Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Point Mutation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,747 records · Page 97Linked to original sources
PubMed1991

Point mutations in the beta-subunit of cytochrome b558 leading to X-linked chronic granulomatous disease.

The NADPH:O2 oxidoreductase of phagocytic leukocytes is an important enzyme for the bactericidal activity of these cells. Cytochrome b558 is a membrane component of this enzyme. In X-linked chronic granulomatous disease (Xb- CGD) the phagocytes are defective in the beta-subunit (gp91-phox) of this cytochrome. We have studied the genetic defect in a group of six X-linked CGD patients characterized by complete or partial loss of cytochrome b558 with the use of the polymerase chain reaction. All patients had a different single point mutation in the gp91-phox gene, indicating that the genetic defect in Xb- CGD is very heterogeneous. In one patient the mutation leads to a premature termination codon. In the other five cases these mutations predict incorporation of a different amino acid. The mutations were with one exception found in the N-terminal half of the protein, suggesting that this part of cytochrome b558 is important for the binding of the heme or for formation of a stable complex with p22-phox. Two histidyl residues were found that might be ligands of the heme iron.

Alleles↗
PubMed1990

Point mutations in the ICR2 motif of brome mosaic virus RNAs debilitate (+)-strand replication.

Sequences at the 5' termini of the genomic RNAs of brome mosaic virus (BMV) and other (+)-stranded RNA viruses have been shown (L.E. Marsh and T.C. Hall, 1987, Cold Spring Harbor Symp. Quant. Biol. 52, 331-341) to resemble the ICRs 1 and 2 (A and B boxes) of tRNA genes, with the complementary sequences at the 3' termini of the (-) strands resembling the ICR2 motif of methionine initiator tRNA genes (L.E. Marsh, G.P. Pogue, and T.C. Hall, 1989, Virology 172, 415-427). In order to examine the role of these sequences in viral replication, point mutations have been introduced into the ICR2-like sequence of a BMV RNA-2 deletion mutant, pRNA delta M/S (parasitic RNA), that does not encode a functional viral protein but replicates in the presence of genomic RNA-1 and -2. Single-base substitutions introduced at positions A7 or T8 of the (+)-sense ICR2-like motif reduced pRNA delta M/S replication by 70-82%, the primary effect being shown by kinetic analyses to be debilitation of (+)-strand synthesis. Whether these motifs act in their (+)-sense orientation in a manner analogous to tRNA genes or through the tRNA(Meti)-like sequence on the 3' (-) strand remains to be determined, but the data clearly demonstrate that the base composition within the ICR-like region of BMV RNAs contributes greatly to (+)-strand promoter function.

Base Sequence↗
PubMed1992

A novel point mutation in the human insulin gene giving rise to hyperproinsulinemia (proinsulin Kyoto).

We have identified a 65-yr-old nonobese Japanese man with diabetes mellitus, fasting hyperinsulinemia (150-300 pM), and a reduced fasting C-peptide/insulin molar ratio of 2.5-3.0. Fasting hyperinsulinemia was also found in his son and daughter. Analysis of insulin isolated from the serum of the proband and his son by reverse-phase high performance liquid chromatography revealed a minor peak coeluting with human insulin and a major peak of proinsulin-like materials. The insulin gene of the patient was amplified by the polymerase chain reaction and the products were sequenced. A novel point mutation was identified in which guanine was replaced by thymine. The substitution gives rise to a new HindIII recognition site and results in the amino acid replacement of leucine for arginine at position 65. These results indicate that the amino-acid replacement prevents recognition of the C-peptide-A chain dibasic protease and results in an elevation of proinsulin-like materials in the circulation. Furthermore, in this family the proinsulin-like materials is due to a biosynthetic defect, inherited as an autosomal dominant trait. Rapid detection of this mutation can be accomplished by HindIII restriction enzyme mapping of polymerase chain reaction-generated DNA, which enables us to facilitate the diagnosis and screening.

Adult↗
PubMed1986

Idiotypic selection of an antibody mutant with changed hapten binding specificity, resulting from a point mutation in position 50 of the heavy chain.

Somatic mutation occurs at a low rate in the rearranged antibody V region genes of the hybridoma line B1-8. delta 1 which expresses an antibody with specificity for the hapten 4-hydroxy-3-nitro-5-iodo-phenylacetyl (NIP). A mutant was selected which had lost a binding site-related idiotope but retained most of its other idiotypic determinants. The mutant had concomitantly lost NIP binding and acquired specificity for dinitrophenylated bovine serum albumin. It carried a single point mutation in position 50 of the heavy chain, resulting in the replacement of an arginine by a glycine.

Amino Acid Sequence↗
PubMed1990

Point mutations impairing cell surface expression of the common beta subunit (CD18) in a patient with leukocyte adhesion molecule (Leu-CAM) deficiency.

The leukocyte adhesion molecules CD11a/CD18, CD11b/CD18, and CD11c/CD18 (Leu-CAM) are members of the integrin receptor family and mediate crucial adhesion-dependent functions in leukocytes. The molecular basis for their deficient cell surface expression was sought in a patient suffering from severe and recurrent bacterial infections. Previous studies revealed that impaired cell surface expression of Leu-CAM is secondary to heterogeneous structural defects in the common beta subunit (CD18). Cloning and sequencing of complementary DNA encoding for CD18 in this patient revealed two mutant alleles, each representing a point mutation in the coding region of CD18 and resulting in an amino acid substitution. Each mutant allele results in impaired CD18 expression on the cell surface membrane of transfected COS M6 cells. One substitution involves an arginine residue (Arg593----cysteine) that is conserved in the highly homologous fourth cysteine-rich repeats of other mammalian integrin subfamilies. The other substitution involves a lysine residue (Lys196----threonine) located within another highly conserved region in integrins. These data identify crucial residues and regions necessary for normal cell surface expression of CD18 and possibly other integrin beta subunits and define a molecular basis for impaired cell surface expression of CD18 in this patient.

Amino Acid Sequence↗
PubMed1991

Diverse point mutations in the human gene for polymorphic N-acetyltransferase.

Classification of humans as rapid or slow acetylators is based on hereditary differences in rates of N-acetylation of therapeutic and carcinogenic agents, but N-acetylation of certain arylamine drugs displays no genetic variations. Two highly homologous human genes for N-acetyltransferase (NAT; arylamine acetyltransferase, acetyl CoA:arylamine N-acetyltransferase, EC 2.3.1.5), NAT1 and NAT2, presumably code for the genetically invariant and variant NAT proteins, respectively. In the present investigation, 1.9-kilobase human genomic EcoRI fragments encoding NAT2 were generated by the polymerase chain reaction with liver and leukocyte DNA from seven subjects phenotyped as homozygous and heterozygous acetylators. Direct sequencing revealed multiple point mutations in the coding region of two distinct NAT2 variants. One of these was derived from leukocytes of a slow acetylator and was distinguished by a silent mutation (codon 94) and a separate G----A transition (position 590) leading to replacement of Arg-197 by Gln; the mutated guanine was part of a CpG dinucleotide and a Taq I site. The second NAT2 variant originated from liver with low N-acetylation activity. It was characterized by three nucleotide transitions giving rise to a silent mutation (codon 161), accompanied by obliteration of the sole Kpn I site, and two amino acid substitutions: Thr for Ile (codon 114) and Arg for Lys (codon 268). Heterozygosity was detected in three NAT2 samples: two were heterozygous for the rapid and one of the allelic variants, and the third was a compound heterozygote of both mutant alleles. The results show conclusively that the genetically variant NAT is encoded by NAT2.

Acetylation↗
PubMed1990

Point mutations at codon 12 of C-K-ras in human endometrial carcinomas.

Samples of human endometrial carcinomas of ranging degree of histological differentiation, and normal endometrial tissues were screened for the presence of single site DNA mutations at codon 12 of the C-K-ras gene using oligonucleotide hybridisation of DNA amplified by the polymerase chain reaction (PCR). Two instances of base substitution were detected; one resulting in a GLY-ALA amino acid replacement in a well differentiated (grade 1) tumour and the second causing a GLY-CYS replacement in a poorly differentiated (grade 3 tumour). The small number of samples tested does not permit conclusions to be drawn with respect to correlations between DNA point mutations and the clinical features, age of patient, tumour receptor status and degree of histological differentiation grading.

Adult↗
PubMed1990

Chromosomal rearrangements and point mutations in the DHFR-TS gene of Plasmodium chabaudi under antifolate selection.

Selection of the rodent malaria Plasmodium chabaudi with low levels of the antifolate drug pyrimethamine has previously been shown by us to result in duplication of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene by a duplication of chromosome 7 and subsequent rearrangements. We have selected this resultant parasite line with large doses of pyrimethamine and analysed the DHFR-TS gene and chromosomes for any changes. Increased drug pressure has resulted in reappearance of a chromosome with the same structure as chromosome 7 from DS the parent line. Sequencing of the DHFR gene from each of the chromosomes has identified a single point mutation that results in a serine to asparagine change at position 106. This is the equivalent mutation that has been identified as the key residue in the mechanism of resistance to pyrimethamine in Plasmodium falciparum. There is no apparent increase in transcription of the DHFR-TS gene and the large increase in resistance is most likely a result of the mutation in the DHFR gene.

Animals↗
PubMed1987

Factor IXAlabama: a point mutation in a clotting protein results in hemophilia B.

Factor IXAlabama is a variant factor IX molecule responsible for a clinically moderate form of hemophilia B. Twenty-five kilobases (kb) of the variant gene, including seven exons coding for the structural protein, were cloned and characterized. The restriction map and the arrangement of coding regions are identical to those of the normal gene. DNA sequence analysis of the coding regions revealed a single base-pair difference between the gene for factor IXAlabama and the normal factor IX gene. An adenine to guanine transition in the first nucleotide of exon d causes the substitution of a glycine codon (GGT) for the normal aspartic acid codon (GAT). This point mutation results in a single amino acid substitution at residue 47 of the zymogen and represents the genetic defect in factor IXAlabama.

Amino Acid Sequence↗
PubMed1990

A unique point mutation in the human vitamin D receptor chromosomal gene confers hereditary resistance to 1,25-dihydroxyvitamin D3.

The syndrome of hereditary resistance to 1,25-dihydroxyvitamin D3 is due to defective function of the vitamin D receptor (VDR). The recent cloning and nucleotide sequence determination of the human VDR chromosomal gene have enabled a direct evaluation of the genetic basis for this disease in affected patients. In this report we employed polymerase chain reaction techniques to amplify the gene exons that encode the DNA-binding domain of the VDR from two 1,25-dihydroxyvitamin D3-resistant patients whose receptors displayed defective binding to nonspecific DNA. Although their families were apparently unrelated, each patient displayed an identical homozygous point mutation within the third exon, a mutation that causes substitution of a glutamine for an arginine residue highly conserved within the entire steroid receptor superfamily. We introduced this base change into the normal VDR cDNA via site-directed mutagenesis, transfected an expression vector containing this cDNA into cells, and examined the functional properties of the resultant VDR expression product. The produced mutant receptor bound 1,25-dihydroxyvitamin D3 with normal affinity, but displayed weak affinity for the nuclear fraction and for heterologous DNA. More importantly, the protein was inactive in promoting transcription in a cotransfection assay employing a chloramphenicol acetyltransferase gene reporter fused down-stream of the VDR-inducible osteocalcin gene promoter-enhancer. These results provide the genetic and functional basis for the phenotype of rickets in this inherited disease.

Amino Acid Sequence↗
PubMedDate not supplied

Haemophilia B caused by a point mutation in a donor splice junction of the human factor IX gene.

Haemophilia B (Christmas disease) is an inherited, recessive, sex-linked, haemorrhagic condition caused by a defect in the intrinsic clotting factor IX. This disease occurs in males at a frequency of approximately 1 in 30,000. Patients differ in the severity of their clinical symptoms, and variation in the clotting activity and in the concentration of factor IX antigen in their plasma has been demonstrated. There is probably heterogeneity in the molecular defects of the factor IX gene causing the disease. Here we study a severely affected, antigen-negative patient, and show that the only significant sequence difference from the normal factor IX gene is a point mutation changing the obligatory GT to a TT within the donor splice junction of exon f. We infer that this change is the cause of the disease in this individual. In addition, we have used oligodeoxynucleotide probes specific for this mutation to demonstrate the feasibility of carrier detection and prenatal diagnosis for relatives of the patient.

Base Sequence↗
PubMed1997

Mutational analysis of ectopic factor VIII transcripts from hemophilia A patients: identification of cryptic splice site, exon skipping and novel point mutations.

Mutational analysis of the gene for clotting factor VIII is complicated by its large size, the high frequency of de novo mutations and its tissue-specific expression. In order to facilitate the search for mutations, we have used a combination of reverse transcription-polymerase chain reaction (RT-PCR) of ectopic factor VIII transcripts, PCR of genomic DNA, single-strand conformation polymorphism analysis and direct sequencing. Here we describe the characterization of seven potentially pathogenic mutations: five of them are novel and the reason for the pathogenicity of the sixth could be determined. Here cDNA analysis revealed the absence of the first 47 bp of exon 16 in approximately 80% of the processed factor VIII mRNA, likely due to activation of a cryptic acceptor splice site within exon 16. The other novel mutations reported here include the skipping of exon 19, which predicts the removal of the corresponding 39 amino acids from the A3 domain, and four missense mutations: W14G, Y620C, W1889L, and Q2087R.

DNA Mutational Analysis↗
PubMed2001

Molecular genetic analysis of an endotoxin nonresponder mutant cell line: a point mutation in a conserved region of MD-2 abolishes endotoxin-induced signaling.

Somatic cell mutagenesis is a powerful tool for characterizing receptor systems. We reported previously two complementation groups of mutant cell lines derived from CD14-transfected Chinese hamster ovary--K1 fibroblasts defective in responses to bacterial endotoxin. Both classes of mutants expressed a normal gene product for Toll-like receptor (TLR)4, and fully responded to stimulation by tumor necrosis factor (TNF)-alpha or interleukin (IL)-1 beta. We identified the lesion in one of the complementation groups in the gene for MD-2, a putative TLR4 coreceptor. The nonresponder phenotype of this mutant was reversed by transfection with MD-2. Cloning of MD-2 from the nonresponder cell line revealed a point mutation in a highly conserved region resulting in a C95Y amino acid exchange. Both forms of MD-2 colocalized with TLR4 on the cell surface after transfection, but only the wild-type cDNA reverted the lipopolysaccharide (LPS) nonresponder phenotype. Furthermore, soluble MD-2, but not soluble MD-2(C95Y), functioned to enable LPS responses in cells that expressed TLR4. Thus, MD-2 is a required component of the LPS signaling complex and can function as a soluble receptor for cells that do not otherwise express it. We hypothesize that MD-2 conformationally affects the extracellular domain of TLR4, perhaps resulting in a change in affinity for LPS or functioning as a portion of the true ligand for TLR4.

Animals↗
PubMed1991

Two point mutations in the hormone-binding domain of the mouse glucocorticoid receptor that dramatically reduce its function.

Mouse lymphoma cell line W7M320b, a mutant WEH17 line, requires higher than normal concentrations of glucocorticoid to elicit the hormone responses that are characteristic of this lineage. Complementary DNA clones representing the glucocorticoid receptor (GR) mRNA were derived from the mutant cells, and the sequences coding for the hormone-binding domain were substituted for the analogous wild-type sequences in a GR cDNA expression vector. The function of the resulting GR proteins was tested by transient expression in COS-7 cells along with a glucocorticoid-inducible reporter gene in the presence of varying concentrations of glucocorticoid. From these assays and DNA sequence analyses, two independent functionally significant point mutations in the GR hormone-binding domain were identified. A mutant GR protein containing the single amino acid substitution, Pro547 to Ala, was still functional as a transcriptional activator, but only at hormone concentrations 100 times higher than those required by the wild-type receptor. A second mutant GR protein with a Cys742 to Gly substitution was unstable and almost completely nonfunctional.

Amino Acid Sequence↗
PubMed1990

Failure of cell surface expression of a class I major histocompatibility antigen caused by somatic point mutation.

The ability to down-regulate major histocompatibility complex class I antigen expression on allografts prior to transplantation would be expected to improve their survival in immunocompetent recipients. In order to identify genetic mechanisms that mediate attenuation of MHC class I antigen expression, we have begun characterizing H-2Kb surface null somatic cell variants derived from an H-2 heterozygous tumor cell line (H-2b X H-2d). These variants have sustained a modification in cell surface MHC phenotype, as evidenced by their failure to be recognized by both anti-H-2Kb antibodies and cytotoxic T lymphocytes. The mutant phenotype for one such variant (designated 69.9.15) was marked by the expression of abundant H-2Kb mRNA and immuno-precipitable H-2Kb protein in cell lysates. The failure in cell surface expression of the H-2Kb antigen was caused by a single base change (G to A transition) in exon 3, encoding the second external domain (alpha 2) of the H-2Kb molecule. The mutation resulted in the substitution of Tyr for Cys at amino acid position 164, thereby disrupting an intrachain disulfide linkage formed between Cys 101 and 164. In contrast to the wild-type H-2Kb gene, DNA-mediated transfer of the mutant H-2Kb gene into mouse L cell fibroblasts failed to result in cell surface expression of the H-2Kb antigen, although both the wild-type and mutant genes were transcribed to equivalent levels. These data indicate that a genetic event as limited as somatic point mutation can abrogate expression of a MHC class I antigen and provide support for the hypothesis that protein folding plays an important role in the cell surface expression of MHC class I molecules.

Animals↗
PubMed1989

Recognition of the HLA class II-peptide complex by T-cell receptor: reversal of major histocompatibility complex restriction of a T-cell clone by a point mutation in the peptide determinant.

Recognition of the HLA DR-peptide complex by an influenza haemagglutinin-specific T-cell clone was examined by assaying a variety of peptide analogues for their ability to be recognized. Consistent with earlier experiments arguing that the peptide blinds the restriction element in a helical conformation, acetylation of the amino terminus and amidation of the carboxy terminus of the natural determinant (residues 307-319) resulted in a peptide that exhibited both greater propensity to form a helix, as judged by circular dichroism, and the ability to stimulate the clone at concentrations approximately two orders of magnitude lower than the native sequence. The peptide was modelled into the potential antigen-combining site of HLA class II based on the ability of analogues containing point mutations to stimulate the T-cell clone. The working model was initially tested by examining the ability of Epstein-Barr-transformed B-cell lines expressing in different DR4 subtypes to present the native haemagglutinin sequence and analogues to the clone. The different alleles could be categorized as high, intermediate, or low responders based on the resulting proliferation. DR4 dw15 was a high-responding allele, dw4, 13, and 14 were intermediate-responding alleles, whereas dw10 was a low responder. Mutation of Gln to Arg at 312 in the haemagglutinin sequence converted the high and intermediate responders to non-responders, while turning the low-responding allele into an intermediate responder. Potential explanations for these effects are discussed in the context of the model of the complex between peptide and the major histocompatibility complex.

Amino Acid Sequence↗
PubMed1991

Point mutation causing a single amino acid substitution in the hormone binding domain of the glucocorticoid receptor in familial glucocorticoid resistance.

Familial glucocorticoid resistance is a hypertensive, hyperandrogenic disorder characterized by increased serum cortisol concentrations in the absence of stigmata of Cushing's syndrome. Our previous studies of the first reported kindred showed a two- to threefold reduction in glucocorticoid receptor-ligand binding affinity in the propositus, and a lesser reduction in affinity in his mildly affected son and nephew. Glucocorticoid receptor cDNA from these three patients was amplified by polymerase chain reaction and sequenced. The cDNA nucleotide sequence was normal, except for nucleotide 2054, which substituted valine for aspartic acid at amino acid residue 641. The propositus was homozygous while the other relatives were heterozygous for the mutation. COS-7 monkey kidney cells were cotransfected with expression vectors for either wild type or Val 641-mutant receptors, together with the reporter plasmid pMMTV-CAT. Dexamethasone increased chloramphenicol acetyltransferase activity in cells expressing wild type receptor, but had no effect in cells expressing Val 641-mutant receptors, despite similar receptor concentrations, as indicated by Western blotting. The binding affinity for dexamethasone of the Val 641-mutant receptor was threefold lower than that of the wild type receptor. These results suggest that glucocorticoid resistance in this family is due to a point mutation in the steroid-binding domain of the glucocorticoid receptor.

Amino Acids↗
PubMed1991

The "Normandy" variant of von Willebrand disease: characterization of a point mutation in the von Willebrand factor gene.

We previously reported a functional defect of von Willebrand factor (vWF) in a new variant of von Willebrand disease (vWD) tentatively named vWD "Normandy." The present work has attempted to characterize the molecular abnormality of this vWF that fails to bind factor VIII (FVIII). The immunopurified vWF from normal and patient's plasma were digested by trypsin and the resulting peptides were compared. The electrophoresis of "vWF Normandy" showed a shift in the band corresponding to a polypeptide from amino acid 1 to 272. Consequently, we performed the molecular analysis of the portion of the vWF gene of this patient encoding this amino acid sequence. Exons 18-24 were amplified by the use of polymerase chain reaction and their nucleotide sequences corresponding to 1.8 kb were determined. Our analysis showed a point mutation C to T at codon 791, resulting in the substitution of Methionine for Threonine at position 28 of the mature vWF subunit. Because this nucleotide substitution destroyed a Mae II restriction site, this mutation was conveniently sought in various individual DNAs. The patterns obtained were consistent with the homozygous and heterozygous state of this mutation in the patient and in her son, respectively, and with its absence in 28 normal individuals. We conclude that Threonine at position 28 in plasma vWF may be crucial for the conformation and FVIII-binding capacity of its cystine-rich N-terminal domain.

Base Sequence↗