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At least 1,081 records · Page 60Linked to original sources
PubMed1994

Gene inactivation in Arabidopsis thaliana is not accompanied by an accumulation of repeat-induced point mutations.

Chromosomal integration of multicopy transgene inserts in higher plants is often followed by loss of expression. We have analysed whether this inactivation can trigger repeat-induced point mutations (RIP) as has been observed in Neurospora crassa. We have previously characterized transgenic lines of Arabidopsis thaliana containing the hygromycin phosphotransferase (hpt) gene either as a unique sequence in plants expressing the gene, or as multimeric, closely linked repeats in clones that were resistant to hygromycin directly after transformation but exhibited gene inactivation in the subsequent generation. At the sequence level, we have determined the mutation frequencies in the promoter and coding regions of active and inactive copies of transgene inserts after passage through three sexual generations. No RIP-like mutations were found in inactivated genes. Comparison of our data with those from Neurospora suggest that sequence divergence within plant repetitive DNA is either much slower than in Neurospora or is generated by a different mechanism.

Arabidopsis↗
PubMed1997

Point mutations in the transmembrane domain of DjlA, a membrane-linked DnaJ-like protein, abolish its function in promoting colanic acid production via the Rcs signal transduction pathway.

DjIA is a novel DnaJ-like protein localized to the inner membrane of Escherichia coli through the single transmembrane domain (TMD) found at the N-terminus. The overproduction of DjIA activates expression of the cps operon, controlling synthesis and export of the extracellular polysaccharide colanic acid via the Rcs/B two-component signal transduction pathway. We now show that both the TMD and the J-region are essential for the induction of cps expression observed with the overproduction of DjIA. Furthermore, we describe the isolation and characterization of different point mutations in the TMD that completely or partially block the induction of cps expression associated with overproduction of DjIA. These mutations were shown not to affect the localization, stability or topology of the mutant DjIA proteins. We propose that these mutations are affecting specific interactions between the TMD of DjIA and its substrate protein(s), for example RcsC, the membrane sensor kinase partner of the Rcs/B signal transduction pathway.

Antibodies, Bacterial↗
PubMed2006

A single point mutation (N514Y) in the human M3 muscarinic acetylcholine receptor reveals differences in the properties of antagonists: evidence for differential inverse agonism.

A single asparagine-to-tyrosine point mutation in the human M muscarinic acetylcholine (mACh) receptor at residue 514 (N514Y) resulted in a marked increase (approximately 300%) in agonist-independent [3H]inositol phosphate ([3H]IPx) accumulation compared with the response observed for the wild-type (WT) receptor. All the antagonists tested were able to inhibit both the WT-M3 and (N514Y)M3 mACh receptor-mediated basal [3H]IPx accumulation in a concentration-dependent manner. However, significant differences in both potency and binding affinity were only seen for those antagonists that possess greater receptor affinity. Despite being transfected with equivalent amounts of cDNA, cells expressed the (N514Y)M3 mACh receptor at levels that were only 25 to 30% of those seen for the WT receptor. Differences in the ability of chronic antagonist exposure to up-regulate (N514Y)M3 mACh receptor expression levels were also seen, with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) producing only 50% of the receptor up-regulation produced by atropine or pirenzepine. Basal phosphorylation of the (N514Y)M3 mACh receptor was approximately 100% greater than that seen for the WT-M3 receptor. The ability of antagonists to decrease basal (N514Y)M3 mACh receptor phosphorylation revealed differences in inverse-agonist efficacy. Atropine, 4-DAMP, and pirenzepine all reduced basal phosphorylation to similar levels, whereas methoctramine, a full inverse agonist with respect to reducing agonist-independent [3H]IPx accumulation, produced no significant attenuation of basal receptor phosphorylation. This study shows that mACh receptor inverse agonists can exhibit differential signaling profiles, which are dependent on the specific pathway investigated, and therefore provides evidence that the molecular mechanism of inverse agonism is likely to be more complex than the stabilization of a single inactive receptor conformation.

Cell Line↗
PubMed2003

Real-time PCR assay for rapid and accurate detection of point mutations conferring resistance to clarithromycin in Helicobacter pylori.

The main cause of failure of Helicobacter pylori eradication therapy is resistance to clarithromycin. The resistance is due to three point mutations in two positions on the 23S rRNA (A2142C, A2142G, and A2143G). Our aim was to develop a rapid and accurate method to detect these mutations directly on biopsy specimens. We developed a real-time PCR that included a simultaneous detection of the amplicons by hybridization of two probes labeled with LC-Red and fluorescein by using the fluorescence resonance energy transfer (FRET) technology and melting curve analysis with the LightCycler thermocycler. The assay was first applied successfully on reference strains, reference plasmids, and H. pylori-negative biopsies. Biopsies from 200 patients having failed a first eradication attempt and for whom the H. pylori strain was available were then tested with the new assay. A result was obtained in 199 cases; a single genotype was detected in 157 cases, two genotypes were detected in 41 cases, and three genotypes were detected in one case. There were, in total, seven discrepancies between the real-time PCR and the phenotypic method of determination of clarithromycin susceptibility, and in an additional four cases the two phenotypic methods were in disagreement. PCR-restriction fragment length polymorphism was applied to a sampling of biopsies, including all of the cases with multiple genotypes and all the cases with discrepant results. Finally, in four cases with discrepant results, the real-time PCR detected the resistant population at a concentration so low that it could not be detected by the phenotypic method, while in three cases other mutations could be involved. This assay had an accuracy at least as satisfactory as that of the phenotypic tests and could be performed within 2 h, allowing it to be used before the administration of therapy in the case of a first H. pylori eradication.

Anti-Bacterial Agents↗
PubMed1994

A new point mutation at nucleotide pair 3291 of the mitochondrial tRNA(Leu(UUR)) gene in a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS).

A new point mutation at nucleotide pair 3291 in the mitochondrial tRNA-Leu(UUR) gene was found in a Japanese MELAS patient. The nucleotides at the mutated site were evolutionarily invariant from humans through sea urchins. The mutant genomes were detected in a heteroplasmic fashion in muscle and blood cells of the proband by means of PCR-RFLP. Among 46 MELAS, 5 MERRF, 23 CPEO and 55 normal controls examined, this is the only patient with the mutation. This is the third mutation associated with MELAS in addition to nucleotides at 3243 and 3271. All three mutations occurred within the tRNA-Lue(UUR) region indicating that the tRNA alteration is responsible for the MELAS phenotype.

Animals↗
PubMed1996

A point mutation in exon 7 of the C1-inhibitor gene causing type I hereditary angioedema.

The polymerase chain reaction and nucleotide sequence analysis have been used to characterize a point mutation in the seventh exon of one allele of the C1-inhibitor gene in a family with type I hereditary angioedema. A single base change (C-->T) at nucleotide 1482 in C1-inhibitor converted the codon for Gln-339 to a premature translation termination codon, TAG. Family studies suggest that this mutation is responsible for type I hereditary angioedema in a studied pedigree.

Amino Acid Sequence↗
PubMed2001

Point mutations identified in Lec8 Chinese hamster ovary glycosylation mutants that inactivate both the UDP-galactose and CMP-sialic acid transporters.

Nucleotide-sugar transporters (NSTs) are critical components of glycosylation pathways in eukaryotes. The identification of structural elements that are involved in NST functions provides an important task. Chinese hamster ovary glycosylation mutants defective in nucleotide-sugar transport provide access to inactive transporters that can define such structure/function relationships. In this study, we have cloned the hamster UDP-galactose transporter (UGT) and identified defects in UGT gene transcripts from nine independent Chinese hamster ovary mutants that belong to the Lec8 complementation group. Reverse transcription polymerase chain reaction with primers that span the UGT open reading frame showed that three Lec8 mutants express a full-length open reading frame, while six Lec8 mutants predominantly express truncated UGT gene transcripts. Sequencing identified different single or triplet nucleotide changes in full-length UGT transcripts from three of the mutants. These mutations translate into three different amino acid changes at positions that are highly conserved in all the known mammalian NSTs. Transfection of a cDNA encoding either of the mutations Delta serine 213 or G281D failed to correct the UDP-galactose transport defect in Lec8 transfectants. Most importantly, introducing these same mutations into the homologous region of the murine CMP-sialic acid transporter caused inactivation of this transporter. Thus, identifying point mutations that inactivate UGT in Lec8 mutants resulted in the discovery of amino acids that are critical to the activity of both UGT and CST, the two most divergent mammalian NSTs.

Amino Acid Sequence↗
PubMed1992

Analysis of the tissue distribution and inheritance of heteroplasmic mitochondrial DNA point mutation by denaturing gradient gel electrophoresis in MERRF syndrome.

MERRF (Myoclonic Epilepsy and Ragged-Red Fibres) syndrome is one of the maternally inherited diseases for which a mitochondrial DNA (mtDNA) point mutation has recently been identified. The mutation is always heteroplasmic, that is normal and mutant mtDNA coexist within the same individual. We studied mtDNA heteroplasmy in two families with MERRF syndrome, using a denaturing gradient gel electrophoresis technique that avoids the errors in the evaluation of wild/mutant mtDNA ratios caused by restriction enzyme cutting in the situation of amplification of a heteroplasmic DNA. In two patients, the proportion of muscle mutant mtDNA was in agreement with the severity of muscle mitochondrial proliferation, energy defect and fibre type I predominance. In nine patients from three generations of one family, mutant mtDNA proportion in leukocytes was in relative agreement with the clinical severity of the disease. Transmission of mutant mtDNA through these three generations did not show any tendency toward homoplasmy. Homogeneity of the mutant mtDNA proportion among different tissues from one patient was demonstrated in brain, liver, muscle and heart but a possibility of divergence of the mutant mtDNA proportion during mitosis was documented in cultured skin fibroblasts.

Adolescent↗
PubMed1999

Detection of point mutations by non-isotopic single strand conformation polymorphism.

We have developed a procedure for nonradioactive single strand conformation polymorphism analysis and applied it to the detection of point mutations in the human tumor suppressor gene p53. The protocol does not require any particular facilities or equipment, such as radioactive handling, large gel units for sequencing, or a semiautomated electrophoresis system. This technique consists of amplification of DNA fragments by PCR with specific oligonucleotide primers, denaturation, and electrophoresis on small neutral polyacrylamide gels, followed by silver staining. The sensitivity of this procedure is comparable to other described techniques and the method is easy to perform and applicable to a variety of tissue specimens.

Female↗
PubMed1998

[Regularities of the induction of point mutation in the yeast Saccharomyces cerevisiae after exposure to gamma-radiation].

A tester system consisting of six isogenic strains was used to study the regularities of the induction of point mutations in the yeast Saccharomyces cerevisiae after exposure to gamma-radiation. This system allowed the identification of all six possible substitutions in the Cys-22 codon of the CYC1 gene encoding iso-1-cytochrome c. The dose dependence of the frequency of these six base-pair substitutions was shown to be linear-quadratic. The pattern of base substitutions did not depend on the doses of gamma-irradiation used (from 125 to 1000 Gy) and predominantly included GC-->AT transitions and AT-->TA transversions. The possible mechanisms of gamma-ray mutagenesis leading to a nonlinear dose dependence were considered, and the spectra of mutations obtained for different yeast genes and for Escherichia coli were compared.

Codon↗
PubMed2003

A novel point mutation (I137T) in the conserved 5-phosphoribosyl-1-pyrophosphate binding motif of hypoxanthine-guanine phosphoribosyltransferase (HPRTJerusalem) in a variant of Lesch-Nyhan syndrome.

We identified a novel point mutation (I137T) in the hypoxanthine-guanine phosphoribosyltransferase (HPRT; EC 2.4.2.8) encoding gene, in a patient with partial deficiency of the enzyme (variant of Lesch-Nyhan syndrome). The mutation, ATT to ACT, resulting in substitution of isoleucine to threonine, occurred at codon 137 (exon 6), which is within the region encoding the binding site for 5-phosphoribosyl-1-pyrophosphate (PRPP). We suggest the mechanism by which the mutation-induced structural alteration of HPRT reduced the affinity of the enzyme for PRPP.

Amino Acid Motifs↗
PubMed1998

Integrated amplification and detection of the C677T point mutation in the methylenetetrahydrofolate reductase gene by fluorescence resonance energy transfer and probe melting curves.

A microvolume fluorimeter integrated with a rapid thermal cycler allows both amplification and point mutation detection from genomic DNA in approximately 30 min. This homogeneous method combines rapid cycle DNA amplification with allele-specific fluorescent probe melting profiles for product genotyping. The amplification reaction includes a primer internally labeled with Cy5 and a 3'-fluorescein-labeled probe that spans the region of interest. During asymmetric amplification, the probe hybridizes to excess Cy5-labeled strand and is observed as fluorescence resonance energy transfer. Resonance energy transfer increases each cycle as product accumulates during amplification. When fluorescence is monitored as the temperature increases through the Tm of the probe/product duplex, a characteristic melting profile for each genotype is obtained. Fluorescence genotyping of the common C677T base substitution in the methylenetetrahydrofolate reductase gene in 110 DNA samples correlated perfectly with genotyping by restriction enzyme digestion and gel electrophoresis. The relatively stable G:T mismatch of this example gave a 3 degrees C difference in Tm from complete Watson-Crick pairing, suggesting that this homogeneous fluorescence method can be used for all single-base mismatches.

Alleles↗
PubMed2005

A point mutation of the ED1 gene in a Japanese family with X-linked hypohidrotic ectodermal dysplasia.

X-linked hypohidrotic ectodermal dysplasia (EDA) is characterized by the hypoplasia or absence of hair, teeth and sweat glands. In this study, the authors investigated the ED1 gene in a Japanese family with X-linked hypohidrotic ectodermal dysplasia. The only affected male fulfils the diagnostic criteria for this disorder. His parents were not consanguineous and both of them were healthy. After informed consent, genomic DNA was isolated from the peripheral blood lymphocytes or oral buccal epithelial cells of all members of the family. A polymerase chain reaction fragment containing exon 9 of the ED1 gene was amplified using primers. The patient's amplified fragment, as well as those from his father, mother and sister, were directly sequenced. The sequence from the patient revealed a point mutation (G1149A) in exon 8 of the ED1 gene, which changes codon 291 from glycine to arginine. Heterozygosity was demonstrated in his mother and sister. This mutation has not been reported previously. The amino acid substitution is predicted to disrupt the transmembrane domain, which strongly implies that this is the disease-causing mutation in the family.

Amino Acid Substitution↗
PubMed1991

Discrimination between recurrent mutation and identity by descent: application to point mutations in exon 11 of the cystic fibrosis (CFTR) gene.

A total of 75 non-delta F508 chromosomes from 59 German cystic fibrosis patients was screened for mutations in exon 11 of the cystic fibrosis (CFTR) gene. These Caucasian patients were found to possess an identical haplotype background for two common mutations (G551D, R553X) consistent with their being identical by descent. However, a different R553X associated haplotype found in American black patients was suggestive of recurrent mutation, a postulate supported by the location of the R553X alteration in a hypermutable CpG dinucleotide. Likelihood estimates for recurrent mutation and identity by descent were compared and strongly supported the hypothesis of recurrent R553X mutation. The ability to distinguish between these two alternatives provides an indication of whether or not the search for mutations should be restricted to chromosomes with similar haplotypes.

Cystic Fibrosis↗
PubMed1999

Molecular dynamics of labeled oligonucleotide probes used for the detection of point mutations in DNA.

The fluorescent label BODIPY 576-589 linked to the 5-end of an oligonucleotide via alkyl chain linkers can be used as a probe to detect point mutations in DNA. We have employed fluorescence anisotropy decay and dynamic fluorescence resonance energy transfer (FRET) in order to investigate the molecular origin of the fluorescence lifetime aberrations of BODIPY in the presence of a mismatched base. The results show that both, an increased flexibility of the alcyl chain linker to the BODIPY molecule as well as relaxation of the secondary structure of the whole complex, contribute to the decreased fluorescence lifetimes reported previously.

Base Pair Mismatch↗
PubMed1996

Hypertension-associated point mutations in the adducin alpha and beta subunits affect actin cytoskeleton and ion transport.

The adducin heterodimer is a protein affecting the assembly of the actin-based cytoskeleton. Point mutations in rat adducin alpha (F316Y) and beta (Q529R) subunits are involved in a form of rat primary hypertension (MHS) associated with faster kidney tubular ion transport. A role for adducin in human primary hypertension has also been suggested. By studying the interaction of actin with purified normal and mutated adducin in a cell-free system and the actin assembly in rat kidney epithelial cells (NRK-52E) transfected with mutated rat adducin cDNA, we show that the adducin isoforms differentially modulate: (a) actin assembly both in a cell-free system and within transfected cells; (b) topography of alpha V integrin together with focal contact proteins; and (c) Na-K pump activity at V(max) (faster with the mutated isoforms, 1281 +/- 90 vs 841 +/- 30 nmol K/h.mg pt., P < 0.0001). This co-modulation suggests a role for adducin in the constitutive capacity of the epithelia both to transport ions and to expose adhesion molecules. These findings may also lead to the understanding of the relation between adducin polymorphism and blood pressure and to the development of new approaches to the study of hypertension-associated organ damage.

Actins↗
PubMed2000

Familial form of hirschsprung disease: nucleotide sequence studies reveal point mutations in the RET proto-oncogene in two of six families but not in other candidate genes.

Hirschsprung disease (HSCR; McKusick 142623) or aganglionic megacolon is a frequent (1 in 5,000 live births) heritable disorder of the enteric nervous system. By haplotyping with a variety of microsatellite markers, by amplifying all 20 exons of the RET proto-oncogene and by applying a direct DNA sequencing protocol, we have analyzed the DNA from HSCR patients in 6 different families. In one family with a joint occurrence of HSCR and FMTC (follicular medullary thyroid carcinoma), we have identified a mutation in codon 609 in one out of 6 cysteine residues encoded in exon 10 of the RET gene. This C609R point mutation has not previously been reported to cause HSCR. In 2 of the HSCR patients described here from different families, we have found a mutation in exon 2 (R77C) and a silent mutation in exon 3 (Y204Y), respectively, in the extracellular part of the RET proto-oncogene. In introns 2 and 17 of the RET proto-oncogene in 2 families, we have detected single nucleotide exchanges that are probably polymorphisms with unknown, if any, relations to HSCR. The DNA sequences of 5 further genes (GDNF, GDNFRalpha, EDN3, EDNRB, and NTN), that may contribute to the development of HSCR, have not shown mutations in the patients analyzed so far. In 2 of the reported families with several affected children and one grandchild, sequence analyses revealed no mutations in the coding regions of any of the candidate genes analyzed.

Adult↗
PubMedDate not supplied

[On control of radioactive-chemical protection and point mutations by inhibitors of free radical reactions].

An opportunity to control the DNA protection against radiation in cells and the formation of 2-desoxyribose residue epimers (sites of potential point mutations) by using a set of free radical scavengers differing by parameters of availability (molecular mask and access window) is discussed. The values of steric probability (efficiency) of formation of a chemical bond between atoms Cn of a 2-desoxyribosyl radical (n from 1 to 5) and H-atom of a scavenger donor group were determined. It was shown that the maximum effect of radioprotection determined, for example, by transformations of C2 radical reached 25% (4-oxy-3,5-di-tert-butyl-alpha-methylbenzylamine) and maximum yield of C4-epimer was 15.5% (L-cysteine).

Animals↗