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At least 1,315 records · Page 73Linked to original sources
PubMed1994

3-Methylcholanthrene inactivates the p53 gene in Syrian hamster embryo fibroblasts by inducing a specific intronic point mutation.

The expression of the tumor suppressor gene p53 was studied in Syrian hamster embryo cells neoplastically initiated with a single dose of 3-methylcholanthrene. Ten randomly selected individual 3-methylcholanthrene-transformed colonies were established in culture independently. Eight of these cell lines contained levels of p53 mRNA similar to those in primary embryo cells (p53+ cell lines), as measured by Northern blot analysis of total RNA, whereas two of them (81C43 and 81C47) showed no detectable levels of p53 mRNA (p53- cell lines). However, Southern blot and karyotype analyses did not reveal any significant changes in copy number or gross rearrangements of the p53 gene in any of the p53- cell lines. A 3-kilobase genomic fragment cloned from p53- cells (81C47) containing both upstream and downstream promoters of the p53 gene was able to drive the expression of a CAT reporter gene when transfected into either p53+ or p53- cells. Furthermore, run-on assays performed on nuclei of p53- cells showed that the p53 gene was transcriptionally active, demonstrating that the genetic defect leading to the lack of p53 expression was not due to alterations in the promoter region. Detection of mRNA species corresponding to p53 mRNA precursors in Northern blot analysis of polyadenylated RNA from both p53+ and p53- cells indicated that the lack of p53 expression was not caused by mutations in the 3' regulatory region of the p53 gene affecting transcription termination and/or polyadenylation of p53 precursor mRNA. PCR amplification and nucleotide sequence analysis of extensive internal regions of the gene revealed that both p53- cell lines were homozygous for the same unique point mutation on the splice acceptor site of the fifth intron, a G to C transversion in the last nucleotide of the intron. The presence of this mutation in both p53- cell lines strongly suggests that it was induced specifically by 3-methyl-cholanthrene treatment and indicates that the resulting splicing malfunction may account for the lack of p53 gene expression.

Animals↗
PubMed2004

Glanzmann thrombasthenia Frankfurt I is associated with a point mutation Thr176Ile in the N-terminal region of alpha IIb subunit integrin.

In this study, we report on the characterization of a patient with Glanzmann thrombasthenia (GT). Immunochemical analysis on platelets from the patient showed that the expression of alpha IIb beta 3 was only 25% of that in normal healthy controls, suggesting a case of GT. Functional analysis revealed a total lack of fibrinogen binding capacity. Molecular genetic analysis of the full-length cDNA sequences of alpha IIb and beta 3 subunits showed a novel point mutation C621T in alpha IIb cDNA, leading to a missense substitution of threonine for isoleucine at position 176. Coexpression of normal beta 3 and mutant alpha IIb(1176) isoform in mammalian cells showed a marked reduction in the expression of alpha IIb beta 3 heterodimer when compared to the wild-type and a decreased intracellular level of alpha IIb. The T176 I mutation is located in the N-terminal region in the W3:1-2 connecting strand of the beta-propeller. These data suggest that the N-terminal alpha IIb domain plays an important structural role in the formation of heterodimer and that it is also involved in fibrinogen binding.

Blood Coagulation Tests↗
PubMed1999

Recessive inheritance of a new point mutation of the PMP22 gene in Dejerine-Sottas disease.

The existence of recessive transmission of Dejerine-Sottas disease, a severe demyelinating neuropathy of childhood, has been questioned, because only heterozygous mutations of the myelin proteins P0 or PMP22 genes have been identified in virtually all patients with this phenotype. We report on a family with 3 affected children with this phenotype, born to clinically and electrophysiologically unaffected parents. All 3 children carried a previously unknown homozygous missense point mutation (Arg157Trp) of the PMP22 gene. The parents were heterozygous for the same mutation. These findings demonstrate the occurrence of recessive transmission in this setting.

Adult↗
PubMed2000

Point mutation of bacterial artificial chromosomes by ET recombination.

Bacterial artificial chromosomes (BACs) offer many advantages for functional studies of large eukaryotic genes. To utilize the potential applications of BACs optimally, new approaches that allow rapid and precise engineering of these large molecules are required. Here, we describe a simple and flexible two-step approach based on ET recombination, which permits point mutations to be introduced into BACs without leaving any other residual change in the recombinant product. Introduction of other modifications, such as small insertions or deletions, is equally feasible. The use of ET recombination to achieve site-directed mutagenesis opens access to a powerful use of BACs and is extensible to DNA molecules of any size in Escherichia coli, including the E. coli chromosome.

Chromosomes, Artificial, Bacterial↗
PubMed1994

A point mutation in the human serum albumin gene results in familial dysalbuminaemic hyperthyroxinaemia.

Using DNA samples obtained from two unrelated patients, diagnosed as having familial dysalbuminaemic hyperthyroxinaemia (FDH), exons 1-14 which span the entire coding region of the human serum albumin (HSA) gene were amplified by the polymerase chain reaction. The sequence of each of the 14 DNA fragments was then determined. In each case a point mutation was identified at nucleotide 653 which causes an Arg to His substitution at amino acid position 218. The substitution was confirmed by amino acid sequencing of a mutant peptide resulting from tryptic digestion of the protein. Abnormal affinity of FDH HSA for a thyroxine (T4) analogue was verified by an adaptation of the procedure used in routine free T4 measurement. The location of the mutation is discussed in relation to other studies on the binding properties of HSA.

Amino Acid Sequence↗
PubMed2001

Insertion/deletion frequencies match those of point mutations in the hypervariable regions of the simian immunodeficiency virus surface envelope gene.

A method for encoding insertions and deletions (indels) has been developed and adapted to the SplitsTree program. Following phylogenetic reconstruction, the relative frequencies of indels were estimated for a large number of in vivo sequence sets corresponding to the env V1 hypervariable region of the simian immunodeficiency virus SIVmac251. The method allowed recovery of many point mutations hitherto lost due to gap stripping. Deletions were as frequent as transversions and were 4- to 8-fold more frequent than insertions, invariably duplications. The high proportion of deletions among mutation events suggests that lentivirus vectors may readily delete parts of their cargo.

Base Sequence↗
PubMed2004

A distinct expression pattern and point mutation of c-kit in papillary renal cell carcinomas.

KIT is expressed not only in tumors derived from hematopoietic stem cells, melanocytes, germ cells, mast cells, and interstitial cells of Cajal, but also in other malignancies such as chromophobe renal cell carcinoma. This pattern of KIT expression prompted us to investigate the expression and mutation of c-kit gene exons 9, 11, 13, 17, and intron 17 in the different subtypes of renal cell carcinomas (n=66) and non-neoplastic kidneys (n=12). We found that KIT showed strong immunoreactivity in the cytoplasm of papillary renal cell carcinomas (100%), but on the cell membranes of chromophobe renal cell carcinomas (100%). Interestingly, a specific point mutation of the c-kit intron 17 (T->A) was found only in papillary renal cell carcinomas (94%). Our study demonstrates that the expression pattern and one mutation of c-kit may distinguish papillary renal cell carcinomas.

Base Sequence↗
PubMed1994

A novel Mediterranean "delta beta-thalassemia" determinant containing the delta (+) 27 and beta (0) 39 point mutations in cis.

The term delta beta-thalassemia with normal HbF has been recently proposed to define heterogenous delta and beta globin gene molecular defects involving the same chromosome in cis. Here, we describe a Sardinian family in which three members showing microcytosis, border-line HbA2 levels and normal HbF proved to be heterozygotes for delta(+) 27 and beta(0) 39 point mutations in cis by allele specific oligonucleotide hybridization as well as by ECO 0 109 I endonuclease digestion and electrophoresis. As some of these beta-thalassemia carriers shows normal HbA2 levels, knowledge of the molecular basis of this novel delta beta-thalassemia silent phenotype would be useful in thalassemia screening and genetic counselling.

Base Sequence↗
PubMed1994

Detection of DNA point mutations with DNA mismatch repair enzymes.

We have developed a simple and reliable procedure to screen gene mutations using DNA mismatch repair (MR) specific mut Y enzyme of Escherichia coli and thymidine DNA glycosylase from HeLa cells. The mut Y enzyme cleaves A of G/A mismatches in DNA duplex and thymidine glycosylase cleaves T at G/T mismatches. Previously, we showed the determination of G:C-->T:A mutations in the N-ras gene in two human tumor samples with mut Y G/A MR enzyme. As low as 1-2% mutant DNAs in a sample of mutant and wild-type DNA can be detected with a synthetic DNA to create G/A mispairing for the assay. In this paper, we simplify the assay, include G/T MR thymidine glycosylase from HeLa cells and evaluate the application for screening DNA point mutations of p53 and ras genes. In this method, DNA fragments amplified from normal and mutated genes by polymerase chain reaction (PCR) were mixed and annealed to create DNA mismatches for cleavage by mismatch repair enzymes. The cleaved products and the substrates were separated by gel electrophoresis and detected by autoradiography. In theory, the enzymes that cut G/A or G/T mispairs will detect the mutations of G:C-->A:T, A:T-->G:C, G:C-->T:A and T:A-->G:C. Several human tumor samples were examined for p53 or K-ras mutations with G/A and G/T mismatch repair enzymes. The reliability of mutation detection was evaluated by comparing the results with reported mutations or confirmed by DNA sequencing of the same PCR-amplified DNA fragments. Our data showed that, following mismatch repair enzyme cleavage, all mutated DNA samples yielded cleaved products with sizes as expected. In addition, our assay is able to characterize the nature of mutation by 5' end-labeling of 32P on mutant or wild-type DNA fragments. The low background, reliability and the determination of the sites of mutations as well as the types of DNA base changes indicate the advantages of the method over other techniques in testing DNA mutants.

Base Sequence↗
PubMed2001

A single point mutation at the 3'-untranslated region of Ran mRNA leads to profound changes in lipopolysaccharide endotoxin-mediated responses.

By functional cDNA expression cloning, we have previously established that Ran is important in lipopolysaccharide (LPS) signaling. This was achieved by functional comparison between two cDNAs, differing by a single base substitution within the 3'-untranslated region of the cDNA. This point mutation results in a striking RNA conformational change. No dramatic difference in total RNA at steady state could be found between the two molecules. However, at the protein level, RanC/d (from 870C mRNA) was 5-10-fold higher than RanT/n (from 870T mRNA) and this difference was not observed in non-hematopoietic cells transduced with the same vectors. This tissue-specific difference correlated with a difference in LPS endotoxin responses in corresponding hematopoietic cells. Importantly, the amounts of Ran- C/d and RanT/n proteins were similar initially but the difference became obvious with time. Both Ran proteins migrated from the cytoplasm to the nucleus, but Ran from RanC/d migrated faster than that of RanT/n. RanT/n protein preferentially remained in the cytoplasm and its overall amount was reduced at steady state, consistent with its degradation by intracellular proteases known to be involved in LPS-mediated signal transduction. As the two proteins are identical, the faster RanC/d nuclear localization and a preferred initial cytoplasmic RanT/n distribution suggest a difference in mRNA intracellular localization between the two molecules, as dictated by their RNA structural difference. By pulse-chase experiments, RanC/d proteins are more resistant to degradation than RanT/n protein; there also appear to have two populations of RanT/n proteins, one may reside in the cytoplasm and the other, in the nucleus. More RanC/d GTPase accumulated in the nuclei would conceivably alter the potency of signal transduction and therefore down-modulate LPS-mediated biological responses.

3' Untranslated Regions↗
PubMed1994

Resistance to thyroid hormone in subjects from two unrelated families is associated with a point mutation in the thyroid hormone receptor beta gene resulting in the replacement of the normal proline 453 with serine.

Resistance to thyroid hormone (RTH) is a condition of impaired tissue responsiveness to thyroid hormone characterized by elevated free thyroid hormone levels in serum accompanied by nonsuppressed TSH. RTH has been associated with mutations in the thyroid hormone receptor (TR) beta gene. We report studies carried out in 9 members of a family (F94) of Jewish ethnic origin and a single subject of Mexican origin. All subjects fulfilling the criteria of RTH (6 of family F94 and one of family F27) had the same point mutation in the T3-binding domain on one of the two alleles of the TR beta gene. This mutation resulted in the replacement of the normal proline-453 with serine (P453S). Nevertheless, the clinical characteristics of affected members of each of the two families differed as did the severity of hormonal resistance in terms of responses to the administration of L-T3. Genetic studies indicate that the same mutation occurred independently in each of the two families.

Adult↗
PubMed1993

A heritable point mutation in an extracellular domain of the TSH receptor involved in the interaction with Graves' immunoglobulins.

The TSH receptor (TSHR) is the central antigen in Graves' disease. Variant receptor proteins, arising from mutations in the TSHR gene, may play a role in the pathogenesis of the disease. Therefore, we analysed the TSHR from a 38-year-old patient affected with autoimmune hyperthyroidism, diffuse goitre and ophthalmopathy. Reverse transcription PCR and DNA amplification followed by DNA sequencing revealed a point mutation (C-->A) at cDNA position 253 in one of two alleles. This leads to the replacement of a proline (CCC) by threonine (ACC) at amino acid position 52 of the predicted receptor protein. Secondary structure predictions indicated a major change of protein structure as a result of the mutation. By using allele-specific PCR, we were able to show that this mutation is heritable. Screening of 50 random individuals revealed that four of them also carried this mutation in the heterozygous state. This study shows the presence of different forms of the TSHR gene in the population. The mutation, which is in a portion of the receptor apparently involved in binding of Graves' immunoglobulins, is discussed as to its possible pathophysiological role in autoimmune hyperthyroidism.

Adult↗
PubMed1994

Impact of point mutations on the structure and thermal stability of ribonuclease T1 in aqueous solution probed by Fourier transform infrared spectroscopy.

We undertook a detailed comparative analysis of the infrared spectra of wild-type ribonuclease T1 and three mutants: two single mutants, Tyr-45-->Trp (Y45W) and Trp-59-->Tyr (W59Y), and a double mutant, Tyr-45-->Trp/Trp-59-->Tyr (Y45W/W59Y). These mutants were selected because they are known to affect the activity of the enzyme. The structural differences were evaluated by using peptide backbone and side-chain "marker" bands as conformation-sensitive monitors. All mutations lead to a decrease of the thermal transition temperature, though the mutation Tyr-45-->Trp affects the Tm to a lesser degree than the replacement of Trp-59 by Tyr, both in the single (W59Y) and in the double (Y45W/W59Y) mutant. Small changes in the protein backbone conformation and in the microenvironment of certain amino acids, induced by the point mutations, could be detected. In particular, we found subtle differences in the hydrogen bonding pattern of the beta-strands in the mutants W59Y and Y45W/W59Y, compared to that in wild-type RNase T1 and in the mutant Y45W. Practically identical spectra in the amide I region were obtained for the double mutant Y45W/W59Y and the single mutant W59Y, demonstrating that it is the change from Trp to Tyr in position 59 (located at the interface between the alpha-helix and a beta-strand) which affects the overall protein conformation. The mutation Tyr to Trp in position 45, on the other hand, has practically no impact on the polypeptide backbone conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspergillus oryzae↗
PubMed1995

Point mutation in the pribnow box, the molecular basis of beta-lactamase overproduction in Klebsiella oxytoca.

Klebsiella oxytoca mutants resistant to a variety of beta-lactams were obtained in vitro on aztreonam. Constitutive beta-lactamase production was much higher in the mutants than in the susceptible strains (75-fold). The only difference observed in these mutants compared with the susceptible strains were point mutations in the Pribnow box: a transversion (G-->T) in the first base for one mutant or a transition (G-->A) in the fifth base of the -10 consensus sequence for the other three mutants. The transcriptional output of the beta-lactamase gene (blaOXY) from the mutants was significantly higher than that of the blaOXY gene from the susceptible strains.

Anti-Bacterial Agents↗
PubMed1999

Detection of DNA polymorphisms and point mutations of high-affinity nerve growth factor receptor (TrkA) in human neuroblastoma.

We have studied nerve growth factor (NGF) receptors in 63 neuroblastoma tissues. We found DNA polymorphisms of TrkA consisting in C-->T transitions in tyrosine kinase domain at 6773, 7232 and 7301 nucleotides. Both C/T alleles were detected in tumors belonging to patients at stages 1, 2, 3 and 4 but not in 4S that expresses only the allele with C-->T. Furthermore, we detected a GT transversion resulting in a Gly-->Val substitution in a stage 4 and a stage 4S samples. We report the first evidence of TrkA polymorphism and point mutations in neuroblastoma.

Humans↗
PubMed2000

Point mutations identify a conserved region of the saccharomyces cerevisiae AFR1 gene that is essential for both the pheromone signaling and morphogenesis functions.

Mating pheromone receptors activate a G protein signal pathway that leads to the conjugation of the yeast Saccharomyces cerevisiae. This pathway also induces the production of Afr1p, a protein that negatively regulates pheromone receptor signaling and is required to form pointed projections of new growth that become the site of cell fusion during mating. Afr1p lacks strong similarity to any well-characterized proteins to help predict how it acts. Therefore, we investigated the relationship between the different functions of Afr1p by isolating and characterizing seven mutants that were defective in regulating pheromone signaling. The AFR1 mutants were also defective when expressed as fusions to STE2, the alpha-factor receptor, indicating that the mutant Afr1 proteins are defective in function and not in co-localizing with receptors. The mutant genes contained four distinct point mutations that all occurred between codons 254 and 263, identifying a region that is critical for AFR1 function. Consistent with this, we found that the corresponding region is very highly conserved in the Afr1p homologs from the yeasts S. uvarum and S. douglasii. In contrast, there were no detectable effects on pheromone signaling caused by deletion or overexpression of YER158c, an open reading frame with overall sequence similarity to Afr1p that lacks this essential region. Interestingly, all of the AFR1 mutants showed a defect in their ability to form mating projections that was proportional to their defect in regulating pheromone signaling. This suggests that both functions may be due to the same action of Afr1p. Thus, these studies identify a specific region of Afr1p that is critical for its function in both signaling and morphogenesis.

Amino Acid Sequence↗
PubMed2006

"Occult" mastocytosis with activating c-kit point mutation evolving into systemic mastocytosis associated with plasma cell myeloma and secondary amyloidosis.

A case of a 70-year-old man presenting with exsudative enteropathy due to light-chain-associated amyloidosis is reported. The diagnosis of systemic mastocytosis associated with IgG/lambda plasma cell myeloma and secondary generalised amyloidosis was carried out by morphological evaluation of bone marrow biopsy. The c-kit point mutation D816Y was detected by molecular analysis. Two years before, a cystadenolymphoma of the left parotid gland had been removed. A moderate increase of loosely scattered spindle-shaped mast cells, a subpopulation of them expressing CD25, an antigen that is not expressed by normal or reactive mast cells, was shown by retrospective analysis carried out on an intraparotideal lymph node. The c-kit mutation D816Y was shown by the molecular analysis of the lymph node. In summary, the notion that systemic mastocytosis may very rarely be associated with B cell neoplasms and that neoplastic mast cell infiltrates may be obscured because of only a minimal increase of atypical mast cells, which are outnumbered by other non-neoplastic cells in the same tissue, is supported by this case. This finding was preliminarily termed "occult" mastocytosis.

Aged↗
PubMed1996

A Point Mutation of Acetylcholinesterase Associated with Azinphosmethyl Resistance and Reduced Fitness in Colorado Potato Beetle

A serine to glycine point mutation of acetylcholinesterase (AChE, EC 1.1.1.7) was identified in an azinphosmethyl-resistant strain of Colorado potato beetle [Leptinotarsa decemlineata (Say)]. The position of the mutation corresponds to Val 238 of the Torpedo AChE and represents the first amino acid residue to form the alpha-helix, alpha-E'1. The predicted secondary structure of the mutation-containing region of AChE suggested that the transition from the turn to the alpha-helix occurs sooner in the sequence when serine is replaced by glycine. Thus, conformational changes in the AChE due to the alpha-helix deformation were expected to impinge upon both the catalytic and the peripheral binding sites, resulting in the modification of the bindings of organophosphorus insecticides and other ligands to these sites. The mutation appeared to be associated with the fitness of the beetle. The intrinsic rate of increase of the azinphosmethyl-resistant (AZ-R) strain was relatively low when the beetles were reared on the Russet Burbank potato cultivar, but was relatively high when they were reared on the NDA 1725-1 potato cultivar. Because these two potato cultivars contain different amounts of steroidal glycoalkaloids (e.g., alpha-solanine and alpha-chaconine), the different fitness of the AZ-R strain on different potato cultivars may be partially attributed to the increased sensitivity of the azinphosmethyl-resistant form of AChE to the inhibition by alpha-solanine and reduced sensitivity to alpha-chaconine as previously reported.

Journal Article↗