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At least 505 records · Page 28Linked to original sources
PubMed1994

Detection of point mutations in the androgen receptor gene using non-isotopic single strand conformation polymorphism analysis. German Collaborative Intersex Study Group.

Point mutations in the androgen receptor gene cause androgen insensitivity syndromes, clinically characterized by masculinization defects in karyotypic males due to endorgan resistance to androgenic steroids. Characterization of these mutations with single strand conformation polymorphism analysis utilizing radioactive PCR can serve as a diagnostic tool for molecular subclassification of these syndromes. It is the basis for genetic counseling and for therapeutic decisions. Here we report an improved non-radioactive single strand polymorphism analysis for rapid detection of androgen receptor gene mutations in affected individuals. In addition to previously reported mutations, 10 patients with clinical features of androgen resistance were studied. DNA was isolated from peripheral blood leucocytes and exons 1 to 8 of the coding region of the androgen receptor gene amplified by PCR. Amplification products were denatured and run on non-denaturing gels. These were subjected to fixation and silver staining. Variations were directly sequenced. In all patients a different point mutation in one of the exons was detected. While one insertion mutation was found in a patient with complete androgen insensitivity, all other mutations cause amino acid substitutions. These data suggest that the described non-radioactive single strand polymorphism analysis is a useful tool for the characterization of androgen receptor gene mutations. The omission of radioisotopes is advantageous in a clinical setting. The mutations described emphasize the clinical and molecular heterogeneity of this disease.

Amino Acid Sequence↗
PubMed1997

[A case of probable Creutzfeldt-Jakob disease with a point mutation of prion protein gene codon 180 and atypical MRI findings].

A case of probable Creutzfeldt-Jakob disease (CJD) is reported. A 70-year-old Japanese woman was admitted with a complaint of amnesia. She initially developed Klüver-Bucy syndrome, hypergraphia, and later showed myoclonus and startle response. She developed akinetic mutism within nine months from the onset. Prion protein gene codon 180 point mutation (Met/Ile) was detected and we diagnosed her as CJD. Serial MRI studies revealed abnormal T2 elongation and thickening limited to the cerebral cortices, which started at bilateral temporal lobes and later extended to frontal, parietal, and occipital lobes with relative sparing of hippocampi and central gyri. Serial EEG did not show periodic synchronous discharge (PSD). Three cases of CJD with a point mutation of codon 180 were reported in the past. There are several common features in the past cases and the present case, i.e. 1) negative familial history, 2) late onset, 3) T2 high intensity at cerebral cortices on MRI, and 4) negative PSD. These may be characteristic features of CJD with a point mutation of codon 180.

Aged↗
PubMed1995

Segregation of mitochondrial DNAs carrying a pathogenic point mutation (tRNA(leu3243)) in cybrid cells.

To investigate the mechanism of segregation of mitochondrial DNAs (mtDNAs) carrying a pathogenic point mutation in the tRNA(leu(UUR)) gene (A to G at position 3243) cytoplasts, derived from a heteroplasmic myoblast clone, were fused to rhoo cells to create cybrid cells carrying different proportions of mutant and wild-type mtDNAs. Although the individual myoblasts used as fusion partners contained predominantly mutant mtDNAs (mean proportion 0.77, range 0.46-0.94), the majority (56%) of the cybrid clones isolated after growth in selective medium were homoplasmic wild-type, indicating preferential replication of wild-type mtDNAs. Long-term culture of heteroplasmic cybrid clones in non-selective medium produced no change in the mean proportion of wild-type mtDNAs but an increase in population variance, consistent with a purely stochastic segregation mechanism. These results are at variance with a previous report suggesting a replicative advantage for mtDNAs carrying the tRNA(leu3243) point mutation.

Adenine↗
PubMed1994

Efficient detection of point mutations on color-coded strands of target DNA.

Presently available methods for screening large genetic regions for unknown point mutations are neither flawless nor particularly efficient. We describe an approach, especially well suited to identifying mutations present in the heterozygous state, that combines several improvements in a protocol called fluorescence-assisted mismatch analysis (FAMA). Appropriate gene regions of the wild-type and the putative mutant allele are simultaneously amplified from genomic DNA by using the polymerase chain reaction, and large DNA fragments, so far up to 800 bp, are end labeled with strand-specific fluorophores. Aliquots are denatured and reannealed to form heteroduplexes and subjected to conventional cytosine- and thymine-specific modifications. Cleavages occurring on opposite strands are detected by denaturing gel electrophoresis using an automated DNA sequencer. Since the DNA fragments derived from the mutant allele are also end labeled, the number of informative mispaired bases is doubled compared to conventional searches using wild-type probes. The sensitivity of detection is also increased, because differential fluorescent end labelling allows the identification and measurement of strand-specific background cleavages at matched cytosine or thymine residues. Automatic superimposition of tracings from different subjects allows mismatch detection at sites that, because of the nature of the bases involved and of the neighboring sequence, are known to be less susceptible to cleavage. The effects of the latter parameters have been studied quantitatively on a series of point mutations found in the human C1-inhibitor gene in patients affected by hereditary angioedema. Dilution experiments have demonstrated that most mutations are detected even when the mutant chromosome is diluted 10-fold or more compared with the normal one.

Angioedema↗
PubMed1996

NIRCA: a rapid robust method for screening for unknown point mutations.

We describe a method for screening for dispersed point mutations, based on the observation that RNase frequently cleaves both strands of base pair mismatches in duplex RNA targets. The mismatched substrates are generated by in vitro transcription of wild-type and mutant templates amplified by the PCR or reverse transcription (RT)-PCR; bacteriophage promoters are incorporated into the PCR primers to permit both strands of the products to be transcribed into RNA. Complementary wild-type and mutant transcripts are hybridized and treated with RNase, and the cleavage products are separated on agarose gels and detected by visualization of the ethidium-stained sample under UV light. The method is thus non-isotopic, and since the cleavage products remain double-stranded during analysis, the labor-intensive RNase inactivation steps required in the original procedure can be eliminated. Also, nonspecific background cleavage is reduced so that longer target regions (1 kb) can be screened in a single step. The Non-Isotopic RNase Cleavage Assay (NIRCA) achieved a detection rate of 88%-90% in blind studies in a Factor IX model system, and it was also used to detect unknown p53 mutations in breast tumor samples. NIRCA provides a rapid method for sensitive, non-isotopic, high-throughput genetic screening.

Base Sequence↗
PubMed2000

[A case of NARP (neurogenic muscle weakness, ataxia, and retinitis pigmentosa) with a T-to-C point mutation at nt 8993 of mitochondrial DNA].

We report a case of NARP with a T-to-C point mutation at nt 8993 of mitochondrial DNA. A 37-year old man with mild mental retardation, retinitis pigmentosa, and clonic-tonic seizure was admitted to our hospital. The neurological examination revealed scanning speech, dystonic neck turning to the left side, and pyramidal tract signs. Serum-, CSF-lactate and pyruvate level were slightly elevated. Brain MRI findings showed cerebral atrophy, cerebellar cortical atrophy accompanied with dilation of forth ventricle, and high intensity lesions in the bilateral lenticular nuclei on T2 weighted images. Nucleotide sequence analysis of the mitochondrial DNA in the leukocytes demonstrated a T-to-C point mutation at nt 8993. To our knowledge, this is the first report of a Japanese patient with NARP associated with the T-to-C mutation at nt 8993 of mt DNA. Mitochondrial DNA analysis should be considered in the differential diagnosis of patients with retinitis pigmentosa and various neurological signs.

Adult↗
PubMed1996

Point mutation of the p53 gene is an infrequent event in untreated prostate cancer.

The p53 gene is known to be one of the frequently altered tumor suppressor genes, involved in the oncogenesis of a wide spectrum of human malignant tumors. We investigated mutational events of the p53 gene in 18 clinically untreated prostate cancers. Direct sequencing analysis demonstrated that 1 of 18 cases harbored point mutation in the highly conserved transcript region. The case showed CAT at codon 273 instead of wild-type CGT, substituting the encoded amino acid form histidine to arginine. The case had previously revealed homozygous loci on 17p, including the p53 locus, by restriction fragment length polymorphism analysis. The other 17 cases harbored neither mutation nor small deletion. It is concluded that point mutation of the p53 gene is a infrequent event in the oncogenesis of untreated prostate cancer.

Base Sequence↗
PubMed2007

Protein- and mRNA-based phenotype-genotype correlations in DMD/BMD with point mutations and molecular basis for BMD with nonsense and frameshift mutations in the DMD gene.

Straightforward detectable Duchenne muscular dystrophy (DMD) gene rearrangements, such as deletions or duplications involving an entire exon or more, are involved in about 70% of dystrophinopathies. In the remaining 30% a variety of point mutations or "small" mutations are suspected. Due to their diversity and to the large size and complexity of the DMD gene, these point mutations are difficult to detect. To overcome this diagnostic issue, we developed and optimized a routine muscle biopsy-based diagnostic strategy. The mutation detection rate is almost as high as 100% and mutations were identified in all patients for whom the diagnosis of DMD and Becker muscular dystrophy (BMD) was clinically suspected and further supported by the detection on Western blot of quantitative and/or qualitative dystrophin protein abnormalities. Here we report a total of 124 small mutations including 11 nonsense and frameshift mutations detected in BMD patients. In addition to a comprehensive assessment of muscular phenotypes that takes into account consequences of mutations on the expression of the dystrophin mRNA and protein, we provide and discuss genomic, mRNA, and protein data that pinpoint molecular mechanisms underlying BMD phenotypes associated with nonsense and frameshift mutations.

Adolescent↗
PubMed1994

Identification of the DNA-binding domains of the switch-activating-protein Sap1 from S.pombe by random point mutations screening in E.coli.

Mating type switching in fission yeast, Schizosaccharomyces pombe, is initiated by a site-specific double-strand break (DSB) at the mat1 locus. The DSB is controlled from a distance by cis- and trans-acting elements. The switch-activating protein, Sap1 binds to the SAS1 cis-acting element which controls the frequency of the DSB at the mat1 locus and, consequently the efficiency of mating type switching. We developed a general method for screening randomly mutagenized expression libraries of DNA-binding protein in E.coli. Sap1 gene was mutagenized by PCR under conditions of reduced Taq polymerase fidelity. The mutated DNA was expressed in E.coli and screened for SAS1-recognition. This method was used to isolated 16 point mutations that abolished SAS1 interaction together with 18 mutations that did not affect binding. The position of these point mutations allowed the identification of three protein domains located in the N-terminal part of Sap1 that are essential for DNA-binding. Deletions and biochemical analysis showed that Sap1 is a dimer both in solution and when bound to SAS1 sequence. The dimerization domain was localized C-terminally to the three domains described above and when used in exess it inhibited DNA binding.

Base Sequence↗
PubMed1997

Capillary electrophoresis for the detection of known point mutations by single-nucleotide primer extension and laser-induced fluorescence detection.

Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) was used to detect known point mutations using the method of single-nucleotide primer extension (SNuPE). Three different point mutations in human mitochondrial DNA associated with Leber's hereditary optic neuropathy (LHON) were detected by annealing a primer immediately 5' to the mutation on the template and extending the primer by one fluorescently labeled dideoxy terminator complementary to the mutation. By using two or more differently labeled terminators, both the mutant and wild type could be simultaneously detected. The advantages of using CE-LIF for detecting SNuPE reactions include speed and ease of analysis, absence of radioactivity, and potential for automation.

Base Sequence↗
PubMed1994

Low incidence of point mutations detected in the p53 tumor suppressor gene from chemically induced rat renal mesenchymal tumors.

Previous studies have shown renal mesenchymal tumors (RMTs) induced in rats by a single intrarenal injection of nickel subsulfide and iron are more pleomorphic and metastatically aggressive than RMTs induced by a single ip injection of methyl(methoxymethyl)nitrosamine (DMN-OMe). While both RMT types contain high levels of K-ras activation, the specific mutational spectra within codon 12 of K-ras are quite different. Nickel subsulfide and iron-induced tumors exhibited codon 12 GGT-->GTT transversions exclusively, while DMN-OMe RMTs showed a wide array of codon 12 mutations, as well as mutations within codons 61 and 63 [K. G. Higinbotham, J. M. Rice, B. A. Diwan, K. S. Kasprzak, C. D. Reed, and A. O. Perantoni, Cancer Res., 52: 4747-4751, 1992; K. G. Higinbotham, J. M. Rice, and A. O. Perantoni, Mol. Carcinog., 5: 136-139, 1992]. In an effort to further correlate carcinogen-specific molecular events in renal tumors, we investigated the p53 tumor suppressor gene in RMTs induced by these two carcinogens for the presence of point mutations. The evolutionarily conserved portion of the coding region of the gene, including part of exon 4 through exon 10, was surveyed for point mutations utilizing single-strand conformation polymorphism and chemical cleavage of mismatches analyses. None (0 of 10) of the nickel subsulfide and iron-induced RMTs and only 1 of 10 DMN-OMe-induced tumors that were evaluated contained point mutations within this portion of the p53 gene. Direct sequencing of the one single-strand conformation polymorphism and chemical cleavage of mismatches-"positive" DMN-OMe-induced RMT revealed a GCC-->GTC (Ala-->Val) transition in codon 345 within exon 10. These results suggest that the different tumorigenic phenotypes exhibited by these two RMTs are not the result of specific mutations or patterns of mutations within the portion of the p53 gene examined and that the mutated p53 tumorigenic pathway, whereby p53 plays a major role in many human neoplasms, does not function in RMTs induced by either agent.

Animals↗
PubMed1998

[Detection of point mutations on the Ki-ras gene using the PCR technique].

Present study was undertaken to detect Ki-ras point mutation at codon 12 in pancreatic adenocarcinomas (CaP) using the polymerase chain reaction-restriction fragment lengths polymorphism (PCR-RFLP). Three modifications of PCR-RFLP were performed with a mismatched primers creating a recognition site with only one allelic from (wild or mutated). Using two-step PCR-RFLP and two modifications of one-step PCR-RFLP we examined 5 resected adenocarcinomas of pancreas, 6 pancreatic juices and one DNA sample from peripheral blood of patient with generalized stadium of CaP. We compare all techniques and conclude, that the very sensitive two step PCR-RFLP is a suitable method for detection point mutations and eliminates the need for either oligonucleotide hybridization or DNA sequencing.

Adenocarcinoma↗
PubMed1992

Generation and analysis of random point mutations in an antibody CDR2 sequence: many mutated antibodies lose their ability to bind antigen.

We have investigated the impact of mutations on the binding functions of the phosphocholine (PC)-specific T15 antibody in the absence of antigen selection pressure. The H chain complementarity determining region 2 (CDR2) sequence of T15 antibody was saturated with point mutations by in vitro random mutagenesis. From the mutant library, 289 clones were screened by direct DNA sequencing. The point mutations generated by this method were randomly distributed throughout the CDR2 region and included all kinds of substitutions. 46 unique mutant antibodies, containing one to four point mutations each, were expressed in SP2/0 myeloma cells. Functional analysis on these antibodies has provided insights into several aspects of somatic mutation. (a) The majority (26/46) of mutant antibodies either lost (20/46) or had reduced (6/46) ability to bind PC-protein conjugates or R36a, a PC-expressing strain of Streptococcus pneumoniae. In contrast, none of the mutant antibodies displayed increased binding for these PC antigens. Taken together with calculations of destructive mutations elsewhere in the V region, the data suggest that somatic mutation may cause extensive wastage among B cells during clonal expansion after antigen stimulation. (b) The frequency of binding-loss mutants increased sharply when a second mutation was introduced into the CDR2 sequence; it appears that, in some cases, two or more mutations are needed to destroy binding. (c) The mutant antibodies were tested for their reactivity to 11 non-PC antigens as well as to three PC analogues. None of the mutants gained new reactivity or changed their ability to discriminate structural analogues, supporting the notion that the major role of somatic mutation is to increase or decrease affinity rather than to create new specificities. (d) Mutations in at least five different positions in CDR2 were deleterious, suggesting that these residues may be essential for antigen binding. Three of these positions are novel in that they had not been identified to be important for binding PC by previous crystallographic analysis. (e) Introduction of mutations into two highly conserved residues in CDR2 did not alter the overall conformation of the V region as judged by antiidiotypic analysis, and, in some cases, did not affect the antigen binding function. The results thus indicate that even nonconservative substitutions of invariant residues need not be deleterious, suggesting that their conservation may be due to reasons other than maintaining antibody structure or specificity.

Amino Acid Sequence↗
PubMed2005

Molecular profile of hyalinizing trabecular tumours of the thyroid: high prevalence of RET/PTC rearrangements and absence of B-raf and N-ras point mutations.

Hyalinizing trabecular tumour (HTT) of the thyroid is a neoplasm of follicular derivation that shares several morphological similarities with papillary thyroid carcinoma (PTC). In this study, we investigated the prevalence of B-raf point mutations, RET/PTC rearrangements and N-ras point mutations in a large HTT series (28 samples). Twenty benign thyroid lesions and 10 PTC served as control cases. A high (47%) prevalence of RET/PTC rearrangements was found in HTT. By contrast, neither B-raf nor N-ras mutations were found in HTT. These findings suggest that, although RET/PTC, N-ras, and B-raf proteins may act along the same signalling cascade, the biological and morphological outcome of their oncogenic activation is not completely overlapping. Thus, in clinical practice, the detection of B-raf mutations in a thyroid follicular tumour may prove to be a valuable tool, supplementing histological examination, and allowing a differential diagnosis between PTC and HTT.

Adult↗
PubMed1996

A point mutation within each of two ATP-binding motifs inactivates the functions of elongation factor 3.

We have investigated how point mutations in the two ATP-binding motifs (G(463)PNGCGK(469)ST and G(701)PNGAGK(707)ST) of elongation factor 3 (EF-3) affect ribosome-activated ATPase activity of EF-3, polyphenylalanine synthesis, and growth of Saccharomyces cerevisiae. The point mutation impaired the ribosome-activated ATPase activity of EF-3, when glycine(463 and 701) and lysine(469 and 707) were replaced with valine and arginine, respectively. Thus, each glycine and lysine residue in both ATP-binding motifs is indispensable for EF-3's binding with ATP and the ensuing generation of ribosome-activated ATPase activity. Additionally, the mutant EF-3s did not catalyze polyphenylalanine synthesis in vitro when each glycine(463 and 701) was replaced with valine. The mutant EF-3s did not support cell growth in TEF3-disrupted S. cerevisiae, when each lysine(469 and 707) and glycine(463) was replaced with arginine and valine, respectively. Thus, each of the two ATP-binding motifs of EF-3 is indispensable for the ribosome-activated ATPase activity of EF-3, which is required for protein synthesis and cell growth in S. cerevisiae.

Adenosine Triphosphatases↗
PubMed2005

Glucocorticoid receptor point mutation V571M facilitates coactivator and ligand binding by structural rearrangement and stabilization.

Two-point mutations in the human glucocorticoid receptor have been studied by computer simulations to rationalize experimental data, where mutants comprising the V571M substitution improve both transcriptional activity and affinity for aldosterone despite large distances between the mutated residue and the coactivator-binding surface and ligand-binding pocket, respectively. Our molecular dynamics simulations show that the V571M mutation modifies the coactivator-binding site defined by helices 3, 4, and 12, and that specific structural rearrangement of the coactivator-binding site correlates well with transactivation data. A similar reorganization of the coactivator-binding cleft is observed in crystallographic structures of the estrogen receptor in the presence of coactivator peptide, compared with structures without peptide, indicating that induced fit for coactivator binding is a general phenomenon for nuclear receptors. These results suggest that the V571M substitution facilitates recruitment of coactivator protein by promotion of a conformational substate reducing the energetic penalty for the induced fit of the receptor-coactivator complex. Furthermore, our simulations of V571M mutants showed reduced fluctuations of residues lining the ligand-binding pocket. This indicates that a reduction of the entropic cost for ligand binding may explain the increased affinity of V571M mutants for certain ligands.

Allosteric Site↗
PubMed1999

Analysis of K-ras codon 12 point mutations using duodenal lavage fluid for diagnosis of pancreatic carcinoma.

We evaluated the diagnostic significance of the K-ras point mutations at codon 12 in duodenal lavage fluid (DLF) compared with pure pancreatic juice (PPJ). The DLF was easily and safely collected by injecting distilled water into the duodenum and then aspirating through the working channel of the endoscope during endoscopic retrograde cholangiopancreatography. Two types of DLF are collected this way: DLF 1 is collected just after insertion of the endoscope into the duodenum and DLF 2 is collected after cholangiopancreatography and/or collection of the PPJ using secretin. Analysis of K-ras mutations was performed using enriched polymerase chain reaction. In patients with pancreatic carcinoma (PC), K-ras mutations were detected in 14 of 23 (60.9%) in DLF 1, 16 of 21 (76.2%) in DLF 2, 14 of 20 (70.0%) in PPJ, and 19 of 21 (90.5%) in either DLF 1 or DLF 2. In patients with noncancerous pancreatic diseases consisting of pancreatic cystic diseases and chronic pancreatitis, the incidence of K-ras mutations was 2 of 21 (9.5%) in DLF 1 and 7 of 19 (36.8%) in DLF 2. These values were lower than that in PPJ, and there was significant difference between the incidence in DLF 1 and PPJ. These results suggested that DLF may provide a new and useful material for analysis of K-ras codon 12 point mutations in the diagnosis of PC.

Adenocarcinoma↗
PubMed1993

Screening for point mutations by semi-automated DNA sequencing using sequenase and magnetic beads.

We have established an improved method for detecting point mutations by semi-automated DNA sequencing of PCR fragments generated from genomic DNA. The method employs magnetic beads to create immobilized single-stranded DNA templates, and the sequencing reaction is performed with Sequenase. This method is superior to sequencing with Taq DNA polymerase because the uniform peak height with Sequenase makes heterozygosity easily detectable as double peaks that are half the normal height. Detection of heterozygosity by this method is illustrated by sequencing a 180-bp fragment of the human apolipoprotein B gene. This fragment contains codon 3500, where a point mutation (3500CGG-->CAG) is found in subjects with the autosomal dominant disease familial defective apolipoprotein B. The nonuniform peak height with Taq DNA polymerase makes it more difficult to detect heterozygosity. This is also illustrated by sequencing a 278-bp fragment of the low-density lipoprotein receptor gene.

Apolipoproteins B↗