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At least 973 records · Page 54Linked to original sources
PubMed1996

Point mutations in the Ki-ras2 gene of codon 12 in the Dunning R-3327 Prostatic Adenocarcinoma system.

Detection of point mutations in the Dunning System with the Ki-ras2 gene in the first and second positions of codon 12 exon I, has been performed using allele-specific oligonucleotide (ASO) primers. PCR generated 94bp templates and genomic DNA extracted from the Dunning cell lines AT-1, AT-3, Mat-Lu, and Mat-Lylu were tested. Our investigation revealed that the first and second positions of codon 12 have undergone either transitions or transversions from the wild type sequence (GG). Dorsal prostate (DP) solid tissues used as controls revealed the wild type configuration as well as transversions at both positions, in addition to a transition in the first position for Mat-Lu. The most aberrant of the Dunning lines (AT-3 and Mat-LyLu) collectively displayed sequence changes (transitions and transversions) with no evidence of the wild type configuration. These findings are supportive of biometric studies (area, shape factor, and motility) along with adhesion and invasion assays done in our laboratory in correlating genotypic and phenotypic properties to metastatic potential.

Adenocarcinoma↗
PubMed1993

Quantification of mitochondrial DNA carrying the tRNA(8344Lys) point mutation in myoclonus epilepsy and ragged-red-fiber disease.

Myoclonus epilepsy and ragged-red-fiber syndrome (MERRF) is caused by a point mutation at nucleotide 8344 in the tRNA(Lys) gene of mitochondrial DNA. We analyzed leukocyte DNA from nine members of a large MERRF family using a new technique, solid-phase minisequencing. Quantitative analysis of the tRNA(8344Lys) mutation showed that the mutated mtDNA comprised from 9 to 72% of the total mtDNA in the leukocytes of these individuals. The minisequencing method is a promising tool for the diagnosis of MERRF. In addition to the identification of the tRNA(8344Lys) mutation, the relative amount of mutated mtDNA can be simultaneously determined in the same assay from one blood sample.

Age Factors↗
PubMed2005

Fabrication of DNA microarrays onto polymer substrates using UV modification protocols with integration into microfluidic platforms for the sensing of low-abundant DNA point mutations.

We describe the microfabrication and operational characteristics of a simple flow-through biochip sensor capable of detecting low abundant point mutations in K-ras oncogenes from genomic DNA, which carry high diagnostic value for colorectal cancers. The biochip consisted of an allele-specific ligase detection reaction (LDR) coupled to a universal array for interrogating multiple mutations simultaneously from a clinical sample. The integrated sensing platform was micro-manufactured from two different polymers, polycarbonate, PC, which was used for the LDRs, and poly(methyl methacrylate), PMMA, which was used to build the microarray. Passive elements were hot embossed into the PC and PMMA microchips and then, the chips assembled into a three-dimensional architecture with the interconnect fabricated from an elastomer, poly(dimethylsiloxane), PDMS, to produce a leak-free connection between the biochips. The array in PMMA was produced using a photomodification process, which involved three steps; (1) UV (254 nm) exposure of the polymer surface; (2) EDC coupling of amine-terminated oligonucleotide probes to the surface (via an amide bond) and; (3) washing of the surface. The LDR/hybridization flow-through biochip performed the entire assay at a relatively fast processing speed: 6.5 min for on-chip LDR, 10 min for washing, and 2.6 min for fluorescence scanning (total processing time=19.1 min) and could screen multiple mutations simultaneously for high throughput applications at a level of one mutant sequence in 100 wild-type sequences.

Biosensing Techniques↗
PubMed1998

Prognostic value of RET proto-oncogene point mutations in malignant and benign, sporadic phaeochromocytomas.

Somatic mutations in the RET proto-oncogene are involved in the pathogenesis of an important subset (40-60%) of sporadic medullary thyroid carcinomas (MTCs) and less frequently (0-31%) in benign, sporadic phaeochromocytomas. Since limited data exist regarding the significance of somatic RET mutations in malignant phaeochromocytomas, we analysed a multicentre series of proven malignant (i.e., metastasised) phaeochromocytomas. Analogous with MTCs, where RET mutations lead to an aggressive behaviour, we hypothesised that somatic mutations would occur more frequently in malignant than in benign phaeochromocytomas. Paraffin-embedded tissue was available from 29 malignant and 27 benign phaeochromocytomas. Exons 10, 11 and 16 were analysed by non-radioactive single-strand conformation polymorphism, heteroduplex gel electrophoresis, restriction enzyme digestion and aberrant band patterns by non-isotopic sequencing. In only 1 of 29 malignant phaeochromocytomas was a mis-sense mutation found (at codon 634 of exon 11), whereas in 15% (4/27) of the benign tumours a point mutation was detected (in 3 tumours in exon 16 at codon 918 and in 1 tumour in exon 10 at codon 618). Absence of these mutations in non-tumourous DNA proved their somatic origin. Contrary to what has been reported for MTCs, oncogenic RET mutations are not associated with an aggressive tumour behaviour in sporadic phaeochromocytomas.

Adolescent↗
PubMed1987

Elevated expression and point mutation of the Ha-ras proto-oncogene in mouse skin tumors promoted by benzoyl peroxide and other promoting agents.

The two-stage skin carcinogenesis model of initiation and promotion in SENCAR mice has been used to examine the effects of various tumor-promoting agents on the expression of the Ha-ras oncogene in early stages of tumorigenesis in vivo. Papillomas were induced in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated SENCAR mouse epidermis by (i) complete promotion with benzoyl peroxide; (ii) complete promotion with 12-O-tetradecanoyl phorbol-13-acetate (TPA); and (iii) two-stage promotion with TPA for 2 weeks followed by mezerein for 9 weeks. Results of Northern blot hybridization analyses show that early papillomas contain significantly elevated levels of Ha-ras polyadenylated [poly(A)+] RNA, irrespective of the type of tumor promotion regimen used. This pattern holds for promoters of the phorbol ester class as well as for the free radical generating agent benzoyl peroxide. Furthermore, digestion of tumor DNA with diagnostic restriction endonucleases demonstrated that 9-week-old papillomas induced by DMBA contained a point mutation in the 61st codon of one allele of the Ha-ras gene. The results represent the earliest stage in the development of a papilloma at which a Ha-ras point mutation has been reported.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1992

Cytotoxic T lymphocytes (CTL) against a transforming gene product select for transformed cells with point mutations within sequences encoding CTL recognition epitopes.

The 94-kD large tumor (T) antigen specified by simian virus 40 (SV40) is sufficient to induce cell transformation. T antigen contains four H-2Db-restricted cytotoxic T lymphocyte (CTL) recognition epitopes that are targets for CTL clones Y-1, Y-2, Y-3, and Y-5. These epitopes have been mapped to T antigen amino acids 207-215 (site I), 223-231 (sites II and III), and 489-497 (site V), respectively. Antigenic site loss variant cells that had lost one or more CTL recognition epitopes were previously selected by coculturing SV40-transformed H-2Db cells with the site-specific Db-restricted CTL clones. The genetic bases for T antigen CTL recognition epitope loss from the variant cells were identified by DNA amplification and direct sequencing of epitope-coding regions from variant cell DNAs. Cells selected for resistance to CTL clone Y-1 (K-1; K-1,4,5; K-3,1) carry deleted SV40 genomes lacking site I, II, and III coding sequences. Point mutations present within the site II/III coding region of Y-2-/Y-3-resistant cell lines specify the substitution of asparagine for lysine as T antigen amino acid 228 (K-2) or phenylalanine for tyrosine at position 230 (K-3). Point mutations identified within independently selected Y-5 resistant populations (K-5 and K-1,4,5) direct the substitution of isoleucine for asparagine at position 496 (K-5) or the substitution of phenylalanine for isoleucine at position 491 (K-1,4,5) of T antigen. Each substitution causes loss of the relevant CTL recognition epitope, apparently by compromising CTL T cell receptor recognition. These experiments identify specific amino acid changes within a transforming protein that facilitate transformed cell escape from site-specific CTL clones while allowing maintenance of cellular transformation. This experimental model system provides unique opportunities for studying mechanisms of transformed cell escape from active immunosurveillance in vivo, and for analysis of differential host immune responses to wild-type and mutant cell-transforming proteins.

Amino Acid Sequence↗
PubMed1994

Cytoplasmic transfer of the mtDNA nt 8993 T-->G (ATP6) point mutation associated with Leigh syndrome into mtDNA-less cells demonstrates cosegregation with a decrease in state III respiration and ADP/O ratio.

A point mutation in the mtDNA-encoded ATP6 gene (T-->G at nt 8993) associated with Leigh syndrome in two pedigrees was found to decrease ADP-stimulated (state III) respiration and the ratio of ADP molecules phosphorylated to oxygen atoms reduced (ADP/O ratio) but did not affect 2,4-dinitrophenol (DNP)-uncoupled respiration, suggesting a defective mitochondrial H(+)-translocating ATP synthase. Intact mitochondria isolated from patient and control lymphoblastoid cell lines were tested for state III, ADP-limited (state IV), and DNP-uncoupled respiration with various substrates. Mitochondria isolated from patient lymphoblasts harboring 95-100% of mtDNAs carrying the nt 8993 T-->G mutation showed state III respiration rates 26-50% lower than controls while having normal DNP-uncoupled rates. This resulted in state III/DNP ratios of 0.52-0.70 in patient mitochondria versus 0.88-0.97 in controls. The ADP/O ratio was also decreased 30-40% in patient mitochondria. Patient lymphoblasts heteroplasmic for the nt 8993 mutation were enucleated by using Percoll gradients and the cytoplasts were fused to mtDNA-deficient (rho 0) cells by electric shock. Cybrid clones homoplasmic for the wild-type nucleotide (T) at nt 8993 gave state III/DNP and ADP/O ratios similar to those of control cybrids, whereas cybrid clones homoplasmic for the mutant nucleotide (G) showed a 24-53% reduction in state III respiration, a state III/DNP ratio of 0.53-0.64, and a 30% decrease in the ADP/O ratio. Thus, the reduced state III respiration rates and ADP/O ratios are linked to the T-->G mutation at nt 8993.

DNA, Mitochondrial↗
PubMed2006

Rapid and sensitive real-time polymerase chain reaction method for detection and quantification of 3243A>G mitochondrial point mutation.

Maternally inherited diabetes and deafness and mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes result from the 3243A>G mitochondrial point mutation. Current methods to detect the presence of the mutation have limited sensitivity and may lead to potential misclassification of patients with low levels of heteroplasmy. Here, we describe development and validation of a rapid real-time polymerase chain reaction (PCR) method for detection and quantification of levels of heteroplasmy in a single assay. Standard curve analysis indicated that the sensitivity of detection was less than 0.1%. Time from sample loading to data analysis was 110 minutes. We tested 293 samples including 23 known positives, 40 known negatives, and 230 samples from patients clinically classified as having type 2 diabetes. All positive samples were correctly detected, and of those samples previously quantified, heteroplasmy levels determined using the real-time assay correlated well (r(2) = 0.88 and 0.93) with results from fluorescently labeled PCR-restriction fragment length polymorphism and pyrosequencing methods. Screening of 230 patients classified as having type 2 diabetes revealed one patient with 0.6% heteroplasmy who had previously tested negative by PCR-restriction fragment length polymorphism. Real-time PCR provides rapid simultaneous detection and quantification of the 3243A>G mutation to a detection limit of less than 0.1%, without post-PCR manipulation.

Adenosine↗
PubMed1996

A point mutation in the extracellular domain activates LET-23, the Caenorhabditis elegans epidermal growth factor receptor homolog.

The let-23 gene encodes a Caenorhabditis elegans homolog of the epidermal growth factor receptor (EGFR) necessary for vulval development. We have characterized a mutation of let-23 that activates the receptor and downstream signal transduction, leading to excess vulval differentiation. This mutation alters a conserved cysteine residue in the extracellular domain and is the first such point mutation in the EGFR subfamily of tyrosine kinases. Mutation of a different cysteine in the same subdomain causes a strong loss-of-function phenotype, suggesting that cysteines in this region are important for function and nonequivalent. Vulval precursor cells can generate either of two subsets of vulval cells (distinct fates) in response to sa62 activity. The fates produced depended on the copy number of the mutation, suggesting that quantitative differences in receptor activity influence the decision between these two fates.

Amino Acid Sequence↗
PubMed1992

Identification of point mutations in the steroid sulfatase gene of three patients with X-linked ichthyosis.

X-linked ichthyosis (XLI) is an inborn error of metabolism caused by steroid sulfatase (STS) deficiency. In more than 80% of XLI patients the enzyme deficiency is due to large deletions involving the entire STS gene and flanking sequences. However, some patients with the classical XLI phenotype and complete STS deficiency do not show any detectable deletions by Southern blot analysis using full-length STS cDNA as a probe. We have studied five unrelated patients who are such "nondeletion" mutants. Western blot analysis using anti-STS antibodies was performed on patients' fibroblast extracts and revealed absence of cross-reacting material. First-strand cDNA synthesis by reverse transcription from patients' RNA isolated from cultured fibroblasts and PCR amplification of overlapping segments of the entire STS polypeptide coding region were performed. Three point mutations were identified by chemical mismatch cleavage, sequenced by dideoxynucleotide chain-termination sequencing and confirmed by allele-specific oligonucleotide hybridization of the patients' genomic DNA. The mutations resulted in the substitution of a tryptophan for an arginine at codon 1319, changing a hydrophobic to a basic hydrophilic amino acid, the substitution of a cysteine for a tyrosine at codon 1542, potentially losing a disulfide bond, and the substitution of a serine for a leucine at codon 1237. These are the first point mutations to be documented in the STS gene and may allow insight into functionally important domains of the protein.

Amino Acids↗
PubMed2001

Seven novel point mutations in the uroporphyrinogen decarboxylase (UROD) gene in patients with familial porphyria cutanea tarda (f-PCT).

In this work, we describe seven novel molecular defects in the uroporphyrinogen decarboxylase gene responsible for familial porphyria cutanea tarda in Italian subjects with reduced erythrocyte URO-D activity. Four of these molecular abnormalities (R142Q, L161Q, S219F, P235S) are missense mutations, one (Q206X) is a nonsense mutation, one (IVS8-1 G>C) is a splicing defect causing the exon 9 deletion and one (1107 G>A) is located in the 3' untranslated region of UROD gene. All the amino acid substitutions fall in conserved regions in several organisms suggesting an important role in catalysis or in the protein structure stabilization. Three of these mutations have been detected in more than one subject. These results suggest a molecular heterogeneity at the UROD locus in Italian PCT patients although recurrent mutations have been identified.

3' Untranslated Regions↗
PubMed1995

Point mutation of a conserved arginine (104) to lysine introduces hypersensitivity to inhibition by glyphosate in the 5-enolpyruvylshikimate-3-phosphate synthase of Bacillus subtilis.

The role of a conserved arginine (R104) in the putative phosphoenol pyruvate binding region of 5-enolpyruvyl shikimate-3-phosphate synthase of Bacillus subtilis has been investigated. Employing site directed mutagenesis arginine was substituted by lysine or glutamine. Native and mutant proteins were expressed and purified to near homogeneity. Estimation of Michaelis and inhibitor constants of the native and mutant proteins exhibited altered substrate-inhibitor binding mode and constants. Mutation R104K hypersensitized the enzyme reaction to inhibition by glyphosate. The role of R104 in discriminating between glyphosate and phosphoenol pyruvate is discussed.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2004

Novel point mutations in the mitochondrial DNA detected in patients with dilated cardiomyopathy by screening the whole mitochondrial genome.

Dilated cardiomyopathy (DCM) is widely accepted as a pluricausal or multifactorial disease. Because of the linkage between energy metabolism in the mitochondria and cardiac muscle contraction, it is reasonable to assume that mitochondrial abnormalities may be responsible for some forms of DCM. We analysed the whole mitochondrial genome in a series of 45 patients with DCM for alterations and compared the findings with those of 62 control subjects. A total of 458 sequence changes could be identified. These sequence changes were distributed among the whole mitochondrial DNA (mtDNA). An increased number of novel missense mutations could be detected nearly in all genes encoding for protein subunits in DCM patients. In genes coding for NADH dehydrogenase subunits the number of mtDNA mutations detected in patients with DCM was significantly increased (p < 0.05) compared with control subjects. Eight mutations were found to occur in conserved amino acids in the above species. The c.5973G > A (Ala-Trp) and the c.7042T > G (Val-Asp) mutations were located in highly conserved domains of the gene coding for cytochrome c oxidase subunit. Two tRNA mutations could be detected in the mtDNA of DCM patients alone. The T-C transition at nt 15,924 is connected with respiratory enzyme deficiency, mitochondrial myopathy, and cardiomyopathy. The c.16189T > C mutation in the D-loop region that is associated with susceptibility to DCM could be detected in 15.6% of patients as well as in 9.7% of controls. Thus, mutations altering the function of the enzyme subunits of the respiratory chain can be relevant for the pathogenesis of dilated cardiomyopathy.

Adult↗
PubMed2000

[Point mutation of thyrotropin receptors as the main cause of autonomously functioning thyroid nodules: experiences in the framework of our Styrian patients].

Autonomously functioning thyroid nodules are frequently associated with mutations of the thyrotropin receptor. We analyzed a part of exon 10 of the thyrotropin receptor gene (base pairs 1762-1976) by direct sequencing and found missense mutations in 5 of 14 cases (codons 629, 631, 632, 633). Histologically, 3 of the 14 nodules were adenomas whereas 11 were hyperplasias. Nodules with mutations did not show significant differences from nodules without mutations with respect to age, histology, size, additional (non-functional) nodules and clinical symptomatology. Our results confirm that thyrotropin receptor mutations are involved in the development of autonomously functioning thyroid nodules. In this context, the terms hyperplasia and neoplasia should be reevaluated.

Adenoma↗
PubMed2001

Biochemical analysis of point mutations in the 5'-3' exonuclease of DNA polymerase I of Streptococcus pneumoniae. Functional and structural implications.

To define the active site of the 5'-3' exonucleolytic domain of the Streptococcus pneumoniae DNA polymerase I (Spn pol I), we have constructed His-tagged Spn pol I fusion protein and introduced mutations at residues Asp(10), Glu(88), and Glu(114), which are conserved among all prokaryotic and eukaryotic 5' nucleases. The mutations, but not the fusion to the C-terminal end of the wild-type, reduced the exonuclease activity. The residual exonuclease activity of the mutant proteins has been kinetically studied, together with potential alterations in metal binding at the active site. Comparison of the catalytic rate and dissociation constant of the D10G, E114G, and E88K mutants and the control fusion protein support: (i) a critical function of Asp(10) in the catalytic event, (ii) a role of Glu(114) in the exonucleolytic reaction, being secondarily involved in both catalysis and DNA binding, and (iii) a nonessential function of Glu(88) for the exonuclease activity of Spn pol I. Moreover, the pattern of metal activation of the mutant proteins indicates that none of the three residues is a metal-ligand at the active site. These findings and those previously obtained with D190A mutant of Spn pol I are discussed in relation to structural and mutational data for related 5' nucleases.

Amino Acid Sequence↗
PubMed2005

A switch 3 point mutation in the alpha subunit of transducin yields a unique dominant-negative inhibitor.

The rhodopsin/transducin-coupled vertebrate vision system has served as a paradigm for G protein-coupled signaling. We have taken advantage of this system to identify new types of constitutively active, transducin-alpha (alphaT) subunits. Here we have described a novel dominant-negative mutation, made in the background of a chimera consisting of alphaT and the alpha subunit of G(i1) (designated alphaT*), which involves the substitution of a conserved arginine residue in the conformationally sensitive Switch 3 region. Changing Arg-238 to either lysine or alanine had little or no effect on the ability of alphaT* to undergo rhodopsin-stimulated GDP-GTP exchange, whereas substituting glutamic acid for arginine at this position yielded an alphaT* subunit (alphaT*(R238E)) that was incapable of undergoing rhodopsin-dependent nucleotide exchange and was unable to bind or stimulate the target/effector enzyme (cyclic GMP phosphodiesterase). Moreover, unlike the GDP-bound forms of alphaT*, alphaT*(R238A) and alphaT*(R238K), the alphaT*(R238E) mutant did not respond to aluminum fluoride (AlF4(-)), as read out by changes in Trp-207 fluorescence. However, surprisingly, we found that alphaT*(R238E) effectively blocked rhodopsin-catalyzed GDP-GTP exchange on alphaT*, as well as rhodopsin-stimulated phosphodiesterase activity. Analysis by high pressure liquid chromatography indicated that the alphaT*(R238E) mutant exists in a nucleotide-free state. Nucleotide-free forms of G alpha subunits were typically very sensitive to proteolytic degradation, but alphaT*(R238E) exhibited a resistance to trypsin-proteolysis similar to that observed with activated forms of alphaT*. Overall, these findings indicated that by mutating a single residue in Switch 3, it is possible to generate a unique type of dominant-negative G alpha subunit that can effectively block signaling by G protein-coupled receptors.

Aluminum Compounds↗