Amber Termination Codon Research Articles

Structural studies of the acidic transactivation domain of the Vmw65 protein of herpes simplex virus using proton NMR

We have overproduced and purified the carboxy-terminal transactivation domain of Vmw65 (VP16) of herpes simplex virus, and studied potential folding of the domain by 1H NMR. Two species of the acidic domain were obtained from the bacterial expression system, and we demonstrate that one of these represents read-through of the natural amber termination codon of the Vmw65 reading frame producing a larger polypeptide. Additional residues in the read-through product were identified by total amino acid analysis and by NMR. Study of the correctly terminated product by 1D NMR gave resonances which were clustered into groups around their random-coil chemical shift positions, and 2D NMR demonstrated that, even in mixed solvents containing up to 80% MeOH, there was very little evidence of secondary structure. Together these results indicate that the isolated acid domain has little if any alpha-helical content of any stable nature. We discuss these results with reference to the demonstrated activity of the acidic domain in a wide variety of polypeptide contexts.

Mutants of Escherichia coli initiator tRNA that suppress amber codons in Saccharomyces cerevisiae and are aminoacylated with tyrosine by yeast extracts.

We recently described mutants of Escherichia coli initiator tRNA that suppress amber termination codons (UAG) in E. coli. These mutants have changes in the anticodon sequence (CAU----CUA) that allow them to read the amber codon and changes in the acceptor stem that allow them to bind to the ribosomal aminoacyl (A) site. We show here that a subset of these mutants suppress amber codons in Saccharomyces cerevisiae and that they are aminoacylated with tyrosine by yeast extracts. Analysis of a number of mutants as substrates for yeast tyrosyl-tRNA synthetase has led to identification of the C1.G72 base pair and the discriminator base A73, conserved in all eukaryotic cytoplasmic and archaebacterial tyrosine tRNAs, as being important for recognition. Our results suggest that the C1.G72 base pair and the discriminator base, in addition to the anticodon nucleotides previously identified [Bare, L.A. & Uhlenbeck, O.C. (1986) Biochemistry 25, 5825-5830] as important in yeast tyrosyl-tRNA synthetase recognition, may comprise the critical identity determinants in yeast tyrosine tRNA.

Adeno-associated viruses having nonsense mutations in the capsid genes: Growth in mammalian cells containing an inducible amber suppressor

When an adeno-associated virus (AAV) genome contained in a recombinant plasmid is transfected into adenovirus-infected cells, infectious AAV particles are efficiently generated. We previously described the construction of a conditional lethal mutant of AAV having an amber termination codon inserted in the rep gene. This mutant was propagated on a monkey kidney cell line (SupD12) having an inducible amber suppressor tRNA ser. We now describe the construction and propagation of two additional conditional lethal mutants of AAV having amber codons affecting all three capsid proteins (AAV Capam) or only the VP1 capsid protein (AAV VP1 am). Suppression of the amber mutations in the capsid proteins was demonstrated directly by immunoblot analysis. The efficiency of amber suppression on the SupD12 cell was about 6 to 10% for AAV VP1 am and 4 to 5% for AAV Capam. The reversion frequency of either mutant was apparently less than 10 −5. On nonsuppressing cells AAV VP1 am exhibited an Lip (Inf) phenotype, whereas AAV Capa m exhibited a Cap phenotype.

A nonsense mutation in the tyrosinase gene of Afghan patients with tyrosinase negative (type IA) oculocutaneous albinism.

We detected a nonsense mutation in the tyrosinase gene of two Afghan sibs with classical tyrosinase negative (type IA) oculocutaneous albinism. The mutation, a single base substitution at codon 178, creates an amber termination codon that truncates the 529 amino acid tyrosinase polypeptide at this position. The patients' parents are first cousins, and the patients are therefore homoallelic for this mutation.

High-level direct expression of semi-synthetic human interleukin-6 in Escherichia coli and production of N-terminus met-free product.

We have developed a direct expression system for high-level production of recombinant human interleukin-6 (rhIL-6) in Escherichia coli. In this system, (i) the natural N-terminal coding region of the hIL-6 gene was replaced by a synthetic sequence containing A-T rich codons, (ii) dual Shine-Dalgarno (SD) sequences were employed, (iii) an A-T rich segment was inserted in front of the initiation codon to avoid putative mRNA secondary structure in the region and (iv) the natural amber termination codon of the hIL-6 gene was changed to an ocher stop codon. The hIL-6 polypeptide, synthesized at a high level, formed cytoplasmic inclusion bodies. After refolding, the N-terminal methionine was removed by aminopeptidase-P in vitro. The purified recombinant hIL-6 had B-cell differentiation activity equivalent to natural IL-6 from a human T-cell culture.

Sequence conservation and divergence of hepatitis δ virus RNA

The complete RNA sequence of the hepatitis δ virus (HDV) obtained from the Nauru Island in the Pacific was determined by cDNA cloning and amplification by polymerase chain reaction (PCR). The sequence showed 14–17% divergence from the two known HDV RNA sequences. There are three highly conserved domains: the region around the autocatalytic cleavage site of the genomic RNA (nucleotides 659 to 772), the region around the autocatalytic cleavage site of the antigenomic-sense RNA (nucleotides 847 to 966), and the region around the middle one-third domain of the open reading frame (ORF) encoding the hepatitis δ antigen on the antigenomic RNA (nucleotides 1267 to 1347). The two autocatalytic activities are required for the cleavage and ligation of HDV RNA during RNA replication. The third conserved domain codes for the RNA-binding domain of HDAg, which specifically interacts with HDV RNA. Three nucleotide changes within the genomic catalytic sequence are present but did not alter the catalytic cleavage activity of the HDV RNA. Microheterogeneity of the RNA sequences was also detected. One of these occurred within the coding region of the δ antigen, creating an amber termination codon in some of the RNA species. Thus, this HDV strain contains two different RNA species, one of which encodes a S antigen of 214 amino acids and the other 195 amino acids. These two protein species were detected by immunoblotting of the patient's plasma. In contrast to other HDV strains, only three ORFs capable of encoding more than 100 amino acids each are present in this HDV RNA. We recommend that oligonucleotides complementary to the highly conserved sequences should be used as primers for PCR in clinical detection assays of hepatitis S virus infection.

Heterogeneity of hepatitis delta antigen

Hepatitis delta antigen (HDAg) is the only known protein encoded by the hepatitis delta virus (HDV). Two HDAg species of different sizes have been detected in the sera and livers of the infected humans, chimpanzees, and woodchucks, even though only one RNA species was previously identified in most of the HDV strains. To study HDAg heterogeneity, we took advantage of the fact that a single base mutation at nucleotide 1015 (C to U), which results in an amber termination codon in the HDAg open reading frame (ORF), eliminates a unique Ncol restriction enzyme site. We screened various HDV cDNA clones and detected sequence heterogeneity of the HDAg-coding region on the basis of the presence or absence of the Ncol site. Five delta hepatitis patients were examined. In every patient, two types of HDAg-coding sequence were detected at nucleotide 1015: one which contains a C and results in an ORF encoding a delta antigen of 214 amino acids, and the other which possesses a U and results in an amber termination codon and a truncated HDAg species of 195 amino acids. The in vitro translation products of these two ORFs comigrated with the two HDAg species from the patient's plasma on SIDS polyacrylamide gels. Polymerase chain reaction (PCR) amplification of the HDV RNA from some patients' sera and subsequent sequencing showed several additional mutations in the HDAg-coding region. These mutations are independent of the C or U nucleotide change at the site of the amber termination codon. These results suggest that the two HDAg species are derived from heterogeneous HDV RNA and are the result of selection, and that microheterogeneity of the HDV RNA exists in every delta hepatitis patient.

Efficient translation of the UAG termination codon in Candida species

Clinical isolates of the dimorphic fungus Candida albicans encode a tRNA that, in a cell-free translation system prepared from the yeast Saccharomyces cerevisiae, efficiently translates the amber (UAG) termination codon. Unusually, the efficiency of this UAG read-through in the heterologous cell-free system is not further enhanced by polyamines. The suppressor tRNA is also able to efficiently translate the UAG codon in the rabbit reticulocyte cell-free system and with efficiencies approaching 100% in a homologous (C. albicans) cell-free system. That the suppressor tRNA is nuclear-encoded is demonstrated by the lack of activity in purified C. albicans mitochondrial tRNAs. Finally, UAG suppressor tRNA activity is also demonstrated in three other pathogenic Candida species, C. parapsilosis, C. guillermondii and C. tropicalis. These results suggest that some, but not all, Candida species have evolved an unusual nuclear genetic code in which UAG is used as a sense codon.

Mutation eliminating mitochondrial leader sequence of methylmalonyl-CoA mutase causes muto methylmalonic acidemia.

Methylmalonyl-CoA mutase (EC 5.4.99.2) is a mitochondrial matrix enzyme whose activity is deficient in the inherited disorder methylmalonic acidemia. Previous studies on primary fibroblast cell lines from patients with methylmalonic acidemia have delineated a variety of biochemical phenotypes underlying this disorder. One cell line with primary mutase apoenzyme deficiency exhibited a particularly unusual phenotype; it expressed an abnormally small and unstable immunoreactive protein, which was not imported by mitochondria. We now report cloning and sequencing of the cDNA encoding this mutant protein. The mutation is a single base change, a cytosine----thymine transition, which introduces an amber termination codon at position 17 within the mitochondrial leader sequence. The immunoreactive protein produced by these cells reflects translation from AUG codons downstream from this termination codon and, hence, lacks a mitochondrial leader peptide. This mutation represents a complex prototype for a class of mutations in which absence of the mitochondrial targeting sequence leads to absence of a functioning gene product.

A specific base transition occurs on replicating hepatitis delta virus RNA

Three independent lines of evidence showed that when an infectious clone of hepatitis delta virus of known sequence was used to initiate genome replication, up to 41% of the genomes were specifically mutated in the amber termination codon (UAG to UGG) for the open reading frame of the delta antigen, thereby increasing the length of the predicted protein from 195 to 214 amino acids. This change was detected only on molecules that participated in RNA-directed RNA synthesis.

Initiation of protein synthesis from a termination codon.

We show that the amber termination codon UAG can initiate protein synthesis in Escherichia coli. We mutated the initiation codon AUG of the chloramphenicol acetyltransferase (CAT) gene to UAG (CATam1) and translated mRNA derived from the mutant CAT gene in E. coli S-30 extracts. A full-length CAT polypeptide was synthesized in the presence of tRNA(fMetCUA), a mutant E. coli initiator tRNA which has a change in the anticodon sequence from CAU to CUA. Addition of purified E. coli glutaminyl-tRNA synthetase substantially stimulated synthesis of the CAT polypeptide. Thus, initiation of protein synthesis with UAG and tRNA(fMetCUA) most likely occurs with glutamine and not methionine. The UAG codon also initiates protein synthesis in vivo. To eliminate a weak secondary site of initiation from AUC, the fifth codon, we further mutagenized the CATam1 gene at codons 2 (GAG----GAC) and 5 (AUC----ACC). Transformation of E. coli with the resultant CATam1.2.5 gene yielded transformants that synthesized CAT polypeptide and were resistant to chloramphenicol only when they were also transformed with the mutant tRNA(fMetCUA) gene. Immunoblot analyses and assays for CAT enzyme activity in extracts from transformed cells indicate that initiation from UAG is efficient, 60-70% of that obtained from AUG. Initiation of protein synthesis from UAG using a mutant initiator tRNA allows tightly regulated expression of specific genes. This may be generally useful for overproduction in E. coli and other eubacteria of proteins which are toxic to these cells.

The genome structure of turnip crinkle virus

The nucleotide sequence of turnip crinkle virus (TCV) genomic RNA has been determined from cDNA clones representing most of the genome. Segments were confirmed using dideoxynucleotide sequencing directly from viral RNA, and the 3′ terminal sequence was confirmed by chemical sequencing of end-labeled genomic RNA. Three open reading frames (ORFs) have been identified by examination of the deduced amino acid sequences and by comparison with the ORFs found in the genome of carnation mottle virus. ORF 1 initiates near the 5′ terminus of the genome and is punctuated by an amber termination codon. Translation of ORF 1 would yield a 28-kDa protein and an 88-kDa read-through product. The read-through domain possesses amino acid sequence similarities with putative viral RNA polymerases. ORFs 2 and 3 encode products of 38 (coat protein) and 8 kDa, respectively, which are expressed from subgenomic mRNAs. The organization of the TCV genome suggests that TCV is closely related to carnation mottle virus and distinct from members classified in other small RNA virus groups, such as the tombus- and sobemoviruses.

Suppression of Amber Codons in Vivo as Evidence That Mutants Derived from Escherichia coli Initiator tRNA Can Act at the Step of Elongation in Protein Synthesis

The absence of a Watson-Crick base pair at the end of the amino acid acceptor stem is one of the features which distinguishes prokaryotic initiator tRNAs as a class from all other tRNAs. We show that this structural feature prevents Escherichia coli initiator tRNA from acting as an elongator in protein synthesis in vivo. We generated a mutant of E. coli initiator tRNA in which the anticodon sequence is changed from CAU to CUA (the T35A36 mutant). This mutant tRNA has the potential to read the amber termination codon UAG. We then coupled this mutation to others which change the C1.A72 mismatch at the end of the acceptor stem to either a U1:A72 base pair (T1 mutant) or a C1:G72 base pair (G72 mutant). Transformation of E. coli CA274 (HfrC Su- lacZ125am trpEam) with multicopy plasmids carrying the mutant initiator tRNA genes show that mutant tRNAs carrying changes in both the anticodon sequence and the acceptor stem suppress amber codons in vivo, whereas mutant tRNA with changes in the anticodon sequence alone does not. Mutant tRNAs with the above anticodon sequence change are aminoacylated with glutamine in vitro. Measurement of kinetic parameters for aminoacylation by E. coli glutaminyl-tRNA synthetase show that both the nature of the base pair at the end of the acceptor stem and the presence or absence of a base pair at this position can affect aminoacylation kinetics. We discuss the implications of this result on recognition of tRNAs by E. coli glutaminyl-tRNA synthetase.

Mapping and characterization of mutations induced by benzo[a]pyrene diol epoxide at dihydrofolate reductase locus in CHO cells.

Chinese hamster ovary cells were mutagenized with benzo[a]pyrene diol epoxide (BPDE), an aromatic hydrocarbon carcinogen, and mutants at the dihydrofolate reductase (dhfr) locus were isolated. Of 15 mutants analyzed by Southern blotting, one contained a large deletion that spanned all six exons of the 25-kb dhfr gene; the remaining mutants exhibited no detectable changes. Three of these putative point mutations were localized by the loss of a restriction site: a SacI site in exon III, an MspI site in exon III, and a KpnI site in exon VI. The affected regions in two of these mutants were cloned and sequenced. The SacI- mutant was caused by a G:C----T:A transversion resulting in an amber termination codon. In the MspI- mutant, the deletion of a single C:G resulted in a frameshift and a downstream ochre termination codon. On the basis of overlapping restriction site sequences, the KpnI- mutant was deduced to be a splicing mutant involving the most 3' G in intron V. The location of these and the remaining 11 putative point mutations was sought using RNA heteroduplex mapping. Mismatched bases between riboprobes complementary to wild-type dhfr mRNA and mutant mRNA molecules were detected in 10 of the 14 mutants analyzed. These mutations mapped to four of the six exons or exon splice sites. Surprisingly, over half of these mutants exhibited greatly reduced (approximately 10-fold) steady-state levels of dhfr mRNA.

Nucleotide sequence of beet western yellows virus RNA.

The nucleotide sequence of the genomic RNA (5641 nt) of beet western yellow virus (BWYV) isolated from lettuce has been determined and its genetic organization deduced. The sequence of the 3'terminal 2208 nt of RNA of a second BWYV isolate, obtained from sugarbeet, was also determined and was found to be very similar but not identical to that of the lettuce isolate. The complete sequence of BWYV RNA contains six long open reading frames (ORFs). A cluster of three of these ORFs, including the coat protein cistron, display extensive amino acid sequence homology with corresponding ORFs of a second luteovirus, the PAV isolate of barley yellow dwarf virus (BYDV) (1,2). The ORF corresponding to the putative viral RNA-dependant RNA polymerase, on the other hand, resembles that of southern bean mosaic virus. There is circumstantial evidence that expression of the BWYV RNA polymerase ORF may involve a translational frameshift mechanism. The ORF immediately following the coat protein cistron may be translated by in-frame readthrough of the coat protein cistron amber termination codon. Similar mechanisms have been proposed for expression of the corresponding ORFs of BYDV(PAV) (1).

High rate of somatic point mutation in vitro in and near the variable-region segment of an immunoglobulin heavy chain gene.

The "silent" allele at the immunoglobulin heavy-chain locus in the pre-B-lymphocyte line 18-81 contains a correctly assembled gene. However, an amber termination codon within the variable-region gene segment prematurely terminates translation into complete heavy chain. Revertants that do produce heavy chain are generated at a high rate, which is termed hypermutation. By DNA sequencing of subclones, we have confirmed that whenever mu chain is produced by the usually silent allele, a true reversion is found in the DNA. Mutations are not confined to the position of the amber termination codon but are also found at other sites in and near the variable-region gene segment.

Nucleotide Sequence of Beet Necrotic Yellow Vein Virus RNA-2

Summary The complete nucleotide sequence of beet necrotic yellow vein virus (BNYVV) RNA-2 has been determined from a study of cloned cDNA. The RNA sequence is 4612 nucleotides in length, excluding the poly(A) tail. There are six long open reading frames (ORFs) in the sequence. The viral coat protein cistron is the ORF nearest to the 5′ terminus and the coat protein amber termination codon is followed by a long in-phase ORF. A corresponding readthrough polypeptide with the coat protein sequence at its N terminus has been detected in previous in vitro translation studies. Four additional ORFs encoding potential polypeptides of mol. wt. 42000 (42K), 12.5K, 14.8K and 14.4K are present in the sequence and evidence is presented that the 42K polypeptide is expressed, probably from a subgenomic messenger RNA. There is extensive homology between sequences near the 3′ termini of RNA-2, RNA-3 and RNA-4 of BNYVV.

In vitro mutagenesis of the putative replicase genes of tobacco mosaic virus.

We have established an in vitro transcription system to produce infectious tobacco mosaic virus (TMV) RNA from a cloned cDNA copy. Using this system, several TMV mutants were transcribed in vitro from cDNA clones mutagenized at or near the leaky amber termination codon of the 130K protein gene, and their infectivity was assayed on tobacco plants. Three (two frame-shift and one non-sense) mutants with an intact 130K but a defective 180K protein gene were not infectious, while two mutants with a one-amino-acid insertion in the 180K protein gene were infectious. When the amber codon of the 130K protein gene was deleted, infectivity was lost. However, when the amber termination codon was replaced with ochre or tyrosine codon, infectivity was retained. Sequence analyses revealed that introduced mutations were retained in progeny viral sequences except in the progeny of the amber-to-tyrosine mutant, which was a mixture of the parental mutagenized virus and a pseudo-revertant with ochre codon.

Translational readthrough of an amber termination codon during synthesis of feline leukemia virus protease.

Feline leukemia virus contains a protease which apparently has the same specificity as murine leukemia virus protease. It cleaves in vitro the Pr65gag of Gazdar-mouse sarcoma virus into the constituent p15, p12, p30, and p10 proteins. We purified the protease and determined its NH2-terminal amino acid sequence (the first 15 residues). Alignment of this amino acid sequence with the nucleotide sequence (I. Laprevotte, A. Hampe, C. H. Sherr, and F. Galibert, J. Virol. 50:884-894, 1984) reveals that the protease is a viral-coded enzyme and is located at the 5' end of the pol gene. As previously found for murine leukemia virus (Y. Yoshinaka, I. Katoh, T. D. Copeland, and S. Oroszlan, Proc. Natl. Acad. Sci. U.S.A. 82:1618-1622, 1985), feline leukemia virus protease is synthesized through in-frame suppression of the gag amber termination codon by insertion of a glutamine in the fifth position, and the first four amino acids are derived from the gag gene.

Murine leukemia virus protease is encoded by the gag-pol gene and is synthesized through suppression of an amber termination codon.

We have purified from Moloney murine leukemia virus (Mo-MuLV) a protease that has the capacity of accurately cleaving the polyprotein precursor Pr65gag into the mature viral structural proteins. Both the NH2- and COOH-terminal amino acid sequences have been determined and aligned with the amino acid sequence deduced from the DNA sequence of Mo-MuLV by other workers. The results show that: (i) the protease is located at the 5' end of the pol gene, and the first four amino acids are overlapped with the 3' end of the gag gene; (ii) the fifth amino acid residue is glutamine, which is inserted by suppression of the UAG termination codon at the gag-pol junction; and (iii) the protease is composed of 125 amino acids with calculated Mr = 13,315, and the COOH terminus of the protease is adjacent to the NH2 terminus of reverse transcriptase. The map order of the gag-pol gene is proposed to be 5'-p15-p12-p30-p10-protease-reverse transcriptase-endonuclease-3'.