAG amber mutation site as determined by the suppression patterns of a set of tester mutants of bacteria and phages (Yamao et al., 1988). In this paper, we screened temperature sensitive mutants of E. coli in which the mischarging suppression was abolished even at the permissive temperature. Four such mutants were obtained and they were identified as the mutants of a structural gene for tryptophanyl-tRNA synthetase (trpS). Authentic trpS mutations, such as trpS5 or trpS18, also restricted the mischarging suppression. These results strongly support the previous prediction that the mutant tRNAs of Su+2, A37A29 and A37C38, are capable of interacting with tryptophanyl-tRNA synthetase and being misaminoacylated with tryptophan in vivo. However, in an assay to determine the specificity of the mutant glutamin tRNAs, we detected predominantly glutamine, but not any other amino acid, being inserted at an amber codon in vivo to any significant degree. We conclude that the mutant tRNAs still accept mostly glutamine, but can accept tryptophan in an extent for mischarging suppression. Since the amber suppressors of Su+7 tryptophan tRNA and the mischarging mutants of Su+3 tyrosine tRNA are charged with glutamine, structural similarity among the tRNAs for glutamine, tryptophan and tyrosine is discussed.     

CbfA, the C-module DNA-binding factor, plays an essential role in the initiation of Dictyostelium discoideum development

We recently isolated from Dictyostelium discoideum cells a DNA-binding protein, CbfA, that interacts in vitro with a regulatory element in retrotransposon TRE5-A. We have generated a mutant strain that expresses CbfA at <5% of the wild-type level to characterize the consequences for D. discoideum cell physiology. We found that the multicellular development program leading to fruiting body formation is highly compromised in the mutant. The cells cannot aggregate and stay as a monolayer almost indefinitely. The cells respond properly to prestarvation conditions by expressing discoidin in a cell density-dependent manner. A genomewide microarray-assisted expression analysis combined with Northern blot analyses revealed a failure of CbfA-depleted cells to induce the gene encoding aggregation-specific adenylyl cyclase ACA and other genes required for cyclic AMP (cAMP) signal relay, which is necessary for aggregation and subsequent multicellular development. However, the cbfA mutant aggregated efficiently when mixed with as few as 5% wild-type cells. Moreover, pulsing cbfA mutant cells developing in suspension with nanomolar levels of cAMP resulted in induction of acaA and other early developmental genes. Although the response was less efficient and slower than in wild-type cells, it showed that cells depleted of CbfA are able to initiate development if given exogenous cAMP signals. Ectopic expression of the gene encoding the catalytic subunit of protein kinase A restored multicellular development of the mutant. We conclude that sensing of cell density and starvation are independent of CbfA, whereas CbfA is essential for the pattern of gene expression which establishes the genetic network leading to aggregation and multicellular development of D. discoideum.     

First position wobble in codon-anticodon pairing: amber suppression by a yeast glutamine tRNA

A 2.4-kb fragment of DNA isolated from the Saccharomyces cerevisiae genome was found to suppress amber mutations when its carrier plasmid was present in high copy number. A 1.2-kb subclone of this fragment was sufficient to confer suppressor activity. Sequencing has established that this fragment carries a normal glutamine tRNA gene. Deletion of this tRNA gene from the subclone resulted in the loss of suppressor activity. The tRNAGln has the anticodon CUG that normally recognizes the glutamine codon CAG. We propose that suppression occurs via an inefficient readthrough of the UAG amber stop codons during translation. Such readthrough requires wobble in the first position of the codon.     

Tetracycline-Regulated Suppression of Amber Codons in Mammalian Cells

As an approach to inducible suppression of nonsense mutations in mammalian cells, we described recently an amber suppression system in mammalian cells dependent on coexpression of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) along with the E. coli glutamine-inserting amber suppressor tRNA. Here, we report on tetracycline-regulated expression of the E. coli GlnRS gene and, thereby, tetracycline-regulated suppression of amber codons in mammalian HeLa and COS-1 cells. The E. coli GlnRS coding sequence attached to a minimal mammalian cell promoter was placed downstream of seven tandem tetracycline operator sequences. Cotransfection of HeLa cell lines expressing a tetracycline transactivator protein, carrying a tetracycline repressor domain linked to part of a herpesvirus VP16 activation domain, with the E. coli GlnRS gene and the E. coli glutamine-inserting amber suppressor tRNA gene resulted in suppression of the amber codon in a reporter chloramphenicol acetyltransferase gene. The tetracycline transactivator-mediated expression of E. coli GlnRS was essentially completely blocked in HeLa or COS-1 cells grown in the presence of tetracycline. Concomitantly, both aminoacylation of the suppressor tRNA and suppression of the amber codon were reduced significantly in the presence of tetracycline.     

Identification of a single base-pair mutation of TAA (Stop codon) → GAA (Glu) that causes light chain extension in a CHO cell derived IgG1

We describe here the identification of a stop codon TAA (Stop) → GAA (Glu) = Stop221E mutation on the light chain of a recombinant IgG1 antibody expressed in a Chinese hamster ovary (CHO) cell line. The extended light chain variants, which were caused by translation beyond the mutated stop codon to the next alternative in-frame stop codon, were observed by mass spectra analysis. The abnormal peptide peaks present in tryptic and chymotryptic LC–MS peptide mapping were confirmed by N-terminal sequencing as C-terminal light chain extension peptides. Furthermore, LC-MS/MS of Glu-C peptide mapping confirmed the stop221E mutation, which is consistent with a single base-pair mutation in TAA (stop codon) to GAA (Glu). The light chain variants were approximately 13.6% of wild type light chain as estimated by RP-HPLC analysis. DNA sequencing techniques determined a single base pair stop codon mutation, instead of a stop codon read-through, as the cause of this light chain extension. To our knowledge, the stop codon mutation has not been reported for IgGs expressed in CHO cells. These results demonstrate orthogonal techniques should be implemented to characterize recombinant proteins and select appropriate cell lines for production of therapeutic proteins because modifications could occur at unexpected locations.     

Isolation of an aggregation-less mutant of Dictyostelium discoideum with the expression of contact site A glycoprotein

We isolated mutants defective in aggregation (aggregation-less) by mutagenizing the "double-bypass" mutant HG592 of Dictyostelium discoideum as the parental strain. One of the mutants expressed the contact site A glycoprotein with an apparent molecular weight of 80 X 10(3) on the cell surface in the normal developmental stage and retained EDTA-stable cell contact as well as EDTA-sensitive cell contact. However, the mutant failed to aggregate on agar plates with bacteria. This mutant was designated HG700. We could not identify any differences between this mutant and the parental strain in levels of adenylate cyclase or extracellular phosphodiesterase activity, or in its chemotaxis toward cAMP. The mutant had greatly decreased the incorporation of [35S] sulfate into the particulate fractions of the cells starved for 6 h. This suggests that the modification by sulfation may crucially affect the mechanism of cell aggregation.     

Studies on the bacteriophage MS2: XLI. Nature of the azure mutation

Azure (or reverse amber) mutants grow normally on wild-type Escherichia coli but not on host strains harbouring a strong UAG suppressor mutation. Three different bacteriophage MS2 azure mutants obtained by treatment with nitrous acid have been characterized at the nucleotide sequence level. The 3′-end fragment of the ³²P-labelled mutant genomes was isolated by DNA:RNA hybridization and treatment with nuclease S₁, and was analyzed by mini-fingerprinting of the RNA. It is known that the wild-type MS2 polymerase gene ends with a UAG codon, followed seven triplets further by an in-phase UAA triplet. All three azure mutants contained an A → G transition in this UAA second stop codon of the polymerase gene, resulting in a second suppressible UAG (amber) codon. Analysis of revertants demonstrated that the azure mutation can be counteracted either by a true site reversion at the second stop or by the creation of a new stop signal for the polymerase gene, either UAA (ochre) or UGA (opal), before or at the first stop, or beyond the second stop. On the basis of these results, a mechanism for the azure mutation is proposed. Silent mutations (one in the coding region, three in the untranslated 3′-terminal sequence) have also been observed in these phage stocks.     

In vitro selection of RNA aptamer against Escherichia coli release factor 1

A pool of 84-nt RNAs containing a randomized sequence of 50 nt was selected against gel-immobilized Escherichia coli release factor 1 (RF-1) responsible for translation termination at amber (UAG) stop codon. The strongest aptamer (class II-1) obtained from 43 clones bound to RF-1, but not to UAA/UGA-targeting RF-2, with Kd = 30+/-6 nM (SPR). A couple of unpaired hairpin domains in the aptamer were suggested as the sites of attachment of RF-1. By binding to and hence inhibiting the action of RF-1 specifically or bio-orthogonally, aptamer class II-1 enhanced the amber suppression efficiency in the presence of an anticodon-adjusted (CUA) suppressor tRNA without practically damaging the protein translation machinery of the cell-free extract of E. coli, as confirmed by the translation of amber-mutated (gfp(amber141) or gfp(amber178)) and wild-type (gfp(wild)) genes of GFP.     

Vinculin is essential for muscle function in the nematode

Actin filaments in the body wall muscle of the nematode Caenorhabditis elegans are attached to the sarcolemma through vinculin-containing structures called dense bodies, Z-line analogues. To investigate the in vivo function of vinculin, we executed a genetic screen designed to recover mutations in the region of the nematode vinculin gene, deb-1. According to four independent criteria, two of the isolated mutants were shown to be due to alterations in the deb-1 gene. First, antibody staining showed that the mutants had reduced levels of vinculin. Second, the sequence of each mutant gene was altered from that of wild type, with one mutation altering a conserved splice sequence and the other generating a premature amber stop codon. Third, the amber mutant was suppressed by the sup-7 amber suppressor tRNA gene. Finally, injection of a cloned wild type copy of the gene rescued the mutant. Mutant animals lacking vinculin arrested development as L1 larvae. In such animals, embryonic elongation was interrupted at the twofold length, so that the mutants were shorter than wild type animals at the same stage. The mutants were paralyzed and had disorganized muscle, a phenotype consistent with the idea that vinculin is essential for muscle function in the nematode.     

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.