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PubMed1998

Point mutation in intron sequence causes altered carboxyl-terminal structure in the aryl hydrocarbon receptor of the most 2,3,7,8-tetrachlorodibenzo-p-dioxin-resistant rat strain.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent dioxin. There are exceptionally wide inter- and intraspecies differences in sensitivity to TCDD toxicity with Han/Wistar (H/W) (Kuopio) rats being the most resistant mammals tested. A peculiar feature of H/W rats is that despite their unresponsiveness to the acute lethality of TCDD, their sensitivity to other biological impacts of TCDD (e.g., CYP1A1 induction) is preserved. The biological effects of TCDD are mediated by the aryl hydrocarbon receptor (AhR). We recently found that the AhR of H/W rats (about 98 kDa) is smaller than the receptor in other rat strains (106 kDa). In the present study, molecular cloning and sequencing of the H/W rat AhR revealed that the reason for its smaller size is a deletion/insertion-type change at the 3' end of exon 10 in the receptor cDNA. This change emanates from a single point mutation at the first nucleotide of intron 10, resulting in altered mRNA splicing. At the protein level, the mutation leads to a total loss of either 43 or 38 amino acids (with altered sequence for the last seven amino acids in the latter case) toward the carboxyl-terminal end in the trans-activation domain of the AhR. H/W rats also harbor a point mutation in exon 10 that will cause a Val-to-Ala substitution in codon 497, but this occurs in a variable region of the AhR. These findings suggest that there is a relatively small region in the AhR trans-activation domain that may be capable of providing selectivity to its function.

Animals↗
PubMed1992

Crude isolation of DNA from unselected human pancreatic tissue and amplification by the polymerase chain reaction of Ki-ras oncogene to detect point mutations in pancreatic cancer.

Human pancreatic tissue obtained during surgery was divided into two samples, one stored in formalin for histology and the other frozen directly for DNA studies. A small bit of the frozen tissue was excised without prior differentiation of malignant from non-malignant tissue and crude DNA prepared. The DNA was used to amplify the Ki-ras oncogenes with the polymerase chain reaction (PCR) using primers for codons 12, 13 and 61. The PCR products were hybridized with oligonucleotides to detect mutations in these codons. No mutations were seen in patients histologically free from pancreatic cancer, having cancer of the papilla of Vater or endocrine pancreatic cancer. In the 9 cases of pancreatic cancer, point mutations were detected in codon 12 in 7 cases and in codon 13 in one patient. Thus, the PCR technique can be used to detect point mutations in Ki-ras oncogenes even in crude DNA without selection of malignant from non-malignant tissue.

Codon↗
PubMed1995

Point mutation of the somatostatin receptor 2 gene in the human small cell lung cancer cell line COR-L103.

The effect of somatostatin (SS) on adrenocorticotrophic hormone (ACTH) secretion from COR-L103 cells derived from a human small cell lung carcinoma was examined. SS at 1 microM had no effect on ACTH secretion from the cells on either short-term or long-term incubation. Studies by the reverse transcription-polymerase chain reaction (RT-PCR) showed that mRNA transcripts of the somatostatin receptor (SSTR) 2, SSTR3 and SSTR4 genes were present in COR-L103 cells. Extra bands were obtained by PCR-single strand conformation polymorphism (SSCP) analysis of the SSTR2 gene Sequence analysis of the SSTR2 gene demonstrated one point mutation in codon 188 of TGG for tryptophan to TGA for a stop codon causing loss of 182 C-terminal amino acid residues of SSTR2. The nucleotide sequences of the SSTR3 and SSTR4 genes in COR-L103 cells were normal. Binding studies using 125I-Tyr11-SS-14 showed specific affinity binding sites on COR-L103 cells and mouse pituitary tumor AtT-20 cells. Octreotide acetate suppressed the binding of 125I-Tyr11-SS-14 to these two cell lines, but the Kd of COR-L103 cells (160 nM) was 60-fold higher than that of AtT-20 cells (2.6 nM). Affinity cross-linking studies using 125I-Tyr11-SS-14 gave three bands of 72 KDa, 55 KDa and 32 KDa from AtT-20 cells, but only two bands of 55KDa and 32kDa from COR-L103 cells. These findings suggest that SSTR2 is not expressed in the plasma membranes of COR-L103 cells due to a point mutation, but that this may have no influence on the effect of SS on ACTH secretion.

Adrenocorticotropic Hormone↗
PubMed2000

A point mutation in the mma3 gene is responsible for impaired methoxymycolic acid production in Mycobacterium bovis BCG strains obtained after 1927.

BCG vaccines are substrains of Mycobacterium bovis derived by attenuation in vitro. After the original attenuation (1908 to 1921), BCG strains were maintained by serial propagation in different BCG laboratories (1921 to 1961). As a result, various BCG substrains developed which are now known to differ in a number of genetic and phenotypic properties. However, to date, none of these differences has permitted a direct phenotype-genotype link. Since BCG strains differ in their abilities to synthesize methoxymycolic acids and since recent work has shown that the mma3 gene is responsible for O-methylation of hydroxymycolate precursors to form methoxymycolic acids, we analyzed methoxymycolate production and mma3 gene sequences for a genetically defined collection of BCG strains. We found that BCG strains obtained from the Pasteur Institute in 1927 and earlier produced methoxymycolates in vitro but that those obtained from the Pasteur Institute in 1931 and later all failed to synthesize methoxymycolates, and furthermore, the mma3 sequence of the latter strains differs from that of Mycobacterium tuberculosis H37Rv by a point mutation at bp 293. Site-specific introduction of this guanine-to-adenine mutation into wild-type mma3 (resulting in the replacement of glycine 98 with aspartic acid) eliminated the ability of this enzyme to produce O-methylated mycolic acids when the mutant was cloned in tandem with mma4 into Mycobacterium smegmatis. These findings indicate that a point mutation in mma3 occurred between 1927 and 1931, and that this mutant population became the dominant clone of BCG at the Pasteur Institute.

Amino Acid Sequence↗
PubMed2001

Evidence for the contribution of point mutations to vlsE variation and for apparent constraints on the net accumulation of sequence changes in vlsE during infection with Lyme disease spirochetes.

In the Lyme disease spirochetes, both the ospE and vlsE gene families have been demonstrated to undergo sequence variation during infection. To further investigate the mechanisms associated with the generation of vls variation, single-nucleotide polymorphism and subsequent DNA sequence analyses were performed on the vlsE gene and its paralog, BBJ51, a related gene with a frameshift mutation. These analyses focused on a series of postinfection clonal populations obtained from mice infected with Borrelia burgdorferi B31MIpc or its clonal derivative, B31MIc53. vlsE, but not BBJ51, was found to undergo sequence changes during infection. Consistent with that reported previously (J.-R. Zhang et al., Cell 89:275-285, 1997) many of the sequence changes appear to have arisen through gene conversion events and to be localized to the variable regions of vlsE. However, analysis of the vlsE nucleotide sequences revealed that some sequence changes were the result of point mutations, as these changes did not have potential contributing sources in the vls cassettes. To determine if sequence changes accumulate in vlsE over long-term infection, the vlsE genes of clonal populations recovered after 7 months of infection in mice were analyzed. While new sequence changes developed, a significant number of these changes resulted in the restoration of the vlsE sequence of the original infecting clone. In addition, we noted that some positions within the variable regions (VR) are stable even though the cassettes possess residues that could contribute to sequence variation through gene conversion. These analyses suggest that the total number of amino acid sequence changes that can be maintained by VlsE levels off during infection. In summary, in this report we demonstrate that the development of point mutations serves as a second mechanism by which vlsE sequence variation can be generated and that the capacity for vlsE variation, while still significant, is less than previously postulated.

Amino Acid Sequence↗
PubMed1993

Ocular pathology of MELAS syndrome with mitochondrial DNA nucleotide 3243 point mutation.

PURPOSE: The authors describe the clinical, histopathologic, and ultrastructural findings in two eyes obtained at autopsy from a 21-year-old woman with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS syndrome). METHODS: The eyes were obtained immediately after death. The right eye was fixed in 10% neutral-buffered formalin and processed for standard histologic examination. The left eye was fixed in a neutral-buffered 2.5% glutaraldehyde solution and processed for transmission electron microscopic examination. The authors compared the histologic and ultrastructural findings with the clinical features recorded photographically. RESULTS: The main clinical ophthalmologic features were bilateral ptosis, chronic external ophthalmoplegia, diffuse choroidal atrophy, atypical pigmentary retinopathy with macular involvement, and patchy atrophy of the iris stroma. Molecular genetic analysis detected a tRNA Leu (UUR) point mutation at position 3243 of mitochondrial DNA (MELAS genotype). Results of histologic and ultrastructural examination showed ragged-red fibers in the rectus muscles, degeneration of photoreceptor outer segments in the macula, hyperpigmentation and atrophy of the retinal pigment epithelium of the macula, atrophy of the iris stroma, early posterior subcapsular cataract, and optic atrophy. The retinal pigment epithelium, inner segments of the photoreceptors, smooth muscle cells of the choroidal and retinal vessels, the dilator and sphincter muscle of the iris, cornea, lens epithelium, and ciliary epithelium all contained many, often enlarged, structurally abnormal mitochondria with occasional paracrystalline inclusions and circular cristae. CONCLUSIONS: The MELAS-associated mitochondrial DNA nucleotide 3243 point mutation can cause a spectrum of ocular signs and symptoms that may be dependent on the patient's age and the amount of mutant mitochondrial DNA in the tissue. MELAS syndrome should be considered in the differential diagnosis of bilateral ptosis, external ophthalmoplegia, and atypical pigmentary retinopathy with macular involvement.

Adult↗
PubMed1993

Introduction of a point mutation into the mouse genome by homologous recombination in embryonic stem cells using a replacement type vector with a selectable marker.

The introduction of small mutations instead of null alleles into the mouse genome has broad applications to the study of protein structure-function relationships and the creation of animal models of human genetic diseases. To test a simple mutational strategy we designed a targeting vector for the mouse proopiomelanocortin (POMC) gene containing a single nucleotide insertion that converts the initial tyrosine codon of beta-endorphin 1-31 to a premature translational termination codon and introduces a unique Hpal endonuclease restriction site. The targeting vector also contains a neo cassette immediately 3' to the last POMC exon and a herpes simplex virus thymidine kinase cassette to allow positive and negative selection. Homologous recombination occurred at a frequency of 1/30 clones of electroporated embryonic stem cells selected in G418 and gancyclovir. 10/11 clones identified initially by a polymerase chain reaction (PCR) strategy had the predicted structure without evidence of concatemer formation by Southern blot analysis. We used a combination of Hpa I digestion of PCR amplified fragments and direct nucleotide sequencing to further confirm that the point mutation was retained in 9/10 clones. The POMC gene was transcriptionally silent in embryonic stem cells and the targeted allele was not activated by the downstream phosphoglycerate kinase-1 promoter that transcribed the neo gene. Under the electroporation conditions used, we have demonstrated that a point mutation can be introduced with high efficiency and precision into the POMC gene using a replacement type vector containing a retained selectable marker without affecting expression of the allele in the embryonic stem cells. A similar strategy may be useful for a wide range of genes.

Alleles↗
PubMed1999

Dysfunctional glucocorticoid receptor with a single point mutation ablates the CCAAT/enhancer binding protein-dependent growth suppression response in a steroid-resistant rat hepatoma cell variant.

We used glucocorticoid-resistant and -sensitive hepatoma cell variants to characterize the mechanism of hepatoma cell resistance to the growth inhibitory effects of glucocorticoids. BDS1 hepatoma cells express transcriptionally active glucocorticoid receptors and undergo a stringent G1 cell cycle arrest in response to glucocorticoids that is dependent on the induced expression of the CCAAT/enhancer binding protein alpha (C/EBPalpha) transcription factor. In contrast, EDR1 hepatoma cells, which express normal levels of glucocorticoid receptors, fail to growth arrest or express C/EBPalpha when treated with glucocorticoids. Ectopic expression of wild-type rat glucocorticoid receptors into EDR1 cells restored the growth suppression response, suggesting a defect in the EDR1 receptor. DNA sequence analysis revealed a single point mutation causing a cysteine-to-tyrosine substitution at amino acid position 457 (C457Y-GR) in the zinc finger region of the glucocorticoid receptor that mediates both receptor-DNA and receptor-protein interactions. Glucocorticoid activation of the alpha1-acid glycoprotein (AGP) promoter, a liver acute-phase response gene, requires receptor-DNA binding as well as an interaction with C/EBPalpha. In contrast to the wild-type glucocorticoid receptor, ectopic expression of C/EBPalpha in EDR1 cells, or coexpression of C/EBPalpha along with the C457Y-GR into receptor-deficient EDR3 cells was required to partially restore glucocorticoid responsiveness of the AGP promoter by the EDR1 glucocorticoid receptor. Constitutive expression of the wild-type glucocorticoid receptor, but not the C457Y-GR mutant, was sufficient to restore the glucocorticoid growth suppression response to receptor-deficient EDR3 cells. Thus, we have identified a glucocorticoid-resistant hepatoma cell variant with a single point mutation in the zinc finger region of the glucocorticoid receptor gene that ablates the glucocorticoid growth suppression response and attenuates transcriptional activation of the AGP promoter.

Animals↗
PubMed1991

The molecular genetics of haemophilia A: screening for point mutations in the factor VIII gene using the restriction enzyme TaqI.

A combination of Southern blotting and the analysis of polymerase chain reaction (PCR) amplified DNA fragments was used to screen the factor VIII genes of 527 haemophilia A patients for point mutations within TaqI restriction sites. Since this "directed search" strategy yielded only four gene lesions, it was concluded that its efficacy is less than that originally predicted. One novel point mutation was however found in a moderately severe haemophiliac; a CGA (Arg) to CTA (Leu) transversion at codon 2209, an evolutionarily conserved residue in the C2 domain of the factor VIII protein. The remaining three detected lesions, CGA (Arg)----TGA (Term) transitions at codons 2116, 2147 and 2307, respectively, have been reported before and are consistent with recurrent mutation at these hypermutable sites. A number of TaqI restriction site polymorphisms/rare variants were also noted. These variants appear to be population-specific but are nevertheless potentially useful in individual cases as intragenic markers for carrier detection and antenatal diagnosis.

Base Sequence↗
PubMed1989

Two novel point mutations correlate with an altered developmental expression of blood coagulation factor IX (hemophilia B Leyden phenotype).

Hemophilia B Leyden is characterized by low levels of factor IX antigen and activity before the age of 15 years, whereas after puberty factor IX levels rise at a rate of about 5% per year. Two distinct point mutations (deletion of A, A----G) were identified at position +13 of the factor IX gene of a Greek and an American patient with hemophilia B Leyden. The nucleotide changes have occurred 32 basepairs downstream of a previously reported point mutation in a Dutch kindred with the same hemophilic phenotype. The results point to the importance of sequences surrounding the putative start site for the constitutive expression of the factor IX gene and to the possible significance of an imperfect direct repeat of DNA.

Age Factors↗
PubMed2001

Novel point mutations, deletions, and polymorphisms in the cathepsin C gene in nine families from Europe and North Africa with Papillon-Lefèvre syndrome.

Papillon-Lefèvre syndrome is an autosomal recessive disorder characterized by palmoplantar keratoderma, periodontitis, and premature loss of dentition. Mutations in the CTSC gene that encodes cathepsin C have been described in families affected with Papillon--Lefèvre syndrome. Cathepsin C is the least understood of the lysosomal cysteine proteases; it has been reported to participate in both intracellular and extracellular cleavage of proteins and activation of serine proteases in immune and inflammatory cells. We report here eight new mutations in Papillon-Lefèvre syndrome families: four deletions and four point mutations, including a missense mutation in the propeptide chain that could help elucidate structure-function relationships in this protein. We also found that the 458C > T mutation, first reported in two families by Hart et al (2000c), was a neutral polymorphism in our families, as suggested by Allende et al (Cathepsin C gene: first compound heterozygous patient with Papillon--Lefèvre syndrome and novel symptomless mutation. Hum Mutat 17:152-153, 2001).

Africa, Northern↗
PubMed1997

A point mutation of human nucleoside diphosphate kinase A found in aggressive neuroblastoma affects protein folding.

The point mutation serine 120 to glycine in the human nucleoside diphosphate kinase A has been identified in several aggressive neuroblastomas (Chang, C. L., Zhu, X. X., Thoraval, D. H., Ungar, D., Rawwas, J., Hora, N., Strahler, J. R., Hanash, S. M. & Radany, E. (1994) Nature 370, 335-336). We expressed in bacteria and purified wild-type and S120G mutant nucleoside diphosphate kinase A. The mutant enzyme had enzymatic and structural properties similar to the wild-type enzyme, whereas its stability to denaturation by heat and urea was markedly reduced. More importantly, upon renaturation of the urea-denatured mutant protein, a folding intermediate accumulated, having the characteristics of a molten globule. It had no tertiary structure, as shown by near UV circular dichroism, whereas the secondary structure was substantially recovered. The hydrophobic probe 8-anilino-1-naphthalene sulfonate bound to the intermediate species with an increase in fluorescence intensity and a blue shift. The hydrodynamic size was between that expected for a folded and an unfolded monomer. Finally, electrophoresis in a transverse urea gradient displayed no renaturation curve, and the protein showed the tendency to aggregate at the lowest urea concentrations. The existence of a molten globule folding intermediates resulting from an altered folding in the mutated protein might be related to the aggressiveness of neuroblastomas.

Antigens, Neoplasm↗
PubMed1986

Molecular basis of hemophilia B: a defective enzyme due to an unprocessed propeptide is caused by a point mutation in the factor IX precursor.

A mutant factor IX, designated factor IXCambridge, was isolated from a patient with hemophilia B. This protein includes an 18-residue propeptide attached to the NH2 terminus of factor IX. A point mutation at residue -1, from an arginine to a serine, precludes cleavage of the propeptide by a processing protease and interferes with gamma-carboxylation of the factor IX, indicating the importance of the leader sequence in substrate recognition by the vitamin K-dependent carboxylase. This represents an example of an enzyme defect due to the presence of a point mutation in a precursor protein (preproenzyme) that is the cause of a human hereditary disease. This defect will serve as a prototype for understanding the molecular basis of some forms of hemophilia and other hereditary enzyme deficiencies.

Amino Acid Sequence↗
PubMed1985

A method to detect and characterize point mutations in transcribed genes: amplification and overexpression of the mutant c-Ki-ras allele in human tumor cells.

A significant percentage of human tumors contain activated ras oncogenes that have acquired oncogenic potential as a result of somatic point mutations at codon 12 or 61 of the encoded ras gene product. We report here a method to detect and characterize mutations in ras genes that is based on the ability of pancreatic ribonuclease (RNase A; EC 3.1.27.5) to cleave RNA heteroduplexes containing single-base mismatches. Using this method, we show that certain human tumor cells contain mutant c-Ki-ras genes, and we define the nature and position of these mutations. At the same time, we describe the presence and estimate the expression of both normal and mutant c-Ki-ras alleles in the same tumor cells. This method should be useful for the diagnostic detection and characterization of single point mutations in expressed genes.

Alleles↗
PubMed1996

Two novel point mutations causing receptor-negative familial hypercholesterolemia in a South African Indian homozygote.

Two novel point mutations have been identified in the low density lipoprotein receptor (LDLR) gene of a South African Indian patient with a clinical diagnosis of homozygous familial hypercholesterolemia (FH). The patient is a compound heterozygote, whose paternally-inherited allele has a single base substitution of A to T at position + 1. This conversion of the initiation codon ATG (methionine) to TTG (leucine) would abolish initiation of translation at the normal site, and consequently the synthesis of any normal LDLR molecules. The second mutation identified is a C to A base change at nucleotide position 1176 in exon 8, which creates a stop codon at cysteine-371. Except for previously-described polymorphisms in specific regions of the LDLR gene, the mutations identified in exons 1 and 8 were the only variants observed by screening enzymatically amplified genomic DNA comprising the entire coding and promoter region of the LDLR gene by combined heteroduplex-single-strand conformation polymorphism analysis and by direct sequencing. Cultured cells from the proband expressed no functional LDLR activity and contained no receptor protein that could be detected by antibody binding. These findings are consistent with the nature of the two base changes identified and provide evidence that the mutations cause FH in the proband and his affected family members. The mutations, designated M-21L and C371X, were absent in 17 apparently unrelated Indian hypercholesterolemics and 200 normal chromosomes screened.

Adolescent↗
PubMed2004

Analysis of BRAF point mutation and RET/PTC rearrangement refines the fine-needle aspiration diagnosis of papillary thyroid carcinoma.

Point mutations in BRAF are genetic hallmarks of papillary thyroid carcinoma (PTC). In this retrospective study, we examined thyroid aspirates and corresponding paraffin-embedded surgical samples for the presence of BRAF mutations. Altogether, we examined 96 cases, including 69 PTCs, 19 follicular adenomas, and eight nontoxic nodular goiters for BRAF; 60 of these samples were also examined for RET/PTC rearrangements. The results were correlated with the cytological diagnosis and the final histopathology. The BRAF mutation (V599E) was detected in 38% of the samples that were PTC on histopathology; RET/PTC was found in 18% of the PTC cases. In all the cases, the presence of the genetic alteration was confirmed in the surgically resected tumor. The identification of BRAF mutation and RET/PTC refined the diagnosis of PTC in five of 15 samples that were considered either indeterminate or insufficient at cytology. No mutation was found in aspirates of follicular adenomas and nontoxic nodular goiters. These results indicate that BRAF mutation and RET/PTC rearrangements are molecular markers of PTC that can be applied to FNA in adjunct to traditional cytology.

Adenoma↗
PubMed2001

Implication of annexin 1 in phagocytosis: effects of n-terminal domain deletions and point mutations of the phosphorylation site Ser-27.

Directed mutagenesis, in the form of deletions and point mutations, was used to investigate the regulatory importance of the N-terminal domain of annexin 1. Wild-type and mutant forms were fused to green fluorescent protein (GFP) to track their localization and introduced in to J-774A.1 cells by transfection. The fusion of annexin 1 to GFP at the N- or C-terminal end did not alter the cellular distribution or co-localization with phagosomes. The effects of mutations were determined according to these characteristics. The prominent effect resulted from S27E mutation which mimics the phosphorylated state of Ser-27. Although still retaining the granular structures in the cytoplasm, S27E annexin 1 failed to associate with the phagosomal protein complex. This suggests an essential regulatory role of the phosphorylation of residue 27 in annexin 1 function.

Animals↗
PubMed2003

[Detection of point mutation in p53 gene by capillary electrophoresis-PCR-SSCP analysis].

OBJECTIVE: To screen the point mutation of p53 gene rapidly by capillary electrophoresis (CE). METHODS: A simple capillary electrophoresis system for the strand conformational polymorphism (SSCP) analysis of genomic DNA was developed by choosing commercially available capillary and gel buffer. This CE-SSCP system was applied to the analysis of twenty PCR products amplified from the exon 7 of the p53 gene of tissue specimens from patients with colon cancer. RESULTS: Five of them (Ca4, Ca6, Ca7, Ca8, Ca14) were found to have mutation within 25 minutes, while the results of the SSCP analysis by PAGE-silver staining techniques only detected mutation of four samples. And sequencing of these PCR products proved that the results of CE-SSCP were correct. CONCLUSION: This simple, accurate and less expensive method could be used in the field of medical research and in the clinical DNA diagnosis of human cancers and other diseases.

Base Sequence↗