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PubMed2004

Readthrough of dystrophin stop codon mutations induced by aminoglycosides.

We report the translational readthrough levels induced by the aminoglycosides gentamicin, amikacin, tobramycin, and paromomycin for eight premature stop codon mutations identified in Duchenne's and Becker's muscular dystrophy patients. In a transient transfection reporter assay, aminoglycoside treatment results show that one stop codon mutation is suppressed significantly better (up to 10% stop codon readthrough) than the others; five show lower but statistically significant suppression (< 2% stop codon readthrough); and two appear refractory to aminoglycoside treatment. Readthrough levels do not substantially vary between different sources of gentamicin, and, for this set of mutations, the efficiency of termination at the premature stop codon mutation does not appear to correlate with disease severity.

Aminoglycosides↗

PubMed1995

Genomic DNA sequencing of mRNA splicing mutants in the hprt gene of Chinese hamster ovary cells.

We have analyzed 41 mRNA-splicing mutants from the hypoxanthine-guanine phosphoribosyl-transferase (hprt) gene of Chinese hamster ovary (CHO) cells. Twenty-two of these mutants produced single cDNA PCR products with a partial or complete exon deletion; 19 mutants produced multiple cDNA PCR products, and most of these products contained one or more deleted exons. The affected exons and surrounding introns were amplified from genomic DNA and sequenced in order to identify mutations causing aberrant splicing. We found acceptor site mutations in 10 mutants, exonic mutations in 8 mutants, and no mutations in 5 mutants. Four mutants from solvent controls did not amplify the appropriate exons and were considered genomic deletion mutants. Our previous work [Manjanatha MG et al. (1994): Mutat Res 308;65-75] showed that nonsense mutants in the hprt gene of CHO cells are associated with multiple cDNA PCR products containing deleted exons and a low abundance of hprt mRNA if the mutation is found in an internal exon. The present results are consistent with these associations being facilitated by instability of mRNA after ribosome termination at nonsense codons.

Animals↗

PubMed1992

Two novel mutations responsible for hereditary type I protein C deficiency: characterization by denaturing gradient gel electrophoresis.

Hereditary protein C (PC) deficiency is usually associated with a high risk of thrombosis. We report the results of a study undertaken to screen for molecular defects in families with hereditary quantitative PC deficiency. Using a strategy combining polymerase chain reaction amplification of selected gene fragments, denaturing gradient gel electrophoresis of the amplification products, and direct sequencing of fragments with altered melting behavior, we studied the PC gene exons and exon/intron junctions of subjects with hereditary type I PC deficiency. Computer simulation of DNA melting was used to design several sets of primers, each containing a GC-clamp, permitting the complete analysis of each amplified exon sequence. Using this procedure, we identified two previously undescribed mutations located in exon VII: a C-to-T substitution generating a nonsense codon in place of Arg 157 in the mature PC and a G-to-A substitution converting Arg 178 to GIn. The two mutations were detected in, respectively, 3 and 2 apparently independent families. This strategy is therefore a valuable tool for screening patients, and the results emphasize its advantages over plasma assays in individuals with a family history of thrombosis.

Adult↗

PubMed1995

Molecular basis of hereditary fructose intolerance: mutations and polymorphisms in the human aldolase B gene.

Mutations in the human aldolase B gene that result in hereditary fructose intolerance have been characterized extensively. Although the majority of subjects have been from northern Europe, subjects from other geographical regions and ethnic groups have been identified. At present 21 mutations have been reported; 15 of these are single base substitutions, resulting in nine amino acid replacements, four nonsense codons, and two putative splicing defects. Two large deletions, two four-base deletions, a single-base deletion, and a seven-base deletion/one-base insertion have been found. This last mutation leads to a defect in splicing and it is likely that one of the small deletions does as well. Regions of the enzyme where mutations have been observed recurrently are encoded by exons 5 and 9. Indeed, the three most common mutations are found in these exons. Two of these prevalent HFI mutations arose from a common ancestor and spread throughout the population by genetic drift. This finding was based on linkage to two sequence polymorphisms, which are among very few informative polymorphic markers that have been identified within the aldolase B gene. Because of the prevalence of a few HFI alleles, and the recent advances in molecular methods for identifying and screening for mutation, the diagnosis of HFI by molecular screening methods should become routine. These molecular diagnostic methods will be extremely beneficial for this often difficult to diagnose and sometimes fatal disease.

Chromosome Mapping↗

PubMed2004

Genotype-phenotype correlation in von Hippel-Lindau families with renal lesions.

von Hippel-Lindau (VHL) disease arises from mutations in the VHL gene and predisposes patients to develop a variety of tumors in different organs. In the kidney, single or multiple cysts and renal cell carcinomas (RCC) may occur. Both inter- and intrafamilial heterogeneity in clinical expression are well recognized. To identify VHL-dependent genetic factors, we investigated the renal phenotype in 274 individuals from 126 unrelated VHL families in whom 92 different VHL mutations were characterized. The incidence of renal involvement was increased in families with mutations leading to truncated protein (MLTP) or large rearrangement, as compared to families with missense changes (81 vs. 63%, respectively; P=0.03). In the latter group, we identified two mutation cluster regions (MCRs) associated with a high risk of harboring renal lesions: MCR-1 (codons 74-90) and MCR-2 (codons 130-136). In addition, the incidence of RCC was higher in families with MLTP than in families with missense changes (75 vs. 57%; P=0.04). Furthermore, mutations within MCR-1 but not MCR-2 conferred genetic susceptibility to develop RCC. Overall, our data argued for a substantial contribution of the genetic change in the VHL gene to susceptibility to renal phenotype in VHL patients.

Adult↗

PubMed2003

Mutational screening of the RB1 gene in Indian patients with retinoblastoma reveals eight novel and several recurrent mutations.

Retinoblastoma is the most common primary intraocular malignancy in children, caused by inactivation of the RB1 gene on chromosome 13. We carried out a mutational screen of the exons and promoter of the RB1 gene in Indian patients with retinoblastoma in order to determine the range of mutations giving rise to disease. Forty-seven patients were screened for mutations in all exons and promoter of the RB1 gene by single strand conformation polymorphism followed by sequencing. Tumors were available from 27 patients (12 bilateral and 15 unilateral retinoblastoma) while only peripheral blood was available from 20 patients, all with bilateral disease. Mutations were found in 22 patients, 9 from the analysis of tumors and 13 from peripheral blood. Eight novel mutations were identified, including 4 single base changes, 2 small deletions and 1 duplication. These are g.64365T>G (Tyr325Ter), g.78131G>A (Trp515Ter), g.150061G>T (Glu587Ter), g.170383C>G (S834X), g.41924A>C (IVS3-2A>C), g.150064ins4, g.160792del22, and g.76940del14 (IVS15 del +20-33). Almost all mutations produced nonsense codons or frameshifts. Recurrent mutations, especially at CpG sites were seen predominantly. Detectable mutations in exons were found in 46% of patients tested. Large deletions, epigenetic changes as well as mutations in non-coding regions may be the cause of disease in the remainder of patients. Knowledge of the full range of mutations can aid in the design of screening tests for individuals at risk.

Child↗

PubMed1982

A novel method for site-directed mutagenesis: its application to an eukaryotic tRNAPro gene promoter.

We present a novel general method for localized mutagenesis. The DNA segment to be mutagenized is inserted in the beta-galactosidase gene of a M13-lac vector, generally causing loss of beta-galactosidase function by generation of frameshifts or nonsense codons. Mutations in the inserted DNA which restore beta-galactosidase function are readily detected and analyzed. The application of this method to the promoter of an eukaryotic (Caenorhabditis elegans) tRNAPro gene has allowed the isolation of several mutants altered in transcription.

Animals↗

PubMed1990

Temperature sensitive synthesis of transfer RNAs in vivo in Saccharomyces cerevisiae.

Dictyostelium discoideum tRNA genes can be expressed efficiently in vivo in yeast, and transcription products are processed to mature tRNAs. However, primary transcripts of a variant tRNA(Val)(UAC) gene are processing deficient under standard growth conditions (30 degrees C), due to a slightly altered 5' flanking region. A stable extended amino acid acceptor stem, which seems to be required to compensate a G5-G68 mismatch, cannot form. This mismatch destabilizes secondary and probably tertiary structures to such an extent that recognition of processing enzyme(s) under normal conditions (30 degrees C) is impaired. Growing yeast cells at reduced temperature (22 degrees C) can phenotypically complement the processing defect. This observation provides a new concept for the temperature dependent expression of protein coding genes which carry a nonsense codon. Translation of corresponding messages can be controlled by products of a temperature sensitive su-tRNA gene. We successfully tested this concept with two amber suppressors derived from a tRNA(Glu)(UUC) gene from D. discoideum. One of the variant tRNA genes codes for a product with a destabilized amino acid acceptor stem. Primary transcripts of this particular su-tRNA(Glu)(CUA) gene are processed only at reduced growth temperatures and consequently function as temperature sensitive suppressors only under these conditions.

Base Sequence↗

PubMed2004

Precore/core promoter mutations and genotypes of hepatitis B virus in chronic hepatitis B patients with fulminant or subfulminant hepatitis.

The association of precore stop codon mutation (A1896), dinucleotide mutation (T1762/A1764) in the basic core promoter of hepatitis B virus (HBV) genome, and genotype of HBV with fulminant or subfulminant hepatitis remains controversial. We studied HBV genotypes as well as mutations in the precore and basic core promoter regions in 18 hepatitis B carriers with fulminant or subfulminant hepatitis. Genotyping of HBV was performed by polymerase chain reaction-restriction fragment length polymorphism. The presence of A1896 in the precore gene and T1762/A1764 in the basic core promoter gene was determined by the polymerase chain reaction and by direct sequencing. Eighteen age- and sex-matched patients with chronic active hepatitis B served as controls. The HBV was of genotype B in 14, genotype C in 3, and unclassified in 1. Precore A1896 mutation occurred in 12 (67%) of the 18 patients. In contrast, the prevalence of basic core promoter mutation was only 17%. Nevertheless, the distribution of HBV genotype and the prevalence of precore A1896 mutation in the fulminant and subfulminant hepatitis patients were similar to those in 18 control patients. In conclusion, the genomic variability of HBV does not seem to contribute to the fulminant and subfulminant exacerbation of chronic hepatitis B in Taiwanese HBV carriers.

Adult↗

PubMed2003

Two novel mutations in the muscle glycogen phosphorylase gene in McArdle's disease.

We report on a Spanish family with myophosphorylase (EC 2.4.1.1) deficiency (McArdle's disease). The proband and his symptomatic sister were compound heterozygous for two novel mutations: a T-to-G transversion in exon 14 (c1722 T>G) that changes a tyrosine to a stop codon (Y573X), and a G-to-A transition in exon 15 (c1827 G>A) that disrupts the consensus signal at the donor splicing site. These findings further expand knowledge of the genetic bases of muscle glycogen phosphorylase deficiency.

Adult↗

PubMedDate not supplied

Characterization of the adr1-1 nonsense mutation identifies the translational start of the yeast transcriptional activator ADR1.

We have characterized a nonsense mutation in the ADR1 gene that identifies the translational start of the ADR1 protein. The ADR1 gene of Saccharomyces cerevisiae is required for synthesis of the glucose-repressible alcohol dehydrogenase (ADH2). The adr1-1 mutation, which inhibits ADH2 expression, was identified as a C to G transversion at base pair +32. This alteration would result in a UGA nonsense codon in place of a serine codon that would lead to termination of the ADR1 polypeptide after the 10th amino acid. The effect of the adr1-1 mutation was partially reversed by UGA-tRNA suppressors, indicating that the adr1-1 mutation affects ADR1 expression at the translational level. These observations establish that the first available AUG in the ADR1 sequence is used as the translational start site of ADR1. Tyrosine or leucine UGA-tRNA-suppressors resulted in levels of adr1-1 activity similar to that found for a serine UGA-tRNA-suppressor, suggesting that serine residue-11 is not essential to ADR1 function. Northern analyses showed that the 5.1 kb ADR1 mRNA was two- to three-fold more abundant when isolated from a strain carrying the ADR1 allele than from an isogenic strain containing the adr1-1 allele. These data confirm that the 5.1 kb mRNA is the ADR1 mRNA and suggest that inhibition of adr1-1 mRNA translation results in more rapid degradation of the adr1-1 mRNA.

Alcohol Dehydrogenase↗

PubMed1994

Production of UV-induced frameshift mutations in vitro by DNA polymerases deficient in 3'-->5' exonuclease activity.

In order to study the conversion of UV lesions into frameshift and base substitution mutations, M13mp2 phage DNA was altered by the addition of extra pyrimidines, or by construction of a nonsense codon preceded by a run of pyrimidines within the beta-galactosidase complementing region. The normal sequence 5' GTC GTT TTA CAA 3' was changed to GTC GTT T TTA CAA (MIDT) or GTC GTT C TTA CAA (MIDC) to study frameshifts and to GTC GTT CTT TAA (OCHRE) to study reversion of the ochre (TAA) codon. Escherichia coli pol I Kf and T7 DNA polymerase mutant enzymes devoid of 3'-->5' exonuclease activity produced UV-induced revertants at higher frequency than did their exonuclease proficient counterparts. Removal of cyclobutane dimers with photolyase before in vitro synthesis did not greatly affect mutant frequency although such treatment led to significantly increased DNA synthesis by the wild-type T7 DNA polymerase on UV-irradiated substrate. Reversions of the in frame ochre sequence GTT CTT TAA produced by the delta 28 T7 DNA polymerase were mainly by base substitution in the TAA codon. About half of the E. coli Kf exo- enzyme ochre revertants had a TTA deletion. Five mutant T7 DNA polymerases with varying exonuclease activity gave revertant frequencies that correlated better with published values of enzyme velocity than with exonuclease activity or with measured bypass synthesis. Our data indicate that loss of proofreading activity increases the frequency of UV-induced frameshifts, but lack of such activity is not sufficient for their production. We suggest that frameshifts occur more frequently when nucleotide addition opposite the lesion is slow. The same lesion can give rise to a different spectrum of mutations depending on the polymerase.

Bacteriophage M13↗

PubMed1994

Genetic implication for an interaction between release factor one and ribosomal protein L7/L12 in vivo.

Strains with mutant alleles of the genes encoding release factor one (RF1), prfA, and ribosomal protein L7/L12, rplL, have been analyzed. The prfA1 allele has previously been shown to be associated with temperature sensitivity for growth at 43 degrees C and increased misreading of the nonsense codons UAG and UAA. Here we show that the prfA1 mutation is a nucleotide substitution that results in an arginine to proline change at amino acid position 137 in RF1. This alters the factor in a domain which may be involved in ribosome-binding. The rplL564 allele codes for a mutant form of protein L7/L12 that has a change in its conserved, translation-factor binding, domain. The rplL564 mutation suppresses the temperature sensitive phenotype and the increased translational misreading associated with the prfA1 allele, while other studied rplL mutant alleles do not. These data are consistent with an interaction between L7/L12 and RF1 in vivo.

Bacterial Proteins↗

PubMed1994

The carboxyl-half of the rotavirus nonstructural protein NS53 (NSP1) is not required for virus replication.

The rotavirus nonstructural protein NS53 (NSVP1), the product of genome segment 5, possesses RNA-binding activity and contains a highly conserved cysteine-rich motif located in the amino-terminal half of the protein. The genome of the bovine rotavirus variant, brvA, lacks a normal segment 5 but includes a novel dsRNA (gene A) of approximately 2600 basepairs (bp) that contains segment 5-specific sequences (F. Hundley, B. Biryahwaho, M. Gow, and U. Desselberger, Virology 143, 88-103, 1985). To gain information about the nature of the rearrangement in gene A and its capacity to encode a protein product, we prepared and sequenced complementary (c)DNA of the gene A RNA. The results showed that gene A is 2693 bp in size and contains a head-to-tail duplication of 1112 bp that originates from the open reading frame (ORF) of gene 5. The duplication begins at nucleotide (nt) 1454, which is 53 nt upstream from the end of the ORF for NS53. Gene A contains a point mutation at nt 808 which results in the presence of a nonsense codon near the middle of the ORF for NS53. Thus the predicted product of gene A is a truncated NS53 of 258 amino acids (aa) (31 kDa), approximately one-half the size of the authentic 491-aa NS53 (58 kDa). Examination of lysates from brvA-infected cells by Western blot assay using an NS53-specific antibody confirmed that the variant encodes only a truncated gene 5 product. Despite the truncation, analysis of the gene A product suggested that it, like full-length NS53, accumulated in association with the cytoskeleton of the infected cell, thus providing evidence that the subcellular localization signal in NS53 resides in the amino terminal half of the protein. Given that brvA is a viable, nondefective mutant, these results demonstrate that the carboxyl-terminal 233 aa of NS53 are not required for rotavirus replication in vitro.

Amino Acid Sequence↗

PubMed1998

Identification of viral genes required for cell-to-cell movement of southern bean mosaic virus.

Inoculation of Vigna unguiculata (cowpea) with transcripts synthesized in vitro from a genome-length cDNA clone of the cowpea strain of southern bean mosaic virus (SBMV-C) resulted in a systemic SBMV-C infection of this host. Capped RNA was about five times more infectious than uncapped RNA as determined by a local lesion assay. The SBMV-C cDNA clone was also used for mutagenesis of the four SBMV-C open reading frames (ORFs). ORF1, ORF3, and coat protein (CP) mutants were not infectious in cowpea. Electroporation of cowpea protoplasts with mutant transcripts demonstrated that the ORF1, ORF3, and CP gene products were not required for SBMV-C RNA synthesis, and the ORF1 and ORF3 gene products were not required for SBMV-C assembly. From these results, it was concluded that the ORF1 and ORF3 proteins and the CP are required for SBMV-C cell-to-cell movement. One of the ORF3 mutants pSBMV2-UAA1833 contained a nonsense codon between the predicted -1 ribosomal frameshift site (SBMV-C nucleotides 1796-1802) and a potential ORF3 translation initiation codon at SBMV-C nucleotide 1895. The lack of infectivity of this mutant suggested that ORF3 was expressed by a -1 ribosomal frameshift in ORF2 rather than by initiation of translation at nucleotide 1895.

Electroporation↗

PubMed1996

Structure and function of rotavirus NSP1.

Studies on the structure and function of the nonstructural proteins (NSP1-NSP5) of rotaviruses are important for dissection of the morphogenesis and replication processes of rotavirus. Above all, NSP1, the product of gene 5, has several interesting features, such as extreme sequence diversity, a highly conserved cysteine-rich region, RNA-binding activity, accumulation on the cytoskeleton, and non-random segregation in reassortment. Recently, comparable NSP1 sequence analysis has been performed on a number of rotavirus strains from various species. Furthermore, characterization of mutants with rearranged NSP1 genes has helped to elucidate the structure-function interaction of NSP1. We isolated and characterized two interesting mutants which have a large deletion including the cysteine-rich region or a nonsense codon at the early portion in the open reading frame (ORF) of the NSP1 gene. In this report, we summarize the structure and function of NSP1.

Amino Acid Sequence↗

PubMed1975

Bacterial mutants able to partly suppress the effect of N mutations in bacteriophage lambda.

A method is described whereby bacterial mutants (sun) may be selected which are able to specifically suppress mutations in the N gene of bacteriophage lambda. The sun mutations seem to be allelic to suA mutations, which suppress the polarity of nonsense codons, since suA mutants have all of the properties of sun mutants and both are genetically linked to the ilv gene. In the light of these experiments and recent data by others, models originally suggested to explain polarity in bacterial operons, are discussed with regard to their possible relevance to the mechanism of N action.

Bacteriophage lambda↗

PubMed1988

The peculiar spectrum of beta-thalassemia genes in Tunisia.

We have determined the spectrum of mutations producing beta-thalassemia (beta-thal) in Tunisia by direct DNA analysis using hybridization with allele-specific oligonucleotide probes and restriction endonuclease assay. In all, 34 unrelated beta-thal patients originating from different parts of the country were available for study. The beta-globin gene cluster of each was subjected to haplotype analysis, and on the basis of this analysis, we tested each patient's DNA for one or more mutations previously shown to be linked to that haplotype. We identified four previously unreported haplotypes and found that this population differs from others in Mediterranean areas in the frequency of the beta-thal haplotypes, the unexpected observation being the high frequency of haplotype IX. Six different point mutations were found, accounting for 62% of beta-thal genes in this Tunisian population. The molecular defects known to be the most frequent in Mediterraneans (nonsense codon 39, IVS1 nt 110, IVS1 nt 6) only make up 37% of the mutant genes. One as yet undescribed mutation (IVS1 nt 2 T----G) was identified by molecular cloning and sequencing. Our results should help the implementation of a prenatal diagnosis program for beta-thal in Tunisia.

Genetic Markers↗