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We examined a patient with cleidocranial dysplasia (CCD) and cleft lip and found a new stop codon mutation in CBFA1. This mutation was a heterozygous C-to-T transition in exon 3 of CBFA1. This nucleotide change converts a CAA codon to a TAA (stop) codon at amino acid position Gln195 in the runt domain of CBFA1.
We identified a novel stop codon mutation in the myophosphorylase gene in a Greek patient with typical symptoms of McArdle's disease. This is the first genetic study of myophosphorylase deficiency in a Greek family, showing that the proband was a compound heterozygous for the common "caucasian" mutation (R49X) and a new nonsense mutation (Y52X), both within exon 1. The new point mutation, a C-to-G transversion at codon 52, converts an encoded tyrosine to a stop codon. Our study confirms that the R49X is also present in the Greek population. The Y52X may represent a private mutation or a common mutation among Greeks. Our data further expand the already remarkable genetic heterogeneity of McArdle's disease. The prevalence of the Y52X mutation in Greek patients with McArdle's disease remains to be determined.
The defective major histocompatibility complex (MHC) DRB6 gene is transcribed into mRNA in human [peripheral blood lymphocytes, transfected and Epstein-Barr virus (EBV)] and chimpanzee EBV cell lines. MHC-DRB6 presents several anomalies, which include stop codons in exon 2, lack of the usual polyadenilation signal of other MHC-DRB genes, and a promoter region and exon 1 taken from a locally inserted retrovirus. The complete cDNA sequences from human DRB6*0201 and three common chimpanzee alleles (Patr-DRB6*0108, Patr-DRB6*0109, Patr-DRB6*0111) have been obtained; two exon 1-exon 2 cDNA sequences from bonobos (Papa-DRB6*0101 and Papa-DRB6*0102) are also shown. In contrast to chimpanzee DRB6 transcripts, the human ones: (1) present an exon 1-exon 2 splicing site that includes the transcription of the first 141 nucleotides of intron 1, rendering a longer exon 1, and (2) show a duplication of exon 6, which would render a longer cytoplasmic tail in a putative DRB6 protein. These two characteristics are found in all the human sequences obtained, regardless of the cellular type tested, and they are not present in any of the chimpanzee alleles reported; consequently, they are human-specific. All the alleles reported here bear stop codons in the three possible reading frames; however, a certain level of expression of DRB6 has been observed by cytofluorometry. This could be due to the presence of a selenocysteine insertion sequence (SECIS) stem-loop structure located at the 3 untranslated region of the DRB6 mRNA, which directs selenocysteine incorporation at UGA codons. DRB6 transcription and translation would be the first gene model of a readingthrough stop codon mechanism in primate MHC. It is also feasible that the DRB6 gene might generate a population of short polypeptides, bound to plasmatic membranes, having non-antigen-presenting functions or which are presented by other MHC molecules as HLA-E presents HLA-G and -B leader sequence-derived peptides.
Nonsense codon readthrough and changed translational reading frame were measured in different growth phases in E. coli. The strains used carry plasmid constructs with a translation assay reporter gene. This reporter gene contains an internal stop codon or a run of U-residues. Termination or frameshifting give rise to stable proteins that can be physically quantified on gels along with the complete protein products. Readthrough of the stop codon UGA by a nearcognate tRNA is several fold higher in active growth than in late exponential phase. In early exponential phase, about 7% of -1 frameshift at a U9 slippery sequence is detectable; upon entry to stationary phase this frameshifting increases to about 40% followed by a decrease in stationary phase. A similar increase is observed in the case of +1 reading frameshift at the U9 sequence, which increases from 13% in early exponential growth phase up to 38% at the beginning of stationary phase followed by a decrease. Thus, the levels of both stop codon readthrough and frameshifting are growth phase dependent, though not in an identical fashion.
Hepatitis B virus (HBV) strains carrying the precore stop-codon mutation (A1896) have been considered among the predisposing factors for reactivation during chemotherapy for malignancies. The role of the T1762/A1764 basic core promoter (BCP) mutations has not been fully evaluated. We aimed to record any changes in HBV serological markers after reactivation, detect the presence of A1896 and BCP mutations and evaluate the type of cytotoxic drugs involved. We retrospectively screened eight patients presenting with HBV reactivation following chemotherapy for malignancies. The chemotherapy regimens used included corticosteroids (CSs), fludarabine and cyclophosphamide/adriamycine. The INNO-LiPA HBV PreCore kit was used for the detection of the A1896 and BCP mutations. Six patients who were hepatitis B surface antigen (HBsAg)-(+)/hepatitis B e antigen (HBeAg)-(-) before chemotherapy, had disease reactivation following a mean of four cycles of chemotherapy. Four survived and two died of hepatic failure. At the time of reactivation, all six patients carried the A1896 and five of them the BCP mutations. The remaining two patients were HBsAg-(-)/anti-HBs-(+)/anti-hepatitis B core (HBc)-(+)/HBeAg-(-) before chemotherapy. One of them reverted to HBeAg-(+) status but remained HBsAg-(-), while the other became HBsAg-(+)/HBeAg-(+), following three and eight cycles of fludarabine treatment, respectively. The former carried the A1896 and the latter the wild-type virus. Both died from causes associated with their haematological disease. All but one of our patients with HBV reactivation during chemotherapy carried the precore stop-codon and BCP mutations. Whether this occurs more frequently in such patients than those carrying the wild-type virus needs further investigation. Fludarabine should be added to the list of drugs inducing HBV reactivation. HBV reactivation following fludarabine treatment occurred in HBsAg-(-) patients who had been anti-HBs-(+).
During protein synthesis, translational release factors catalyze the release of the polypeptide chain when a stop codon on the mRNA reaches the A site of the ribosome. The detailed mechanism of this process is currently unknown. We present here the crystal structures of the ribosome from Thermus thermophilus with RF1 and RF2 bound to their cognate stop codons, at resolutions of 5.9 Angstrom and 6.7 Angstrom, respectively. The structures reveal details of interactions of the factors with the ribosome and mRNA, including elements previously implicated in decoding and peptide release. They also shed light on conformational changes both in the factors and in the ribosome during termination. Differences seen in the interaction of RF1 and RF2 with the L11 region of the ribosome allow us to rationalize previous biochemical data. Finally, this work demonstrates the feasibility of crystallizing ribosomes with bound factors at a defined state along the translational pathway.
The human pathogen Candida albicans translates the standard leucine-CUG codon as serine. This genetic code change is mediated by a novel ser-tRNA(CAG), which induces aberrant mRNA decoding in vitro, resulting in retardation of the electrophoretic mobility of the polypeptides synthesized in its presence. These non-standard decoding events have been attributed to readthrough of the UAG and UGA stop codons encoded by the Brome Mosaic Virus RNA 4, which codes for the virion coat protein, and the rabbit globin mRNAs, respectively. In order to fully elucidate the behaviour of the C. albicans ser-tRNA(CAG) towards stop codons, we have used other cell-free translation systems and reporter genes. However, the reporter systems used encode several CUG codons, making it impossible to distinguish whether the slow migration of the polypeptides is caused by the replacement of leucines by serines at the CUG codons, readthrough, or a combination of both. Therefore, we have constructed new reporter systems lacking CUG codons and have used them to demonstrate that aberrant mRNA decoding in vitro is not a result from stop codon readthrough or any other non-standard translational event. Our data show that a single leucine to serine replacement at only one of the four CUG codons encoded by the BMV RNA-4 gene is responsible for the aberrant migration of the BMV coat protein on SDS-PAGE, suggesting that this amino acid substitution (ser for leu) significantly alters the structure of the virion coat protein. The data therefore show that the only aberrant event mediated by the ser-tRNA(CAG) is decoding of the leu-CUG codon as serine.
The genes for the capsid protein CP and the nucleic acid-binding 12K protein (pr12) of potato virus M (PVM) constitute the 3' terminal gene cluster of the PVM RNA genome. Both proteins are presumably translated from a single subgenomic RNA. We have identified two translational strategies operating in pr12 gene expression. Internal initiation at the first and the second AUG codon of the pr12 coding sequence results in the synthesis of the 12K protein. In addition the protein is produced as a CP/12K transframe protein by ribosomal frameshifting. For these studies parts of the CP and pr12 coding sequences including the putative frameshift region were introduced into an internal position of the beta-glucuronidase gene. Mutational analyses in conjunction with in vitro translation experiments identified a homopolymeric string of four adenosine nucleotides which together with a 3' flanking UGA stop codon were required for efficient frameshifting. The signal AAAAUGA is the first frameshift signal with a shifty stop codon to be analyzed in the eukaryotic system. Substitution of the four consecutive adenosine nucleotides by UUUU increased the efficiency of frameshifting, while substitution by GGGG or CCCC dramatically reduced the synthesis of the transframe protein. Also, UAA and UAG could replace the opal stop codon without effect on the frameshifting event, but mutation of UGA to the sense codon UGG inhibited transframe protein formation. These findings suggest that the mechanism of ribosomal frameshifting at the PVM signal is different from the one described by the 'simultaneous slippage' model in that only the string of four adenosine nucleotides represents the slippery sequence involved in a -1 P-site slippage.
We present a patient with an unusual course of hepatitis B e antigen (HBeAg)-negative chronic hepatitis B who had repeated reactivations of his disease progressing to cirrhosis with terminal liver failure. Each flare up presented like an acute hepatitis with very high titres of hepatitis B virus (HBV) and high inflammatory activity followed by rapid clearance of viraemia. The pre-core genome of HBV isolated from sera during 5 years of follow up was analysed. Direct sequencing of polymerase chain reaction (PCR) products derived from consecutive sera showed a rare pre-core stop-codon mutation at nucleotide (nt.) 1897 G --> A with an accompanying mutation nt. 1857 C --> T as well as a stop-codon mutation nt. 1896 G --> A. By cloning and sequencing of PCR products the mutant strain with mutation nt. 1897 was shown to predominate over viral strains with a mutation nt. 1896 during the course of disease, although the stop-codon mutation nt. 1896 in general is observed more frequently. Both mutations allow viral replication by stabilizing the encapsidation signal 'epsilon'. This allowed HBV replication at a very high level as observed during flare ups. The absence of HBeAg may be responsible for the massive cytotoxic T-cell response towards hepatocytes which might explain the rapid progression to liver cirrhosis although no, or very little, HBV replication was observed for long periods. However, there is no clear explanation as to why the nt. 1897 mutant strain overwhelmed the other virus strains.
Pre-mRNA splicing involves recognition of a consensus sequence at the 5' splice site (SS). However, only some of the many potential sites that conform to the consensus are true ones, whereas the majority remain silent and are not normally used for splicing. We noticed that in most cases the utilization of such a latent intronic 5' SS for splicing would introduce an in-frame stop codon into the resultant mRNA. This finding suggested a link between SS selection and maintenance of an ORF within the mRNA. Here we tested this idea by analyzing the splicing of pre-mRNAs in which in-frame stop codons upstream of a latent 5' SS were mutated. We found that splicing with the latent site is indeed activated by such mutations. Our findings predict the existence of a checking mechanism, as a component of the nuclear pre-mRNA splicing machine, to ensure the maintenance of an ORF. This notion is highly important for accurate gene expression, as perturbations that would lead to splicing at these latent sites are expected to introduce in-frame stop codons into the majority of mRNAs.
During a physical checkup, a 42-year-old Japanese man with liver dysfunction was diagnosed as having beta-thalassemia. Using molecular biological techniques including PCR, we investigated the chemical basis of the hematological disorder. We found that a frameshift attributable to the insertion of a thymidine into or following the TTT sequence of codon 42 transformed codon 43 (GAG) into a stop codon (TGA). We believe that this mutation has not been previously reported.
A mutation of the porphobilinogen (PBG) deaminase gene that produces the cross-reacting immunological material (CRIM)-negative type of acute intermittent porphyria (AIP) has been identified in one of 43 unrelated patients with this form of the disorder. The mutation is a C----T transition that abolishes a PstI recognition site in exon 9 of the gene and converts a codon for glutamine to a stop codon.
Expression of mycoplasma sequences in Escherichia coli is often hindered by an unusual mycoplasmal codon usage pattern: the UGA stop codon is utilized for tryptophan. This may result in the truncation of cloned proteins and may prevent the detection of products of many cloned genes. To circumvent this translation barrier, we have developed an expression system for the production of mycoplasma proteins in E. coli. The efficiency of an opal suppressor tRNA (trpT176) was augmented with other suppressor mutations (prfB3 or rrsB(SuUGA-delta C1054)) which influence termination events. System efficacy was analyzed by employing suppressor mutations in the expression of TGA-containing sequences from the P1 protein-encoding gene of Mycoplasma pneumoniae.
The efficiency of translation termination in yeast can vary several 100-fold, depending on the context around the stop codon. We performed a computer analysis designed to identify yeast open reading frames (ORFs) containing a readthrough motif surrounding the termination codon. Eight ORFs were found to display inefficient stop codon recognition, one of which, PDE2, encodes the high-affinity cAMP phosphodiesterase. We demonstrate that Pde2p stability is very impaired by the readthrough-dependent extension of the protein. A 20-fold increase in readthrough of PDE2 was observed in a [PSI+] as compared with a [psi-] strain. Consistent with this observation, an important increase in cAMP concentration was observed in suppressor backgrounds. These results provide a molecular explanation for at least some of the secondary phenotypes associated with suppressor backgrounds.
The C-module-binding factor, CMBF, is a nuclear DNA-binding protein which was originally identified through its specific binding to a promoter element within the retrotransposable element TRE5-A of Dictyostelium discoideum AX2 cells. In order to analyse putative physiological functions of CMBF for the TRE5-A-hosting D. discoideum cells, we used a novel strategy to create mutant cell lines which stably underexpressed functional CMBF. An amber (UAG) translation stop codon was introduced into the chromosomal copy of the CMBF-encoding gene (cbfA), and an amber suppressor tRNA gene was expressed in the same mutant cells. Due to the low efficiency of translation stop codon suppression in this system all recovered cell lines expressed <20 % of wild-type CMBF levels. The mutant cell lines displayed strong growth phenotypes when plated on their natural food source, bacteria. We show evidence that growth reduction was due to impaired phagocytosis of bacteria in the mutants. All obtained mutants showed a strong developmental defect which was defined by the formation of very small fruiting bodies. The strength of the developmental phenotype appeared to depend upon the residual CMBF levels maintained in the mutants. We propose that CMBF is a general transcription regulator which supports the normal expression of several genes required for the maintenance of high proliferation rates of D. discoideum amoebae as well as proper aggregation and development. Our results demonstrate that amber stop codon suppression may be a useful strategy to stably underexpress proteins whose coding genes cannot be successfully disrupted by homologous recombination.
Sixteen Danish unrelated thrombophilic families with plasma protein S deficiency of type 1 (or III) are currently under investigation in our laboratory for defects in the protein S alpha gene. The present paper describes a part of this work, which deals with the identification and phenotypical presentation of two unique mutations in exon XII of the protein S alpha gene in four of these families. The mutations were identified by SSCP screening followed by nucleotide sequence analysis or by direct nucleotide sequence analysis. The mutation found in one family (D) is a novel deletion of an A in either the codon for Gly448 (GGA) or Ile449 (ATI) resulting in a frameshift and a premature stop codon at position 454. The other mutation shared by three families (F, G and J) is a previously reported C-->T transition within a hypermutable CG dinucleotide sequence converting Arg410 (CGA) to Stop (TGA). All affected individuals are heterozygotes for their mutation and in each family the protein S genotype, the plasma protein S phenotype (not shown for Family J) and the clinical phenotype cosegregate. The two mutations can fully explain the abnormal protein S phenotype since premature stop codons are known to disrupt gene function of the mutated allele. Analysis of protein S mRNA from platelets showed that both mutations result in a marked reduction in the amount of protein S mRNA from the mutated alleles indicating that the mutations exert their deleterious effects on gene expression at the transcriptional level. The Arg410-->Stop mutation in Families F, G and J is in all instances linked to a G at the site of a common neutral dimorphism in the codon for Pro626 (CCA/G) in exon XV. This indicates that the mutation in these families could have arisen in a common ancestor. The Arg410 (CGA)-->Stop (TGA) mutation is also seen in exon XII of the normal protein S alpha gene. This gives rise to the speculation as to whether the mutation in the protein S alpha gene is the result of an interaction with the protein S beta gene leading to double homologous unequal crossing-over or gene conversion of a short DNA sequence. However, this is unlikely since none of the 7 other protein S beta-specific nucleotides are present in the mutated exon XII sequence of the protein S alpha gene. The common Arg506-->Gln Leiden mutation in coagulation factor V is not an additional risk factor for thrombosis in any of the four families studied.
The mRNAs of the nad6 and ccmC genes of Arabidopsis and cauliflower were found to be processed upstream of the inframe stop codons. This result was confirmed by northern hybridization and by RT-PCR. There is no evidence that an alternative stop codon is created post-transcriptionally, either by RNA editing or by polyadenylation. The non-stop mRNAs are found in the high molecular weight polysomal fractions, suggesting that they are translated. Using antibodies directed against CcmC, the corresponding protein was detected in Arabidopsis mitochondrial extracts. These observations raise the question of how the plant mitochondrial translation system deals with non-stop mRNAs.
BACKGROUND: Prokaryotic translational release factors, RF1 and RF2, catalyse protein release at UAG/UAA and UGA/UAA stop codons, respectively. Mutations in RF1 and RF2 are known to cause non-sense suppression for UAG (amber) and UGA (opal) codons, respectively, and they do not exert a reciprocal ('cross') suppression phenotype. We aimed to isolate RF mutants of such cross-suppression activity, which we designated 'Csu' phenotype in this paper. RESULTS: Using a lacZ (UAG) reporter, we selected amber suppressor alleles occurring in the plasmid-bearing RF2 gene of Salmonella typhimurium. Of nine such RF2 csu alleles, five were mis-sense mutations and four were non-sense mutations. The former mis-sense mutants retained the RF2 activity and catalysed UGA termination both in vivo and in vitro. RF2 C-terminal deletions equivalent to the non-sense alleles exerted amber suppression as well as opal suppression activity. Moreover, the equivalent RF1 segments also showed both the suppression phenotypes. CONCLUSIONS: All the csu mutations were mapped at the C-terminal half of RF2 and are strikingly coincident with the highly conservative amino acids, suggesting that they affect the conserved function of bacterial RFs. We propose here that there should be an 'initial binding' step of RFs to the ribosome, preceding stop codon recognition ('initial binding' hypothesis) and that the N-terminal RF domain(s), that are truncated or affected by the csu mutations, are responsible for this step and interfere with the proper functioning of cognate release factors on the ribosome.