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Results for “Codon, Nonsense”

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PubMed1996

End-plate acetylcholine receptor deficiency due to nonsense mutations in the epsilon subunit.

We describe a congenital myasthenic syndrome associated with severe end-plate (EP) acetylcholine receptor (AChR) deficiency not associated with an EP myopathy, and with evidence of immature AChR, containing the gamma instead of the epsilon subunit (gamma-AChR) at the EPs. Molecular genetic analysis of AChR-subunit genes revealed two mutations in the epsilon-subunit gene: insertion of a thymine after epsilon nucleotide 1101 (epsilon 11O1insT) that generates a nonsense codon directly, and insertion of a guanine after epsilon nucleotide 1293 (epsilon 1293insG) that generates three missense codons followed by a nonsense codon. Each mutation predicts truncation of the epsilon subunit at the level of the long cytoplasmic loop, between the third (M3) and fourth (M4) membrane spanning domains. The propositus' asymptomatic son carries epsilon 1293G, indicating that the two mutations are heteroallelic. Expression of AChR harboring either mutation in human embryonic kidney (HEK) fibroblasts was markedly reduced. Single-channel activity recorded from HEK cells expressing epsilon 11O1insT-AChR was infrequent but resembled activity of wild-type AChR channels in amplitude and open duration. No channel activity could be recorded from HEK cells expressing epsilon 1293insG-AChR. Expression of gamma-AChR at the EPs may serve as the means of phenotypic rescue from potentially fatal nonsense mutations in the epsilon-subunit gene.

Amino Acid Sequence↗

PubMed2005

beta-globin gene cluster haplotypes associated with beta-thalassemia on Corsica island.

In the Corsican population, the incidence of beta-thalassemia traits is reported to be 3.1%. We have investigated the 2 more important beta-thalassemia mutations present in the Corsican population: beta0-39 and beta+IVS1-110. Seven polymorphic sites in the beta-globin gene cluster were analyzed from a sample of 43 non-related beta-thalassemia heterozygotes and of 47 nonrelated healthy individuals, from Central Corsica (Corte). Among the 43 Corsican patients analyzed, the nonsense codon is predominant (88.40%), whereas the beta+IVS1-110 mutation, the most common of beta-thalassemia in the eastern part of the Mediterranean basin, is underrepresented (2.33%). The other individuals did not show positive for the two tested mutations (9.27%). The beta0-39 mutation in the studied population shows a strong association with haplotype II (18.7%) and a weaker association with haplotypes I (2.3%) and VII (2.1%). The strong association of the beta0-39 mutation with haplotype II was also found in Sardinia, suggesting that the mutation on the two islands have the same origin. In the present study all the data concerning frequencies of the mutations and of sequence haplotypes, support the hypothesis of a western Mediterranean origin of the beta0-39 mutation. For the first time, this paper analyzes the association of beta-globin gene cluster haplotypes with the 2 more frequent beta-thalassemia mutations in an isolated population in the centre of Corsica (Corte), which presents certain genetic peculiarities. However, the analysis of beta-haplotypes will be very useful for the genetic epidemiological study in this region.

Codon, Nonsense↗

PubMed1997

Phenotypic heterogeneity of mutational changes at a conserved nucleotide in 16 S ribosomal RNA.

RNA sites that contain unpaired or mismatched nucleotides can be interaction sites for other macromolecules. C1054, a virtually universally conserved nucleotide in the 16 S (small subunit) ribosomal RNA of Escherichia coli, is part of a highly conserved bulge in helix 34, which has been located at the decoding site of the ribosome. This helix has been implicated in several translational events, including peptide chain termination and decoding accuracy. Here, we observed interesting differences in phenotype associated with the three base substitutions at, and the deletion of, nucleotide C1054. The phenotypes examined include suppression of nonsense codons on different media and at different temperatures, lethality conditioned by temperature and level of expression of the mutant rRNA, ribosome profiles upon centrifugation through sucrose density gradients, association of mutant 30 S subunits with 50 S subunits, and effects on the action of tRNA suppressor mutants. Some of our findings contradict previously reported properties of individual mutants. Particularly notable is our finding that the first reported 16 S rRNA suppressor of UGA mutations was not a C1054 deletion but rather the base substitution C1054A. After constructing deltaC1054 by site-directed mutagenesis, we observed, among other differences, that it does not suppress any of the trpA mutations previously reported to be suppressed by the original UGA suppressor. In general, our results are consistent with the suggestion that the termination codon readthrough effects of mutations at nucleotide 1054 are the result of defects in peptide chain termination rather than of decreases in general translational accuracy. The phenotypic heterogeneity associated with different mutations at this one nucleotide position may be related to the mechanisms of involvement of this nucleotide, the two-nucleotide bulge, and/or helix 34 in particular translational events. In particular, previous indications from other laboratories of conformational changes associated with this region are consistent with differential effects of 1054 mutations on RNA-RNA or RNA-protein interactions. Finally, the association of a variety of phenotypes with different changes at the same nucleotide may eventually shed light on speculations about the coevolution of parts of ribosomal RNA with other translational macromolecules.

Base Sequence↗

PubMed2001

Spontaneous second-site suppressors of the filamentation defect of prr1Delta mutants define a critical domain of Rim101p in Candida albicans.

In response to changes in ambient pH the opportunistic pathogen Candida albicans differentially expresses a number of genes. The response to pH affects morphological differentiation and virulence. The pathway controlling the pH response terminates in the zinc-finger containing transcription factor encoded by RIM101/PRR2. By analogy to the pH response pathway of Aspergillus nidulans, PRR1 of C. albicans encodes a protein that is presumably required to convert Rim101p from an inactive to an active form by proteolytic removal of a C-terminal peptide. A prr1Delta mutant is compromised in its ability to differentiate into the filamentous form. Spontaneous phenotypic revertants of a prr1Delta mutant were selected by their ability to form filamentous colonies. These mutants were also found to be defective in pH-dependent gene expression. Each of the eight mutants examined contained a heterozygous dominant mutation at the RIM101 locus. This was demonstrated genetically in all of the mutants, and directly by sequence determination of both alleles in two of the mutants. The mutant alleles conferred the ability to filament to a prr1Delta mutant, thus demonstrating that they were directly responsible for suppressing the filamentation defect. Seven of the mutant alleles contained a 1-bp substitution and one contained two substitutions at adjacent positions. The mutations were clustered within a 90-bp region near the 3'-end of the gene. In all cases the mutation generated a nonsense codon that resulted in premature termination of Rim101p; the mutant proteins were truncated by 75-104 amino acids. The results define a critical region in the C-terminal region of Rim101p and are consistent with the proposed proteolytic activation of Rim101p.

Alleles↗

PubMed2005

Mechanistic links between nonsense-mediated mRNA decay and pre-mRNA splicing in mammalian cells.

Nonsense-mediated mRNA decay (NMD) generally involves nonsense codon recognition by translating ribosomes at a position approximately 25 nts upstream of a splicing-generated exon junction complex of proteins. As such, NMD provides a means to degrade abnormal mRNAs that encode potentially deleterious truncated proteins. Additionally, an estimated one-third of naturally occurring, alternatively spliced mRNAs is also targeted for NMD. Given the extraordinary frequency of alternative splicing together with data indicating that naturally occurring transcripts other than alternatively spliced mRNAs are likewise targeted for NMD, it is believed that mammalian cells routinely utilize NMD to achieve proper levels of gene expression.

Animals↗

PubMed2005

Transcription promotes guanine to thymine mutations in the non-transcribed strand of an Escherichia coli gene.

Transcription of DNA opens the chromatin, causes topological changes in DNA and transiently exposes the two strands to different biochemical environments. Consequently, it has long been argued that transcription may promote damage to DNA and there are data in Escherichia coli and yeast supporting a correlation between high transcription and mutations. We examined the transcription-dependence of the reversion of a nonsense codon (TGA) in E. coli and found that there was a strong dependence of mutations on transcription in strains defective in the repair of 8-oxoguanine in DNA. Under conditions of high transcription there was a three to five-fold increase in mutations that changed TGA in the non-transcribed strand to a sense codon. Furthermore, in both mutY and mutM mutY backgrounds the mutations were overwhelmingly G:C to T:A. In contrast, when the TGA was in the transcribed strand in relation with the inducible promoter, high transcription decreased the rate of reversion. Similar results were obtained in a strain defective in the transcription-repair coupling factor, Mfd, suggesting that transcription dependent increase in base substitutions does not require transcription-dependent DNA repair. However, Mfd does modulate the magnitude of the mutagenic effect of transcription. These data are consistent with a model in which the non-transcribed strand is more susceptible to oxidative damage during transcription than the transcribed strand. These results suggest that the magnitudes of individual base substitutions and their relative numbers in other studies of mutational spectra may also be affected by transcription.

Codon, Nonsense↗

PubMed2001

Studies toward the site specific incorporation of sugars into proteins: synthesis of glycosylated aminoacyl-tRNAs.

A series of glycosylated serine derivatives was synthesized from peracetylated sugars and Fmoc-protected serine; these were chemically esterified with the tris-(tetrabutylammonium) salt of pdCpA. The fully protected and deprotected glycosylated aminoacyl pdCpAs were ligated enzymatically to an abbreviated tRNA (tRNA-C(OH)) to provide the title compounds that are key intermediates in the elaboration of glycoproteins using readthrough of a nonsense codon.

Codon, Nonsense↗

PubMed2001

Synthesis and application of caged peptides and proteins.

Caged compounds have covalently attached groups that are rapidly cleaved upon exposure to UV light. Attachment of photolabile groups makes the molecule inert until photolysis releases it in its bioactive form. When caged compounds are applied to the experimental system in advance, the concentration jump of biologically active substances can be brought about immediately in a limited area upon irradiation with pulsed and focused UV light. Therefore, caged compounds of low molecular weight, which are commercially available, have been used effectively to study the mechanisms of temporal biological phenomena, such as muscle contraction, intracellular signaling, and neurotransmission. Because many proteins and peptides play important roles in these phenomena, their caged derivatives should serve as powerful tools to clarify complex biological systems. To prepare caged proteins and peptides, several groups have improved upon a chemical modification method, as well as developed two new methods: (1) nonsense codon suppression and (2) solid-phase peptide synthesis. In this review, we summarize recent advances made in the design, preparation, and application of caged peptides and proteins.

Biological Availability↗

PubMed1998

Flash decaging of tyrosine sidechains in an ion channel.

A nonsense codon suppression technique was employed to incorporate ortho-nitrobenzyl tyrosine, "caged tyrosine," in place of tyrosine at any of three positions (93, 127, or 198) in the alpha subunit of the muscle nicotinic ACh receptor (nAChR) expressed in Xenopus oocytes. The ortho-nitrobenzyl group was then removed by 1 ms flashes at 300-350 nm to yield tyrosine itself while macroscopic currents were recorded during steady ACh exposure. Responses to multiple flashes showed (1) that each flash decages up to 17% of the tyrosines and (2) that two tyrosines must be decaged per receptor for a response. The conductance relaxations showed multiple kinetic components; rate constants (<0.1 s(-1) to 10(3) s(-1)) depended on pH and the site of incorporation, and relative amplitudes depended on the number of prior flashes. This method, which is potentially quite general, (1) provides a time-resolved assay for the behavior of a protein when a mutant sidechain is abruptly changed to the wild-type residue and (2) will also allow for selective decaging of sidechains that are candidates for covalent modification (such as phosphorylation) in specific proteins in intact cells.

Acetylcholine↗

PubMed1999

Coupling of transcription and translation in Dictyostelium discoideum nuclei.

The nuclei of Dictyostelium discoideum cells have been found to contain polyribosomes active in protein synthesis. mRNA molecules enter nuclear polyribosomes while they are still being synthesized. "Non sense mediated mRNA decay" occurs in the nucleus, through the interaction of the mRNAs containing a nonsense codon with newly formed nuclear ribosomes, rather than with cytoplasmic ribosomes, as previously generally supposed.

Animals↗

PubMed2004

Epigenetic regulation of translation reveals hidden genetic variation to produce complex traits.

Phenotypic plasticity and the exposure of hidden genetic variation both affect the survival and evolution of new traits, but their contributing molecular mechanisms are largely unknown. A single factor, the yeast prion [PSI(+)], may exert a profound effect on both. [PSI(+)] is a conserved, protein-based genetic element that is formed by a change in the conformation and function of the translation termination factor Sup35p, and is transmitted from mother to progeny. Curing cells of [PSI(+)] alters their survival in different growth conditions and produces a spectrum of phenotypes in different genetic backgrounds. Here we show, by examining three plausible explanations for this phenotypic diversity, that all traits tested involved [PSI(+)]-mediated read-through of nonsense codons. Notably, the phenotypes analysed were genetically complex, and genetic re-assortment frequently converted [PSI(+)]-dependent phenotypes to stable traits that persisted in the absence of [PSI(+)]. Thus, [PSI(+)] provides a temporary survival advantage under diverse conditions, increasing the likelihood that new traits will become fixed by subsequent genetic change. As an epigenetic mechanism that globally affects the relationship between genotype and phenotype, [PSI(+)] expands the conceptual framework for phenotypic plasticity, provides a one-step mechanism for the acquisition of complex traits and affords a route to the genetic assimilation of initially transient epigenetic traits.

Biological Evolution↗

PubMed2006

Recombinant expression of selectively sulfated proteins in Escherichia coli.

Although tyrosine sulfation is a post-translational modification widespread across multicellular eukaryotes, its biological functions remain largely unknown. This is in part due to the difficulties of synthesizing selectively sulfated proteins. Here we report the selective incorporation of sulfotyrosine into proteins in bacteria by genetically encoding the modified amino acid in response to the amber nonsense codon TAG. Moreover, we show that this strategy enables direct expression in Escherichia coli of sulfo-hirudin, previously inaccessible through recombinant methods. The affinity of sulfo-hirudin toward human thrombin is enhanced more than tenfold over that of desulfo-hirudin, suggesting that sulfo-hirudin may offer clinical advantages for use as an anticoagulant. This general approach to the biosynthesis of sulfated proteins should facilitate further study and application of tyrosine sulfation.

Amino Acyl-tRNA Synthetases↗

PubMed2006

Efficient incorporation of unnatural amino acids into proteins in Escherichia coli.

We have developed a single-plasmid system for the efficient bacterial expression of mutant proteins containing unnatural amino acids at specific sites designated by amber nonsense codons. In this system, multiple copies of a gene encoding an amber suppressor tRNA derived from a Methanocaldococcus jannaschii tyrosyl-tRNA (MjtRNATyrCUA) are expressed under control of the proK promoter and terminator, and a gene encoding the desired mutant M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS) is expressed under control of a mutant glnS (glnS') promoter.

Amino Acids↗

PubMed2002

SLC7A9 mutations in all three cystinuria subtypes.

BACKGROUND: Cystinuria is an inherited disorder of cystine and dibasic amino acid transport in kidney. Subtypes are defined by the urinary cystine excretion patterns of the obligate heterozygous parents: Type I/N (fully recessive or silent); Type II/N (high excretor); Type III/N (moderate excretor). The first gene implicated in cystinuria (SLC3A1) is associated with the Type I urinary phenotype. A second cystinuria gene (SLC7A9) was recently isolated, and mutations of this gene were associated with dominant (non-Type I) cystinuria alleles. Here we report genotype-phenotype studies of SLC7A9 mutations in a cohort of well-characterized cystinuria probands and their family members. METHODS: Individual exons of the SLC7A9 gene were screened by single strand conformation polymorphism (SSCP) analysis and sequencing of abnormally migrating fragments. RESULTS: Seven mutations were identified. A single bp insertion (799insA) was present in four patients: on Type III alleles in two patients and on Type II alleles in two patients. These results suggest that Type II and Type III may be caused by the same mutation and, therefore, other factors must influence urinary cystine excretion. A 4bp deletion in intron 12 (IVS12+4delAGTA) and a missense mutation (1245G-->A, A354T) were identified on Type III alleles. A nonsense codon (1491G-->T, E436X) and a possible splicing mutation (IVS9-17G-->A) were seen in a Type I/III patient, but the mutations could not be assigned to particular alleles. Of additional interest were two missense mutations (316T-->C, I44T and 967C-->T, P261L) linked to Type I alleles. CONCLUSION: Our results provide evidence that some SLC7A9 mutations may be associated with fully recessive (Type I) forms of cystinuria. We also demonstrate SLC7A9 mutations in dominant Types II and III cystinuria. The finding of SLC7A9 mutations in all three subtypes underscores the complex interactions between specific cystinuria genes and other factors influencing cystine excretion. A simpler phenotypic classification scheme (recessive and dominant) for cystinuria is warranted.

Adult↗

PubMed2006

A genetically encoded fluorescent amino acid.

The ability to introduce fluorophores selectively into proteins provides a powerful tool to study protein structure, dynamics, localization, and biomolecular interactions both in vitro and in vivo. Here, we report a strategy for the selective and efficient biosynthetic incorporation of a low-molecular-weight fluorophore into proteins at defined sites. The fluorescent amino acid 2-amino-3-(5-(dimethylamino)naphthalene-1-sulfonamide)propanoic acid (dansylalanine) was genetically encoded in Saccharomyces cerevisiae by using an amber nonsense codon and corresponding orthogonal tRNA/aminoacyl-tRNA synthetase pair. This environmentally sensitive fluorophore was selectively introduced into human superoxide dismutase and used to monitor unfolding of the protein in the presence of guanidinium chloride. The strategy described here should be applicable to a number of different fluorophores in both prokaryotic and eukaryotic organisms, and it should facilitate both biochemical and cellular studies of protein structure and function.

Alanine↗

PubMed2003

A functional interaction between ribosomal proteins S7 and S11 within the bacterial ribosome.

In this study, we used site-directed mutagenesis to disrupt an interaction that had been detected between ribosomal proteins S7 and S11 in the crystal structure of the bacterial 30 S subunit. This interaction, which is located in the E site, connects the head of the 30 S subunit to the platform and is involved in the formation of the exit channel through which passes the 30 S-bound messenger RNA. Neither mutations in S7 nor mutations in S11 prevented the incorporation of the proteins into the 30 S subunits but they perturbed the function of the ribosome. In vivo assays showed that ribosomes with either mutated S7 or S11 were altered in the control of translational fidelity, having an increased capacity for frameshifting, readthrough of a nonsense codon and codon misreading. Toeprinting and filter-binding assays showed that 30 S subunits with either mutated S7 or S11 have an enhanced capacity to bind mRNA. The effects of the S7 and S11 mutations can be related to an increased flexibility of the head of the 30 S, to an opening of the mRNA exit channel and to a perturbation of the proposed allosteric coupling between the A and E sites. Altogether, our results demonstrate that S7 and S11 interact in a functional manner and support the notion that protein-protein interactions contribute to the dynamics of the ribosome.

Amino Acid Sequence↗

PubMed2004

Rare autosomal recessive cardiac valvular form of Ehlers-Danlos syndrome results from mutations in the COL1A2 gene that activate the nonsense-mediated RNA decay pathway.

Splice site mutations in the COL1A2 gene of type I collagen can give rise to forms of Ehlers-Danlos syndrome (EDS) because of partial or complete skipping of exon 6, as well as to mild, moderate, or lethal forms of osteogenesis imperfecta as a consequence of skipping of other exons. We identified three unrelated individuals with a rare recessively inherited form of EDS (characterized by joint hypermobility, skin hyperextensibility, and cardiac valvular defects); in two of them, COL1A2 messenger RNA (mRNA) instability results from compound heterozygosity for splice site mutations in the COL1A2 gene, and, in the third, it results from homozygosity for a nonsense codon. The splice site mutations led to use of cryptic splice donor sites, creation of a downstream premature termination codon, and extremely unstable mRNA. In the wild-type allele, the two introns (IVS11 and IVS24) in which these mutations occurred were usually spliced slowly in relation to their respective immediate upstream introns. In the mutant alleles, the upstream intron was removed, so that exon skipping could not occur. In the context of the mutation in IVS24, computer-generated folding of a short stretch of mRNA surrounding the mutation site demonstrated realignment of the relationships between the donor and acceptor sites that could facilitate use of a cryptic donor site. These findings suggest that the order of intron removal is an important variable in prediction of mutation outcome at splice sites and that folding of the nascent mRNA could be one element that contributes to determination of order of splicing. The complete absence of pro alpha 2(I) chains has the surprising effect of producing cardiac valvular disease without bone involvement.

Adult↗

PubMed1997

Extensive RNA editing and possible double-stranded structures determining editing sites in the atpB transcripts of hornwort chloroplasts.

Three nonsense codons and an unusual initiation codon were located within the putative coding region of the atpB gene of chloroplast DNA of the hornwort Anthoceros formosae. Nucleotide sequencing of cDNA prepared from transcripts revealed extensive RNA editing. The unusual initiation codon ACG was changed to AUG and three nonsense codons were converted into sense codons. In total 15 C residues of the genomic DNA were replaced by U residues in the mRNA sequences, while 14 U residues were replaced by C residues. This is the highest number of editing events for a chloroplast mRNA reported so far. Partial editing was also shown in a cDNA clone where 23 sites were edited but six sites remained unedited, representing the existence of premature mRNA. The expected two-dimensional structure of the mRNA shows the existence of a sequence complementary to every editing site, which can produce continuous base pairing longer than 5 bp, suggesting that mispairing in the double strand is the site determinant for RNA editing in Anthoceros chloroplasts. Comparison of the cDNA sequence with other chloroplast genes suggests that the mechanism arose in the first land plants and has been reduced during evolution.

Amino Acid Sequence↗