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At least 181 records · Page 10Linked to original sources
PubMed2005

Increased prevalence of the BRCA2 polymorphic stop codon K3326X among individuals with familial pancreatic cancer.

Germline BRCA2 mutations predispose to the development of pancreatic cancer. A polymorphic stop codon in the coding region of BRCA2 (K3326X) has been described, and although an initial epidemiological study suggested it was not disease causing, subsequent studies have been inconclusive. To investigate the biological significance of the K3326X polymorphism, we determined its prevalence in patients with sporadic and familial pancreatic cancer. Using a case-control design, we studied 250 patients with resected sporadic pancreatic adenocarcinomas, 144 patients with familial pancreatic adenocarcinoma, 115 spouses of patients with pancreatic cancer, and a disease control group of 135 patients without a personal history of cancer who had undergone cholecystectomy for non-neoplastic disease. The K3326X polymorphism was detected using heteroduplex analysis and DNA sequencing. The BRCA2 K3326X polymorphism was significantly more prevalent in individuals with familial pancreatic cancer: 8/144 (5.6%) vs 3/250 controls (1.2%) (odds ratio, 4.84; 95% CI, 1.27-18.55, P<0.01). One K3326X carrier with familial pancreatic cancer carried an alteration (IVS 16-2A>G) suspected to be deleterious. Excluding this case did not alter the significance of the association (OR: 4.24, P<0.01). In contrast, there was no difference in prevalence among individuals with sporadic pancreatic cancer - 7/250 (OR: 2.37, 95% CI: 0.61-9.27). The increased prevalence of the BRCA2 K3326X polymorphism in patients with familial pancreatic cancer suggests that this polymorphism is deleterious and contributes to pancreatic cancer risk.

Adenocarcinoma↗
PubMed2005

A recurrent duodenal gastrointestinal stromal tumor with a frameshift mutation resulting in a stop codon in KIT exon 13.

Gastrointestinal stromal tumors (GISTs) are a specific and rare subset of human gastrointestinal tract tumors. Most GISTs show gain-of-function mutations of KIT, mainly in exon 11, that always maintain the reading frame. We report on data from a 43-year-old Japanese man with recurrent duodenal GIST and a frameshift mutation in KIT exon 13 together with an in-frame deletion in KIT exon 11 detected by genomic DNA sequencing. Deletion of 48 base pairs of KIT exon 11, which preserved the reading frame, was identified in both primary and recurrent tumors, whereas deletion of one nucleotide of codon 642 of KIT exon 13, which changed the reading frame and induced a novel stop codon at amino acid 644, was found only in the recurrent tumor. The predicted protein resulting from the latter would lack part of the kinase domain. To the best of our knowledge, this is the first documentation of a GIST with a frameshift mutation of KIT.

Adult↗
PubMed1997

An HLA-B null allele (B*1526N) with a stop codon in exon 3 generated by a point mutation.

An HLA-B null allele was identified in a Japanese family during histocompatibility testing for bone marrow transplantation. The propositus was a healthy Japanese woman with three children, and her parents were cousins. Serological HLA typing of the family members indicated that the propositus was homozygous for the A24-Cw4-B blank (B null)-DR4.2-DQ3 haplotype. Total RNA was extracted from peripheral blood of the propositus was converted to first-strand cDNA using reverse transcriptase. The cDNA was amplified by the polymerase chain reaction (PCR) using HLA-B locus-specific primers. The PCR product showed no change in size upon polyacrylamide gel electrophoresis (PAGE) compared to that of normal controls, suggesting that HLA-B gene mRNA was normally expressed. The nucleotide sequence of the cDNA was the same as that of B*1501 except at nucleotide 369 or codon 123, where C was replaced with A; TAC encodes Tyr whereas TAA is a stop codon. This point mutation may have truncated the HLA-B molecule of the propositus, resulting in the negative results we obtained with anti-HLA-B sera.

Adult↗
PubMed1997

Folding of an mRNA pseudoknot required for stop codon readthrough: effects of mono- and divalent ions on stability.

Unfolding of an mRNA pseudoknot that induces ribosome suppression of the gag gene stop codon in Moloney murine leukemia virus has been studied by UV hyperchromicity and calorimetry. The pseudoknot melts in two steps, corresponding to its two helical stems. The total enthalpy of denaturation is approximately 170 kcal/mol, approximately the value expected for the secondary structure. At low salt concentrations (<50 mM KCl) the unfolding transitions are not two-state, but they approach two-state behavior at higher salt concentrations. The structure is preferentially stabilized by smaller alkali metal ions (Li+ > Na+ > K+ > Rb+ > Cs+) and by NH4+; the same preferences are exhibited by one of the stems in the context of a hairpin. Divalent metal ions are not required to fold the pseudoknot but do stabilize it further. To examine divalent ion effects over a wide concentration range, urea was used to lower the RNA unfolding temperature and was shown not to affect characteristics of the pseudoknot unfolding in other respects. The pseudoknot binds divalent ions somewhat more tightly than a hairpin but shows only weak selectivity for different size ions. It is suggested that a region of "intermediate" divalent ion binding affinity, in between highly ligated specific sites and purely delocalized ion binding in character, is created by the pseudoknot fold but that nonspecific, delocalized ion binding contributes at least half the free energy of pseudoknot stabilization by Mg2+.

Animals↗
PubMed2005

Changes of primary sequence and secondary structure proximal to the 5' end of the stop codon substantially increases the expression of the variable region of an antibody in E. coli.

The sequence context at the 5' end of the stop codon may influence the efficiency of termination and translation. To increase the expression of a designed variable region of an antibody (named as VH5) against tumor necrosis factor alpha (TNFalpha), two nucleotides (TC) at 25 and 26 nucleotides (nt) upstream of termination codon were substituted with AG, respectively. The free energy of 70 nt (arbitrarily defined from the 32 nt upstream of termination codon to 38 nt downstream) was changed from -13.5 kcal mol-1 to -17.3 kcal mol-1. The expression level was increased from 1+/-0.3% to 10+/-1.2% of total cellular protein. Although the precise mechanism of this phenomenon remains to be elucidated, this report provides an alternative means to increase the expression of a foreign gene in E. coli.

5' Flanking Region↗
PubMed2004

Latent splice sites and stop codons revisited.

The accuracy of the data we reported in an RNA Letter to the Editor earlier this year on the possible relationship between stop codons and splicing is questioned by Miriami et al. (this issue). We reply here that we see no inaccuracy in our data presentation and offer a possible explanation for their interpretation.

Codon, Terminator↗
PubMed1994

Homozygous protein S deficiency due to a one base pair deletion that leads to a stop codon in exon III of the protein S gene.

Homozygous protein S (PS) deficiency is a very rare disorder that causes purpura fulminans in affected newborns. This report describes the molecular genetic abnormality of a severe PS deficient child who developed purpura fulminans shortly after birth. The mutation was identified as a deletion of one adenine in codon 43 of exon III of the PROS 1 gene. This mutation results in a frameshift and a novel stop codon at position 45. The proband was apparently homozygous and his mother heterozygous for this mutation. The proband's father was not available for study. The single base pair deletion predicts a truncated translation product, where Lys 43 and Tyr 44 have been replaced by Asn 43 and Thr 44. This putative protein (predicted mw of 5.696 daltons) contains only the gammacarboxyglutamic acid (Gla) domain and the aromatic stack.

Amino Acid Sequence↗
PubMed1990

Rapid detection and further characterization of infection with hepatitis B virus variants containing a stop codon in the distal pre-C region.

Recently, hepatitis B virus (HBV) replication in the absence of HBe antigenaemia has been attributed to HBV variants with a TAG stop codon in the distal pre-C region associated with one or two point mutations. We describe here a rapid detection method for the diagnosis of such HBeAg-negative HBV variants using selective oligonucleotide hybridization. The entire pre-C region was amplified by the polymerase chain reaction and hybridized under stringent conditions with non-mutated (M0), one (M1) and two (M2) point-mutated oligonucleotide probes. Of the 15 HBeAg-positive (group I) and 20 HBeAg-negative (group II) serum samples studied, 14 samples in group I and one sample in group II hybridized with M0 only and 18 samples in group II hybridized with M1 or M2, or both. The remaining two samples (from groups I and II, respectively) failed to hybridize with any of the three probes. DNA sequencing confirmed mixed distal pre-C sequences in samples hybridizing with more than one probe and also revealed novel mutations in the distal pre-C region of the two samples which failed to hybridize with any of the probes. The latter sample had a +2 frameshift and hence represented a new type of HBeAg-negative HBV variant. This method may therefore prove useful in the diagnosis of infections by HBeAg-negative HBV variants resulting from common mutations in the pre-C region, as well as for the identification of less common variants with novel mutations in the same region.

Base Sequence↗
PubMed2002

A novel nonstop mutation in the stop codon and a novel missense mutation in the type II 3beta-hydroxysteroid dehydrogenase (3beta-HSD) gene causing, respectively, nonclassic and classic 3beta-HSD deficiency congenital adrenal hyperplasia.

We investigated two novel point mutations in the human type II 3beta-hydroxysteroid dehydrogenase (3beta-HSD) gene causing a mild and a severe form of 3beta-HSD deficiency congenital adrenal hyperplasia. The first is a nonstop mutation in the normal stop codon 373 of the gene in exon IV [TGA (Stop) --> TGC (Cys) = Stop373C) identified from one allele of a female child with premature pubarche whose second allele had an E142K mutation. The Stop373C mutation predictably results in an open reading frame and a mutant-type (MT) II 3beta-HSD protein containing 467 amino acid residues, compared with the 372 amino acid residues of wild-type (WT) protein. The second is a homozygous missense mutation in codon 222 [CCA (Pro) --> ACT (Thr) = P222T] in the gene identified from a female neonate with salt-wasting disorder. The pcDNA vectors containing the constructs of WT II 3beta-HSD cDNA, WT cDNA with the open reading frame (WT cDNA(+)), MT Stop373C with the open reading frame (Stop373C(+)) and MT P222T cDNA were transfected in COS-I and 293T cells and expressed a similar amount of 3beta-HSD mRNA. The enzyme activity in intact cells using pregnenolone and dehydroepiandrosterone as substrate in the medium (1 micromol/liter) was identical between the WT cDNA and the WT cDNA(+), but was decreased to 27% of the WT enzymes at 6 h by MT Stop373C(+) enzyme, and was undetectable by P222T enzyme. In the homogenates of the cells, both MT Stop373C(+) and P222T enzyme activities and enzymes were undetectable despite clear detection of WT enzyme activities and WT enzymes. LH response to an LHRH analog stimulation in the pubertal female with the Stop373C/E142K genotypes and in a pubertal female with compound 273/318 frameshift genotypes were comparable to and higher than control females, respectively. In conclusion, a structurally lengthy MT II 3beta-HSD enzyme due to a nonstop mutation was relatively detrimental in intact cells causing the nonclassic phenotype of 3beta-HSD deficiency. A missense P222T mutation was seriously detrimental, causing the classic phenotype of 3beta-HSD deficiency. The undetectable Stop373C and P222T enzymes on Western blottings, together with the respective in vivo and in vitro data, suggest that a relative instability of Stop373C enzyme and a profound instability of the P222T enzyme are likely the detrimental molecular mechanisms. The increased LH in the female with the frameshift genotype and the appropriate LH response in the female with the nonstop genotype correlated with predictably severe and mild ovarian type II 3beta-HSD deficiency, respectively.

3-Hydroxysteroid Dehydrogenases↗
PubMed2003

Severe prekallikrein (Fletcher factor) deficiency due to a compound heterozygosis (383Trp stop codon and Cys529Tyr).

We investigated a family with prekallikrein deficiency, using both standard coagulation tests and molecular biology techniques. The propositus was found to be a compound heterozygote for a Trp383 stop codon and a Cys529Tyr point mutation. The former mutation was located in exon 11, the latter in exon 14. The propositus inherited the first defect from his father and the second from his mother. Both parents had slightly low prekallikrein levels, but the combination of the two genetic defects produced a phenotype characterized by an extremely low prekallikrein activity and antigen. The propositus' plasma showed a progressive reduction in APTT when incubated for a long time. Conversely, plasma deficient in factor XII, factor XI or high molecular weight kininogen (HMWK) failed to show shortening of the APTT. No circulating anticoagulant was found because the patient's APTT was fully corrected by pooled nor-mal and factor XII-, factor XI- or HMWK deficient plasma. No associated abnormality was apparent in the propositus or his parents. As expected, no tendency for bleeding was noted even after tonsillectomy.

Adolescent↗
PubMed2000

The 17 nucleotides downstream from the env gene stop codon are important for murine leukemia virus packaging.

We have identified a previously unknown nucleotide sequence important for the packaging of murine leukemia virus. This nucleotide sequence is located downstream from the stop codon of the env gene but does not overlap the polypurine tract. Deletion of 17 bp from this region resulted in a more than 10-fold decrease in viral titer. Consistent with this result, the deletion mutant showed a 20- to 30-fold drop in the amount of virion RNA in the culture supernatant. The total amount of virion protein in the culture supernatant was comparable for the deletion mutant and the parental virus, suggesting that the mutant construct could release the empty viral particles. These results suggested that the packaging signal sequence might be present at the two extreme sites of the viral genome, one in the region around the splice donor sequence downstream from the 5' long terminal repeat (LTR) and the other immediately upstream from the 3' LTR. Implications for gene therapy, especially in regard to construction of retroviral vectors and packaging constructs, are discussed.

3T3 Cells↗
PubMed2006

Introduction of translation stop codons into the viral glycoprotein gene in a fish DNA vaccine eliminates induction of protective immunity.

A highly efficacious DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was mutated to introduce two stop codons to prevent glycoprotein translation while maintaining the plasmid DNA integrity and RNA transcription ability. The mutated plasmid vaccine, denoted pIHNw-G2stop, when injected intramuscularly into fish at high doses, lacked detectable glycoprotein expression in the injection site muscle, and did not provide protection against lethal virus challenge 7 days post-vaccination. These results suggest that the G-protein itself is required to stimulate the early protective antiviral response observed after vaccination with the nonmutated parental DNA vaccine.

Animals↗
PubMed2001

Analysis of TSC2 stop codon variants found in tuberous sclerosis patients.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations to the TSC1 and TSC2 tumour suppressor genes. We detected two sequence changes involving the TSC2 stop codon and investigated the effects of these changes on the expression of tuberin, the TSC2 gene product, and on the binding between tuberin and the TSC1 gene product, hamartin. While elongation of the tuberin open reading frame by 17 amino acids did not interfere with tuberin-hamartin binding, a longer extension prevented this interaction. Our data illustrate how functional protein assays can assist in the verification and characterisation of disease-causing mutations.

Animals↗
PubMed2003

Guanidine reduces stop codon read-through caused by missense mutations in SUP35 or SUP45.

Sup35 and Sup45 are essential protein components of the Saccharomyces cerevisiae translation termination factor. Yeast cells harbouring the [PSI(+)] prion form of Sup35 have impaired stop codon recognition (nonsense suppression). It has long been known that the [PSI(+)] prion is not stably transmitted to daughter cells when yeast are grown in the presence of mM concentrations of guanidine hydrochloride (GuHCl). In this paper, Mendelian suppressor mutations whose phenotypes are likewise hidden during growth in the presence of millimolar GuHCl are described. Such GuHCl-remedial Mendelian suppressors were selected under conditions where [PSI(+)] appearance was limiting, and were caused by missense mutations in SUP35 or SUP45. Clearly, anti-suppression caused by growth in the presence of GuHCl is not sufficient to distinguish missense mutations in SUP35 or SUP45, from [PSI(+)]. However, the Mendelian and prion suppressors can be distinguished by subsequent growth in the absence of GuHCl, where only the nonsense suppression caused by the [PSI(+)] prion remains cured. Recent reports indicate that GuHCl blocks the inheritance of [PSI(+)] by directly inhibiting the activity of the protein remodelling factor Hsp104, which is required for the transmission of [PSI(+)] from mother to daughter cells. However, the nonsense suppressor activity caused by the GuHCl-remedial sup35 or sup45 suppressors does not require Hsp104. Thus, GuHCl must anti-suppress the sup35 and sup45 mutations via an in vivo target distinct from Hsp104.

Alleles↗
PubMed2006

Construction of a positive selection marker by a lethal gene with the amber stop codon(s) regulator.

A novel positive selection marker for Escherichia coli transformation was developed. The marker consisted of a DNA fragment encoding the C-terminal ribonuclease domain (CRD) of colicin E3 (colE3) and one or more amber stop codons between the initiation codon and the E3-CRD coding sequence. The toxicity of the marker was controlled by the suppressor activity the host cells possessed. This allowed both effective selection and propagation of the vector possessing the maker by selecting appropriate hosts from among those widely distributed: sup+ strains for selection and sup0 strains for propagation respectively. The insert DNA fragment was introduced onto the vector by replacing the marker DNA. The transformants harboring the vector with an insert grew, but those without an insert were effectively removed by the killing activity of E3-CRD encoded on the marker DNA. The marker was also successfully applied to lambda phage display vector.

Codon, Terminator↗
PubMed2002

Competition between SsrA tagging and translational termination at weak stop codons in Escherichia coli.

SsrA is a tmRNA involved in tagging polypeptides on stalled ribosomes. The resulting fusion proteins are then degraded. We purified endogenous SsrA-tagged proteins by means of a genetically engineered SsrA and identified some of them. Analysis of the proteins suggested that they are tagged at their C-terminal extremities. One of them, ribokinase, is expressed from a messenger with a poorly efficient stop codon, leading to translational recoding events. A change in the ribokinase coding sequence from a weak to a strong translational stop sequence (UGAc to UAAu) annihilated SsrA tagging. Translational termination by UGA recruits the translational release factor (RF) 2. We observed that SsrA tagging of ribokinase was inversely correlated with RF2 activity, revealing a dynamic competition between translational termination and SsrA tagging.

Codon, Terminator↗
PubMed2006

Severe nemaline myopathy caused by mutations of the stop codon of the skeletal muscle alpha actin gene (ACTA1).

Most nemaline myopathy patients have mutations in the nebulin (NEB) or skeletal muscle alpha-actin (ACTA1) genes. Here we report for the first time three patients with severe nemaline myopathy and mutations of the ACTA1 stop codon: TAG>TAT (tyrosine), TAG>CAG (glutamine) and TAG>TGG (tryptophan). All three mutations will cause inclusion of an additional 47 amino acids, translated from the 3' UTR of the gene, into the mature actin protein. Western blotting of one patient's muscle demonstrated the presence of the larger protein, while expression of one of the other mutant proteins fused to EGFP in C2C12 cells demonstrated the formation of rod bodies.

3' Untranslated Regions↗
PubMed2001

In vivo regulation of oxidative phosphorylation in cells harboring a stop-codon mutation in mitochondrial DNA-encoded cytochrome c oxidase subunit I.

The mechanisms that regulate oxidative phosphorylation in mammalian cells are largely unknown. To address this issue, cybrids were generated by fusing osteosarcoma cells devoid of mitochondrial DNA (mtDNA) with platelets from a patient with a stop-codon mutation in cytochrome c oxidase subunit I (COX I). The molecular and biochemical characteristics of cybrids harboring varying levels of mutated mitochondrial DNA were studied. We found a direct correlation between the levels of mutated COX I DNA and mutated COX I mRNA, whereas the levels of COX I total mRNA were unchanged. COX I polypeptide synthesis and steady-state levels were inversely proportional to mutation levels. Cytochrome c oxidase subunit II was reduced proportionally to COX I, indicating impairment in complex assembly. COX enzymatic activity was inversely proportional to the levels of mutated mtDNA. However, both cell respiration and ATP synthesis were preserved in cells with lower proportions of mutated genomes, with a threshold at approximately 40%, and decreased linearly with increasing mutated mtDNA. These results indicate that COX levels in mutated cells were not regulated at the transcriptional, translational, and post-translational levels. Because of a small excess of COX capacity, the levels of expression of COX subunits exerted a relatively tight control on oxidative phosphorylation.

Adenosine Triphosphate↗