Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Stop codon”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,027 records · Page 57Linked to original sources
PubMed1997

Expression in BALB/c and C57BL/6 mice of Rt6-1 and Rt6-2 ADP-ribosyltransferases that differ in enzymatic activity: C57BL/6 Rt6-1 is a natural transferase knockout.

Several proteins with NAD+:arginine ADP-ribosyltransferase (ART) activity are expressed in T cells and affect their function. Rat T cells that express the ART designated RT6 are determinants of the expression of autoimmune diabetes. In the mouse, a 35-kDa ecto-ART modulates the proliferation and functional activity of CTL. Here we report on mouse ARTs designated Rt6-1 and Rt6-2 in BALB/c and C57BL/6 mice. mRNAs for Rt6-1 and Rt6-2 were found in spleen, thymus, and intestinal tissue of both strains, but Rt6-1 mRNA in C57BL/6 mice was detected only at low levels. Rt6-1 and Rt6-2 cDNAs from both strains were cloned and sequenced. Predicted amino acid sequences of Rt6-2 were identical in both strains, but there was an in-frame stop codon in the sequence of Rt6-1 in C57BL/6 mice not present in BALB/c mice. Recombinant C57BL/6 Rt6-2 and BALB/c Rt6-1 proteins expressed in COS1 cells exhibited ART activity and were documented to be glycosylphosphatidylinositol-linked membrane proteins. COS-1 cells transfected with a C57BL/6 Rt6-1 cDNA construct expressed a truncated protein consistent in size with that predicted by the presence of the stop codon. This approximately 21-kDa protein appeared not to be glycosylphosphatidylinositol linked to the cell surface and lacked ART activity. C57BL/6 Rt6-1 therefore appears to be a naturally occurring ART knockout. The expression of Rt6-1 and Rt6-2 mRNAs in lymphoid tissues suggests that these ARTs may regulate immune system functions. Expression of Rt6-2 or another redundant ART may compensate for the lack of enzymatically active Rt6-1 in C57BL/6 mice.

ADP Ribose Transferases↗
PubMed1994

Nonsense-mediated mRNA decay in Xenopus oocytes and embryos.

Mutant mRNAs carrying a premature stop codon have a reduced half-life in the cells of many species, probably due to the presence of "surveillance" pathways, which selectively target such mRNAs for degradation. It is reported here that this phenomenon may also occur in Xenopus. In vitro-synthesised transcripts encoding a Xenopus POU-domain protein, XLPOU-60, are stable after injection into the oocyte and embryo. However, introduction of a premature stop codon into these transcripts results in their rapid degradation following injection. In contrast, mutant transcripts with additional or deleted codons but retaining a correct reading frame are stable. These results suggest that RNA stability should be considered when designing control mRNAs for Xenopus injection experiments.

Animals↗
PubMed1999

Wild-type and mutant alleles of the Aspergillus nidulans developmental regulator gene brlA: correlation of variant sites with protein function.

The DNA sequences of two wild-type and eleven mutant alleles of the developmental regulator gene brlA from Aspergillus nidulans, which encodes a zinc-finger protein, were characterized. Variant sites were located on rescued plasmids or PCR products based either on their meiotic map position or the use of denaturing gradient gel electrophoresis. Mutations in three null mutants, one of which is partially suppressible, encode premature stop codons. Two environmentally sensitive mutants were characterised by substitution of leucines required for stabilisation of alpha-helices in each of the two putative zinc-finger domains. A third zinc-finger substitution is predicted to disrupt recognition of a guanine residue in the DNA target. The mutations in four other leaky mutants map C-terminal to the zinc fingers; one minimally leaky mutant has a premature stop codon, which results in the removal of the last 38 residues of the protein product.

Alleles↗
PubMed2006

Cryo-EM visualization of transfer messenger RNA with two SmpBs in a stalled ribosome.

In eubacterial translation, lack of a stop codon on the mRNA results in a defective, potentially toxic polypeptide stalled on the ribosome. Bacteria possess a specialized mRNA, called transfer messenger RNA (tmRNA), to rescue such a stalled system. tmRNA contains a transfer RNA (tRNA)-like domain (TLD), which enters the ribosome as a tRNA and places an ORF into the mRNA channel. This ORF codes for a signal marking the polypeptide for degradation and ends in a stop codon, leading to release of the faulty polypeptide and recycling of the ribosome. The binding of tmRNA to the stalled ribosome is mediated by small protein B (SmpB). By means of cryo-EM, we obtained a density map for the preaccommodated state of the tmRNA.SmpB.EF-Tu.70S ribosome complex with much improved definition for the tmRNA-SmpB complex, showing two SmpB molecules bound per ribosome, one toward the A site on the 30S subunit side and the other bound to the 50S subunit near the GTPase-associated center. tmRNA is strongly attached to the 30S subunit head by multiple contact sites, involving most of its pseudoknots and helices. The map clarifies that the TLD is located near helix 34 and protein S19 of the 30S subunit, rather than in the A site as tRNA for normal translation, so that the TLD is oriented toward the ORF.

Base Sequence↗
PubMed1991

Amber codon suppression: the in vivo and in vitro analysis of two C-hordein genes from barley.

A 1420 bp genomic fragment (lambda-hor1-17) encompassing a Hor-1 gene encoding a C-hordein polypeptide is presented. The deduced amino acid sequence is 261 residues long. It comprises a 20 amino acid signal peptide, unique NH2- and COOH-terminal regions and a coding region comprised of pentapeptide (PQQPY) and octapeptide (PQQPFPQQ) repeat motifs. The 431 bp of 5' non-coding region contains a 'TATA box' at -105, a 'CACA box' (-181 to -201) and a -300 prolamin element. In the 3' non-coding region there are two putative polyadenylation signals located 88 and 142 bp downstream of the stop codon. The structure of lambda-hor1-17 is compared with that of another gene (lambda-hor1-14) encoding a C-hordein polypeptide, which contains an amber codon interrupting the ORF. A functional assay in which the 5' non-coding regions of the two genes were fused to the beta-glucuronidase (GUS) gene demonstrated that both genes were transcriptionally active and that circa 430 bp of the C-hordein promoters were sufficient to drive the expression of the GUS gene in developing barley endosperms. It also demonstrated that both promoters had transcriptional efficiencies comparable with that of the 35S CaMV promoter. The in vitro translation of the coding region of lambda-hor1-14 in the wheat germ system showed that the premature stop codon could be partially suppressed. The suppression was also demonstrated in a transient expression assay in vivo using isolated barley endosperms.

Amino Acid Sequence↗
PubMed1995

A reporter gene to analyse the hypermutation of immunoglobulin genes.

The affinity maturation of antibodies is driven by somatic hypermutation which is localized to specific segments of the coding genes. The information available on this process derives from studies in vivo. With the intention of developing new approaches, we have constructed a fusion gene between a kappa chain and a selectable neomycin resistance gene, neor. The neor gene, which includes the SV40 small t intron and polyadenylation site, but not the upstream elements nor its first 12 amino acids, is an in-frame substitution of the FR2-CDR3 fragment of a rearranged V kappa OX1-J kappa 5 gene. Expression of neor activity is therefore dependent on the upstream immunoglobulin sequence. A stop codon was placed in the CDR1 region so that only mutants survive treatment with geneticin sulphate (G418). The effectiveness of the system was tested by transfecting the NS0 myeloma cell line and isolating spontaneous mutants. Neomycin-resistant clones arose at an estimated rate of 1 x 10(-8)/cell division, and over 90% were authentic structural mutants. Unlike the somatic hypermutations, the majority arose by in-frame deletions including the stop codon, although up to 30% involved a point mutation. The reporter gene was then modified by substituting all the sequences downstream of the J kappa 5 with others known to be required for full hypermutation in vivo. Different cell lines were transfected and G418-resistant clones analyzed. No significant increase in the rate of reversion or in the generation of point mutations versus deletions was detected, even using conditioned culture medium. In the presence of azacytidine however, a mutant involving multiple events (single base addition and deletion plus two point mutations) was detected. The reporter gene system therefore seems suitable to test culture conditions and modifications of the host cells aimed at the derivation of an in vitro assay of somatic hypermutation.

Amino Acid Sequence↗
PubMed1992

Three genes for the human high affinity Fc receptor for IgG (Fc gamma RI) encode four distinct transcription products.

Three distinct but closely related classes of receptors that bind the Fc portion of immunoglobulin G (Fc gamma RI, -II, and -III) have been identified in humans. Only Fc gamma RI has high affinity for ligand and has a unique third extracellular domain (EC3). We have characterized three genes for human Fc gamma RI (A, B, and C). Each gene consists of six exons, spans 9.4 kilobase pairs, and localizes to chromosome 1. Although they are remarkably similar, genes B and C are notably different from A; in-frame stop codons are present in the EC3 domain of genes B and C, and deletions occur in a splice donor sequence of gene B. Four distinct Fc gamma RI transcripts were analyzed. One transcript, from gene A, would encode a transmembrane receptor with three external domains. A second transcript, an alternatively spliced product of gene B, would encode a two-external domain transmembrane receptor. Two transcripts, from genes B and C, have stop codons in EC3 and would be predicted to generate secreted receptors.

Amino Acid Sequence↗
PubMed2001

Translation at higher than an optimal level interferes with coupling at an intercistronic junction.

In pairs of adjacent genes co-transcribed on bacterial polycistronic mRNAs, translation of the first coding region frequently functions as a positive factor to couple translation to the distal coding region. Coupling efficiencies vary over a wide range, but synthesis of both gene products at similar levels is common. We report the results of characterizing an unusual gene pair, in which only about 1% of the translational activity from the upstream gene is transmitted to the distal gene. The inefficient coupling was unexpected because the upstream gene is highly translated, the distal initiation site has weak but intrinsic ability to bind ribosomes, and the AUG is only two nucleotides beyond the stop codon for the upstream gene. The genes are those in the filamentous phage IKe genome, which encode the abundant single-stranded DNA binding protein (gene V) and the minor coat protein that caps one tip of the phage (gene VII). Here, we have used chimeras between the related phage IKe and f1 sequences to localize the region responsible for inefficient coupling. It mapped upstream from the intercistronic region containing the gene V stop codon and the gene VII initiation site, indicating that low coupling efficiency is associated with gene V. The basis for inefficient coupling emerged when coupling efficiency was found to increase as gene V translation was decreased below the high wild-type level. This was achieved by lowering the rate of elongation and by decreasing the efficiency of suppression at an amber codon within the gene. Increasing the strength of the Shine-Dalgarno interaction with 16S rRNA at the gene VII start also increased coupling efficiency substantially. In this gene pair, upstream translation thus functions in an unprecedented way as a negative factor to limit downstream expression. We interpret the results as evidence that translation in excess of an optimal level in an upstream gene interferes with coupling in the intercistronic junction.

Bacteriophage IKe↗
PubMed1997

Mosaic hepatitis B virus core particles allow insertion of extended foreign protein segments.

Because of its particular immunological properties, the core protein of hepatitis B virus (HBcAg) has become one of the favoured 'virus-like particles' for use as a carrier of foreign epitopes. A new strategy to construct core particles presenting extended foreign protein segments was established based on the introduction of a linker containing a translational stop codon between sequences encoding a C-terminally truncated HBcAg (HBcAg delta) and a foreign protein sequence. Expression in an Escherichia coli suppressor strain allowed the simultaneous synthesis of both HBcAg delta and a read-through fusion protein containing a part of the hantavirus nucleocapsid protein. After purification, the presence of core-like mosaic particles with HBc and hantavirus antigenicity was demonstrated by electron microscopy and immunological tests. This strategy of partial stop codon suppression should improve the use of HBcAg as a carrier of foreign epitopes by allowing insertion of long foreign sequences into particle-forming proteins. The resulting mosaic particles should be of general interest for further vaccine developments.

Amino Acid Sequence↗
PubMed1990

Expression of the amino-terminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein.

A human liver cDNA library was screened with a synthetic oligonucleotide, complementary to the 5' region of human transferrin mRNA, as a hybridization probe. The full-length human cDNA clone isolated from this screen contained part of the 5' untranslated region, the complete coding region for the signal peptide and the two lobes of transferrin, the 3' untranslated region, and a poly(A) tail. By use of oligonucleotide-directed mutagenesis in vitro, two translational stop codons and a HindIII site were introduced after the codon for Asp-337. This fragment was inserted into two different expression vectors that were then introduced into Escherichia coli. As judged by NaDodSO4-polyacrylamide gel electrophoresis and Western blot analysis, however, recombinant hTF/2N was undetectable in bacteria transformed by these plasmids. Concurrently, we developed a plasmid vector for the expression of recombinant hTF/2N in eukaryotic cells. In this case, a DNA fragment coding for the natural signal sequence, the hTF/2N lobe, and the two stop codons was cloned into the expression vector pNUT, such that the expression of hTF/2N was controlled by the mouse metallothionein promoter and the human growth hormone termination sequences. Baby hamster kidney cells containing this hTF/2N-pNUT plasmid secreted up to 20 mg of recombinant hTF/2N per liter of tissue culture medium. Recombinant hTF/2N was purified from the medium by successive chromatography steps on DEAE-Sephacel, Sephadex G-75, and FPLC on Polyanion SI. The purified protein was characterized by NaDodSO4-PAGE, urea-PAGE, amino-terminal sequence analysis, UV-visible spectroscopy, iron-binding titration, and proton NMR.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2002

Mapping functionally important motifs SPF and GGQ of the decoding release factor RF2 to the Escherichia coli ribosome by hydroxyl radical footprinting. Implications for macromolecular mimicry and structural changes in RF2.

The function of the decoding release factor (RF) in translation termination is to couple cognate recognition of the stop codon in the mRNA with hydrolysis of the completed polypeptide from its covalently linked tRNA. For this to occur, the RF must interact with specific A-site components of the active centers within both the small and large ribosomal subunits. In this work, we have used directed hydroxyl radical footprinting to map the ribosomal binding site of the Escherichia coli class I release factor RF2, during translation termination. In the presence of the cognate UGA stop codon, residues flanking the universally conserved (250)GGQ(252) motif of RF2 were each shown to footprint to the large ribosomal subunit, specifically to conserved elements of the peptidyltransferase and GTPase-associated centers. In contrast, residues that flank the putative "peptide anticodon" of RF2, (205)SPF(207), were shown to make a footprint in the small ribosomal subunit at positions within well characterized 16 S rRNA motifs in the vicinity of the decoding center. Within the recently solved crystal structure of E. coli RF2, the GGQ and SPF motifs are separated by 23 A only, a distance that is incompatible with the observed cleavage sites that are up to 100 A apart. Our data suggest that RF2 may undergo gross conformational changes upon ribosome binding, the implications of which are discussed in terms of the mechanism of RF-mediated termination.

Amino Acid Motifs↗
PubMed1998

Skipper, an LTR retrotransposon of Dictyostelium.

The complete sequence of a retrotransposon from Dictyostelium discoideum , named skipper , was obtained from cDNA and genomic clones. The sequence of a nearly full-length skipper cDNA was similar to that of three other partially sequenced cDNAs. The corresponding retrotransposon is represented in approximately 15-20 copies and is abundantly transcribed. Skipper contains three open reading frames (ORFs) with an unusual sequence organization, aspects of which resemble certain mammalian retroviruses. ORFs 1 and 3 correspond to gag and pol genes; the second ORF, pro, corresponding to protease, was separated from gag by a single stop codon followed shortly thereafter by a potential pseudoknot. ORF3 (pol) was separated from pro by a +1 frameshift. ORFs 2 and 3 overlapped by 32 bp. The computed amino acid sequences of the skipper ORFs contain regions resembling retrotransposon polyprotein domains, including a nucleic acid binding protein, aspartyl protease, reverse transcriptase and integrase. Skipper is the first example of a retrotransposon with a separate pro gene. Skipper is also novel in that it appears to use stop codon suppression rather than frameshifting to modulate pro expression. Finally, skipper and its components may provide useful tools for the genetic characterization of Dictyostelium.

Amino Acid Sequence↗
PubMed2000

Sequence analysis of human endogenous retrovirus clone 4-1 in systemic lupus erythematosus.

Human endogenous retroviruses (HERV) have emerged as a possible cause of systemic lupus erythematosus (SLE). We previously detected serum antibodies to the gag region of HERV clone 4-1 in patients with SLE, but not in normal volunteers. In the present study, we detected clone 4-1 messenger RNA (mRNA) in peripheral blood mononuclear cells (PBMC) from SLE patients and performed sequence analysis of the cDNA or genomic DNA from clone 4-1 in these patients. Clone 4-1 mRNA was detected in all of the SLE patients tested, although it was not found in normal controls. Sequence analysis of clone 4-1 in these SLE patients revealed inactivation of the stop codons in part of the gag region. In addition, a computer search of current sequence libraries revealed that the clone 4-1 gag genomic DNA from SLE patients was more highly homologous with the clone 4-1 sequence in chromosome 11 from normal individuals when compared with the sequence of clone 4-1 integrated in the other chromosomes. It is possible that transcription of clone 4-1 from chromosome 11 occurs in SLE, and that the stop codon inactivation contributes to the translation of clone 4-1 gag proteins in patients with this disease.

Adult↗
PubMed2004

Dial tm for rescue: tmRNA engages ribosomes stalled on defective mRNAs.

Ribosomes translate genetic information encoded by mRNAs into protein chains with high fidelity. Truncated mRNAs lacking a stop codon will cause the synthesis of incomplete peptide chains and stall translating ribosomes. In bacteria, a ribonucleoprotein complex composed of tmRNA, a molecule that combines the functions of tRNAs and mRNAs, and small protein B (SmpB) rescues stalled ribosomes. The SmpB-tmRNA complex binds to the stalled ribosome, allowing translation to resume at a short internal tmRNA open reading frame that encodes a protein degradation tag. The aberrant protein is released when the ribosome reaches the stop codon at the end of the tmRNA open reading frame and the fused peptide tag targets it for degradation by cellular proteases. The recently determined NMR structures of SmpB, the crystal structure of the SmpB-tmRNA complex and the cryo-EM structure of the SmpB-tmRNA-EF-Tu-ribosome complex have provided first detailed insights into the intricate mechanisms involved in ribosome rescue.

Alanine↗
PubMed2003

Spontaneous mutations of the flavonoid 3'-hydroxylase gene conferring reddish flowers in the three morning glory species.

Among the Ipomoea plants, both Ipomoea nil and Ipomoea tricolor display bright blue flowers, and Ipomoea purpurea exhibits dark purple flowers. While all of these flowers contain cyanidin-based anthocyanin pigments, the mutants of I. nil, I. purpurea, and I. tricolor carrying the magenta, pink, and fuchsia alleles, respectively, produce reddish flowers containing pelargonidin derivatives, and all of them are deficient in the gene for flavonoid 3'-hydroxylase (F3'H). The magenta allele in I. nil is a nonsense mutation caused by a single C to T base transition generating the stop codon TGA, and the cultivar Violet carries the same mutation. Several tested pink mutants in I. purpurea carry inserts of the 0.55-kb DNA transposable element Tip201 belonging to the Ac/Ds superfamily at the identical site. No excision of Tip201 from the F3'H gene could be detected, and both splicing and polyadenylation patterns of the F3'H transcripts were affected by the Tip201 integration. The fuchsia allele in I. tricolor is a single T insertion generating the stop codon TAG, and the accumulation of the F3'H transcripts was drastically reduced by the nonsense-mediated RNA decay. Spontaneous mutations in Ipomoea, including a possible founder mutation in the pink allele, are also discussed.

Amino Acid Sequence↗
PubMed2002

A novel nonsense mutation at E106 of the 2B rod domain of keratin 14 causes dominant epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) is classified into three main types and is caused, in most cases, by missense mutations in the genes encoding keratin (K) 5 and K14. In this study, we clinically, ultrastructurally, immunohistochemically, and molecularly studied a patient with a dominant EBS, Köbner type. Using sequence analysis of genomic DNA, a novel K14 nonsense mutation was identified. A heterozygous mutation G1231T of KRT14 was found to be associated with the disease in the patient. The mutation created a premature stop codon (amino acid codon 411, residue 106 of the 2B helix) in the K14 molecule. This residue lies in a highly conserved region and was recently found to be absolutely required for molecular stability and intermediate filament assembly in K5 and K14. The E411X (E106X) heterozygous ablation, missing the last 16 amino acid residues of the 2B and the entire tail domain of K14, led to disease but did not result in clumping of keratin filaments. It is the first premature stop codon mutation of K14 found in dominant EBS.

Adult↗
PubMed1998

A ribosomal function is necessary for efficient splicing of the T4 phage thymidylate synthase intron in vivo.

Splicing of the group I intron of the T4 thymidylate synthase (td) gene was uncoupled from translation by introducing stop codons in the upstream exon. This resulted in severe splicing deficiency in vivo. Overexpression of a UGA suppressor tRNA partially rescued splicing, suggesting that this in vitro self-splicing intron requires translation for splicing in vivo. Inhibition of translation by the antibiotics chloramphenicol and spectinomycin also resulted in splicing deficiency. Ribosomal protein S12, a protein with RNA chaperone activity, and CYT-18, a protein that stabilizes the three-dimensional structure of group I introns, efficiently rescued the stop codon mutants. We identified a region in the upstream exon that interferes with splicing. Point mutations in this region efficiently alleviate the effect of a nonsense codon. We infer from these results that the ribosome acts as an RNA chaperone to facilitate proper folding of the intron.

Bacteriophage T4↗
PubMed2000

Sequence variations of precore/core and precore promoter regions of hepatitis B virus in patients with or without viral reactivation during cytotoxic chemotherapy.

Reactivation of the hepatitis B virus (HBV) is a well-described complication among cancer patients undergoing cytotoxic chemotherapy. Mutations in the preC/C and the preC promoter regions of HBV have been reported in some patients who developed this condition. A G-to-A mutation at nt 1896 in the preC/C region (HBeAg negative/ anti-HBe positive) has been associated with more severe liver disease than that caused by wild type virus. In addition, it has been suggested that patients with these mutations may be more likely to reactivate than those with the wild type virus. Whether or not such mutations were present before the commencement of or developed during the course of cytotoxic chemotherapy is not known. In this study, 28 cancer patients (consisting of 14 consecutive patients who developed HBV reactivation and another 14 who had no reactivation during cytotoxic chemotherapy) are reported. The objectives were firstly, to determine the prechemotherapy HBeAg status and nucleotide sequences of the preC/C and preC promoter regions of HBV in order to determine if these parameters affected the rate of reactivation, and secondly, for those who developed reactivation, to determine whether the mutations were present before chemotherapy or developed during, possibly as a result of, cytotoxic chemotherapy. HBV DNA was amplified by PCR and nucleotide sequencing performed on samples taken prior to chemotherapy and at the time of reactivation. Results revealed that 16 of the 28 patients were HBeAg negative/anti-HBe positive. Of these 16, four (57%) of the seven patients who had nt 1896 mutation, but only one (17%) of the six who had the wild type HBV genome, developed reactivation. Three had no detectable HBV DNA. In the majority of cases, the type of virus, i.e. wild/mutant at preC/C, that was detected during the reactivation was identical to that detected in the pretreatment samples. With respect to the preC promoter region, the two commonest mutations detected were at nt 1762 (A to T) and nt 1764 (G to A). When this region was translated into amino acid sequences, stop codons leading to truncated X protein at carboxyl terminus were found in four patients, three of whom developed HBV reactivation. We conclude that chronic HBV carriers who are HBeAg negative/anti-HBe positive with nt 1896 mutation (G to A) may be more likely to develop HBV reactivation during cytotoxic chemotherapy than those with the wild type virus. Cytotoxic chemotherapy does not appear to select out mutant HBV, or to be consistently mutagenic in patients who develop HBV reactivation. The occurrence of stop codons in the amino acid sequences of the X protein in three patients who developed HBV reactivation, including one who was detected only at the time of reactivation, is of particular interest, as such mutant viruses remain replication competent.

Adult↗