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PubMed1985

Transforming activity of the c-Ha-ras oncogene having two point mutations in codons 12 and 61.

A recombinant plasmid carrying the human c-Ha-ras gene with two point mutations in codons 12 and 61 was constructed and its transforming activity on mouse NIH 3T3 cells was compared with those of genes with a single mutation in either codon 12 or 61. Quantitative analyses revealed that the gene with two mutations had essentially the same transforming activity as the genes with single mutations. These results indicate that a single mutation of the c-Ha-ras gene in either codon 12 or 61 is sufficient to activate the gene and that neither of the two mutation sites involved in activation of the gene needs to be intact for transforming activity.

Animals↗
PubMed1993

Cyclobutane thymine dimers in a ras proto-oncogene hot spot activate the gene by point mutation.

ras proto-oncogenes with a cyclobutane-type thymine photodimer (cis-syn or trans-syn isomer) were constructed by replacement of a portion of the gene with a chemically synthesized fragment. When the genes were transfected by the calcium phosphate method into mouse NIH3T3 cells, they induced focus-formation, indicating that both photoproducts were mutagenic in mammalian cells. Sequence analysis of the ras gene fragments derived from the transformed cells showed that the genes were activated by a point mutation. The mutations detected most frequently were 3'-T-->A for the cis-syn isomer and 5'-T-->A for the trans-syn isomer. In contrast, a different trend of mutations was observed when a primer on a DNA template with a cis-syn dimer was extended in vitro by either DNA polymerase beta or alpha.

3T3 Cells↗
PubMed2004

Large-scale prediction of protein geometry and stability changes for arbitrary single point mutations.

We have developed a method to both predict the geometry and the relative stability of point mutants that may be used for arbitrary mutations. The geometry optimization procedure was first tested on a new benchmark of 2141 ordered pairs of X-ray crystal structures of proteins that differ by a single point mutation, the largest data set to date. An empirical energy function, which includes terms representing the energy contributions of the folded and denatured proteins and uses the predicted mutant side chain conformation, was fit to a training set consisting of half of a diverse set of 1816 experimental stability values for single point mutations in 81 different proteins. The data included a substantial number of small to large residue mutations not considered by previous prediction studies. After removing 22 (approximately 2%) outliers, the stability calculation gave a standard deviation of 1.08 kcal/mol with a correlation coefficient of 0.82. The prediction method was then tested on the remaining half of the experimental data, giving a standard deviation of 1.10 kcal/mol and covariance of 0.66 for 97% of the test set. A regression fit of the energy function to a subset of 137 mutants, for which both native and mutant structures were available, gave a prediction error comparable to that for the complete training set with predicted side chain conformations. We found that about half of the variation is due to conformation-independent residue contributions. Finally, a fit to the experimental stability data using these residue parameters exclusively suggests guidelines for improving protein stability in the absence of detailed structure information.

Amino Acids↗
PubMed1995

A point mutation in the mitochondrial DNA of diabetes-prone BHE/cdb rats.

Mitochondrial DNA was extracted from hepatic tissue of 50- and 300-day-old male BHE/cdb and Sprague-Dawley rats. The complete gene for the F0ATPase subunits 6 and 8 was sequenced. Four nucleotide substitutions were found: three of the substitutions were silent; the other substitution at position 523 was not. Its codon dictates the substitution of asparagine for aspartic acid in a critical location (in the polar pocket) of the F0ATPase. It is possible that this point mutation may explain previously reported decreases in ATP synthesis efficiency in BHE/cdb rats compared to Sprague-Dawley rats.

Aging↗
PubMed1993

Somatic point mutations in the translocated bcl-2 genes of non-Hodgkin's lymphomas and lymphocytic leukemias: implications for mechanisms of tumor progression.

The t[14;18] chromosomal translocation is the most common cytogenetic abnormality found in hematolymphoid malignancies. The t[14;18] fuses the bcl-2 gene at 18q21 with the immunoglobulin heavy-chain locus at 14q32, resulting in deregulated expression of bcl-2 and production of high levels of its encoded 26-kD protein in the majority of non-Hodgkin lymphomas. Recent data indicate that somatic point mutations frequently occur in translocated bcl-2 alleles, possibly because of the somatic hypermutation mechanism that is associated with the immunoglobulin gene loci and that normally contributes to antibody diversity. In some cases, these mutations can affect the open reading frame of the bcl-2 gene and thereby alter Bcl-2 proteins. Here, we review the currently available data about the incidence, biological effects, and possible clinical importance of somatic mutations within the translocated bcl-2 genes of human lymphomas and leukemias.

Alleles↗
PubMed1993

A point mutation in a Drosophila GABA receptor confers insecticide resistance.

Vertebrates and invertebrates both have GABA (gamma-aminobutyric acid) as a major inhibitory neurotransmitter. GABAA receptors in vertebrates assemble as heteromultimers to form an integral chloride ion channel. These receptors are targets for drugs and pesticides and are also implicated in seizure-related diseases. Picrotoxinin (PTX) and cyclodiene insecticides are GABAA receptor antagonists which competitively displace each other from the same binding site. Insects and vertebrates showing resistance to cyclodienes also show cross-resistance to PTX. Previously, we used a field-isolated Drosophila mutant Rdl (Resistant to dieldrin) insensitive to PTX and cyclodienes to clone a putative GABA receptor. Here we report the functional expression and novel pharmacology of this GABA receptor and examine the functionality of a resistance-associated point mutation (alanine to serine) within the second membrane-spanning domain, the region thought to line the chloride ion channel pore. This substitution is found globally in Drosophila populations. This mutation not only identifies a single amino acid conferring high levels of resistance to the important GABA receptor antagonist PTX but also, by conferring resistance to cyclodienes, may account for over 60% of reported cases of insecticide resistance.

Alanine↗
PubMed1998

A point mutation within exon 5 of the WT1 gene of a sporadic unilateral Wilms' tumor alters gene function.

The Wilms' tumor suppressor gene, wt1, encodes a zinc-finger transcription factor, WT1, that represses transcription of a number of growth-promoting genes and inhibits cell growth. The transcripts of wt1 undergo two alternative splicing events, giving rise to four isoforms of mRNA in constant ratios. The first alternative splice introduces an extra exon 5, which encodes 17 amino acid residues inserted between the transcription regulatory domain and the DNA binding domain of WT1. Previously, we demonstrated that the 17-amino acid domain functioned as a transcription repressor when it was fused with the DNA binding domain of WT1. We have now identified a point mutation within exon 5 of wt1 in a sporadic unilateral Wilms' tumor patient. The mutation changes the last of the 17 amino acids from asparagine to serine. The protein isoform of WT1 carrying this mutation exhibited a 2-3-fold lower transcription-repressing activity than wild-type WT1 in transient cotransfection assays. The mutation also decreased growth-inhibiting activity of WT1 in two osteosarcoma cell lines, U2OS and Saos-2. By diminishing transcription-repressing and growth-inhibiting activities of WT1, this naturally occurring mutation within exon 5 of wt1 may disturb the normal function of the protein and lead to the uncontrolled cell growth characteristic of Wilms' tumor.

Alternative Splicing↗
PubMed1995

Absence of N-RAS point mutations in peripheral blood cells of patients with aplastic anaemia and paroxysmal nocturnal haemoglobinurea.

The myelodysplastic syndromes (MDS) have a significant frequency of evolution into acute myeloid leukaemia (AML). Approximately 30% of MDS patients show activating mutations of the N-RAS proto-oncogene, and these patients are at increased risk of leukaemic evolution. Long-term survivors of aplastic anaemia (AA) and paroxysmal nocturnal haemoglobinurea (PNH) are also at significant risk of developing AML. We have screened peripheral blood DNA from 42 AA patients and 15 PNH patients for the presence of N-RAS point mutations. No mutations were detected in these samples, indicating that the mechanisms of evolution into AML may be different from those in MDS.

Acute Disease↗
PubMed1993

Analysis of the SRY gene in 22 sex-reversed XY females identifies four new point mutations in the conserved DNA binding domain.

The open reading frame of the SRY gene has been examined in a series of 22 XY females with clinically defined pure gonadal dysgenesis by direct sequencing of biotinylated PCR product bound to streptavidin coated beads. Amongst the 22 XY females examined, five (two of whom are sisters) were found to have single base changes all within the highly conserved DNA binding (or HMG box) domain. In the remaining 17 cases, the SRY gene sequence was indistinguishable from that found in normal males. In three of the XY females with point mutations, the altered amino acids occur in highly conserved positions leading to non-conservative changes (Arg to Gly at position 5, Met to Thr at position 21 and Arg to Trp at position 76). Examination of the SRY gene from the father's Y chromosome has shown that the mutations at position 5 in patient SHM60 and position 21 in patient HN31 have arisen de novo. In the case of the two sibs, both have identical mutations where a C to T transition in codon 17 has created a TAG termination signal, thus suggesting that the deceased father is likely to be a gonadal mosaic for the mutation. In the case of the mutations at positions 17 and 76, the fathers are not available for investigation and so it has not been possible to determine whether the changes are de novo. These data indicate that the majority of XY females with pure gonadal dysgenesis owe their sex-reversed phenotype to mutations in as yet uncharacterised segments of the SRY gene, or, at other loci acting early in the sex-determining pathway.

Adolescent↗
PubMed1996

Identification of two novel point mutations in the human protein S gene associated with familial protein S deficiency and thrombosis.

Individuals with thrombosis who were believed to possess associated familial protein S deficiency were analyzed for mutations in the protein S gene by a two-step process. First, the individuals were analyzed for protein S Pro626 A/G dimorphism in both their genomic DNA and reverse-transcribed (RT) polymerase chain reaction (PCR)-amplified cDNA from peripheral blood cell mRNA. If a heterozygote expressed both alleles at the mRNA level at this site in genomic DNA, a search for point mutations was made by direct cDNA sequencing. RT-PCR amplification of exons 1-6 with mRNA from two twin sisters, each of whom has severe type I protein S deficiency, revealed both larger and smaller fragments in addition to the expected 504-base pair fragment in normal individuals. A donor splicesite mutation at position +4 of the 5' end of intron A was subsequently identified in both sisters and their mother. This mutation would lead to incorrect precursor mRNA splicing and the observed cDNA products. Translation of the altered mRNAs would result in a truncated protein without biological activity. In a second family, cDNA sequencing revealed a T-->G mutation at codon 603 (Ile-->Ser) in exon 15 of the protein S gene in an individual with protein S deficiency (mixed type) and a history of thrombosis. The same mutation was also detected in the proband's mother and grandmother, both of whom also exhibit protein S deficiency and thrombotic disease. This mutation occurs within a disulfide loop of protein S that is believed to be responsible for binding to C4b binding protein and may result in greater affinity between protein S and C4b, consequently leading to thrombotic disease.

Adult↗
PubMed2001

A point mutation in the alpha-actinin-4 gene generates an antigenic peptide recognized by autologous cytolytic T lymphocytes on a human lung carcinoma.

We have identified an antigen recognized on a human large cell carcinoma by an autologous tumor-specific CTL clone that was derived from mononuclear cells infiltrating the primary tumor. The antigenic peptide is presented by HLA-A2 molecules and is encoded by the alpha-actinin-4 gene, which is expressed ubiquitously. In the tumor cells, a point mutation generates an amino-acid change that is essential for recognition by the CTLS: The mutation was not found in alpha-actinin-4 cDNA sequences from about 50 lung carcinoma cell lines, suggesting that it is unique to this patient. Although he did not receive chemotherapy or radiotherapy, the patient has been without evidence of tumor since the resection of the primary lesion in 1996. Using tetramers of soluble HLA-A2 molecules loaded with the mutated antigenic peptide, anti-alpha-actinin-4 CTLs could be derived from blood samples collected from the patient in 1998 and 2000. It is possible that these CTLs, recognizing a truly tumor-specific antigen, play a role in the clinical evolution of this lung cancer patient.

Actinin↗
PubMed1993

Abnormal behavior associated with a point mutation in the structural gene for monoamine oxidase A.

Genetic and metabolic studies have been done on a large kindred in which several males are affected by a syndrome of borderline mental retardation and abnormal behavior. The types of behavior that occurred include impulsive aggression, arson, attempted rape, and exhibitionism. Analysis of 24-hour urine samples indicated markedly disturbed monoamine metabolism. This syndrome was associated with a complete and selective deficiency of enzymatic activity of monoamine oxidase A (MAOA). In each of five affected males, a point mutation was identified in the eighth exon of the MAOA structural gene, which changes a glutamine to a termination codon. Thus, isolated complete MAOA deficiency in this family is associated with a recognizable behavioral phenotype that includes disturbed regulation of impulsive aggression.

Aggression↗
PubMed2004

Selective circular oligonucleotide probes improve detection of point mutations in DNA.

The synthesis of disulfide-cross-linked circular oligonucleotides, employing two different approaches, was accomplished. Several circular oligomers, which bear a C(5)-aminoalkyl-tethered thymidine unit, were labeled with photoluminescent europium(III) chelates. All circular structures were thoroughly characterized with denaturing PAGE and electrospray-ionization mass spectrometry. It was demonstrated that the disulfide cross-linking, resulting in circularization, considerably increases the enzymatic stability of phosphodiester oligonucleotides. In addition, UV melting experiments, followed, where possible, by extraction of thermodynamic parameters, revealed that several circular oligomers appear to be more selective towards their complementary targets than their corresponding linear precursors. Finally, the mixed-phase hybridization experiments have demonstrated that use of circular probes indeed improves the selectivity in the detection of DNA point mutations.

DNA, Circular↗
PubMed1994

Point mutations define a mIgM transmembrane region motif that determines intersubunit signal transduction in the antigen receptor.

Ag binding to the membrane Ig (mIg) substructure of the B cell Ag receptor leads to activation of cytoplasmic effector molecules including blk, fyn, lyn, and/or lck tyrosine kinases that are associated with receptor's dimeric Ig-alpha/Ig-beta transducer substructure. The structural basis of the apparent intermolecular transmission of this information within the receptor complex is unknown. Here we report that conservative point mutation of a sequence, S584-K597, at the cytoplasmic end of the predicted transmembrane spanning domain of the mIgM heavy chain (mu) ablates Ag-activated signal transduction, while having no detectable effect on association of mIgM with Ig-alpha/Ig-beta heterodimers. Specifically, mutation of serine584 to alanine, tyrosine587 to phenylalanine, threonine592 to valine, or lysine597 to isoleucine completely abrogated Ag-induced signal transduction leading to protein tyrosine phosphorylation and Ca2+ mobilization. Interestingly, mutants in the more peripheral of these residues, serine584 to alanine and lysine597 to isoleucine, remained responsive to a monoclonal antireceptor Ab (b-7-6) and all mutants remained responsive to polyclonal antireceptor Ab. These data implicate the polar sequence, -Y587STTVT592-, in transfer of information from ligand binding to transducer substructures within this heterooligomeric receptor complex. They further indicate that receptor activation by ligands that bind with high affinity and/or to constant region mIg epitopes is less dependent on the integrity of this motif.

Amino Acid Sequence↗
PubMed1997

A novel point mutation in the insulin gene giving rise to hyperproinsulinemia.

A 58-yr-old obese white Caucasian male type 2 diabetic, entered into the UK Prospective Diabetes Study, was found to have raised fasting total proinsulin levels 708 pmol/L(-1) (normal range, 3-16 pmol/L(-1)) and normal specific plasma insulin level 29 pmol/L(-1) (normal range, 21-75 pmol/L(-1)). Immunoreactive plasma insulin, measured by RIA, was 503 pmol/L(-1). DNA was extracted, the insulin gene amplified by the PCR, and by direct sequencing, a novel point mutation, G1552C, was identified, which resulted in the substitution of proline (CCT) for arginine (CGT) at position 65. This prevented cleavage of the C-peptide A-chain dibasic cleavage site (lys-arg) by the processing protease in the pancreatic beta-cells. The plasma proinsulin and insulin levels were in accord with expression of both the wild-type and the mutant alleles. The G1552C mutation was not linked with diabetes, because it was present in a 37-yr-old nondiabetic daughter and not in a 35-yr-old daughter who had had gestational diabetes.

Arginine↗
PubMed1997

Modulation of a recombinant invertebrate gamma-aminobutyric acid receptor-chloride channel complex by isoflurane: effects of a point mutation in the M2 domain.

1. Inhalational anaesthetics modulate ligand-gated ion channels at clinical concentrations. In this paper we address submolecular mechanisms for gamma-aminobutyric acid (GABA) receptor modulation by isoflurane. 2. Wild-type Drosophila melanogaster homo-oligomeric GABA receptors were characterized and compared with an ion-channel mutant (alanine substituted to a serine in M2) by means of two-electrode voltage-clamp in membrane-invariant Xenopus oocytes. 3. Both channel receptor isoforms generated outwardly rectifying, bicuculline-insensitive currents with reversal potentials characteristic of a chloride current. 4. As previously shown, the point mutation in the M2 domain conferred a profound resistance to the blocking action of 10 microM picrotoxinin (PTX): circa 7 fold reduction at the GABA EC20. 5. Isoflurane, 195-389 microM, enhanced GABA conductance in both receptor variants by significantly increasing the affinity of the agonist for its receptor without changing Hill slope or maximal response. Relative potencies were statistically indistinguishable. 6. Isoflurane concentration-response curves (on circa GABA EC25) demonstrated that enhancement was effected at around 100-195 microM for both receptor subtypes, but a dramatic divergence was evident at concentrations above 400 microM: wild-type receptors exhibited concentration-dependent block, whilst mutant conductances continued to increase over the same concentration range, showing no tendency to saturate (up to 3330 microM). 7. The above divergence was not attributable to differential desensitization: neither wild-type nor mutant conductance desensitized significantly (P > 0.05) in the absence or presence of anaesthetic. 8. This work demonstrates that modulatory sites for anaesthetic are present on a relatively primitive insect ion channel. 9. The depression of GABA response at high isoflurane concentrations, in WT receptors, (typical of a variety of anaesthetic agents) may reflect low affinity channel block via the PTX site. 10. The non-saturable enhancement of chloride conductances, when the PTX site is mutated, is not consistent with topical proposals that inhalational anaesthetics (stereoselectively) occupy a finite number of sites on these membrane spanning proteins.

Animals↗
PubMed1994

Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand.

Mice homozygous for lpr (lymphoproliferation) or gld (generalized lymphoproliferative disease) develop lymphadenopathy and suffer from autoimmune disease. The lpr mice have a mutation in a cell-surface protein, Fas, that mediates apoptosis. Fas ligand (FasL) is a tumor necrosis factor (TNF)-related type II membrane protein and binds to Fas. Here, mouse Fasl gene was isolated and localized to the gld region of mouse chromosome 1. Activated splenocytes from gld mice express Fasl mRNA. However, FasL in gld mice carries a point mutation in the C-terminal region, which is highly conserved among members of the TNF family. The recombinant gld FasL expressed in COS cells could not induce apoptosis in cells expressing Fas. These results indicate that lpr and gld are mutations in Fas and Fasl, respectively, and suggest important roles of the Fas system in development of T cells as well as cytotoxic T lymphocyte-mediated cytotoxicity.

Amino Acid Sequence↗
PubMed2002

Resistance-associated point mutations of organophosphate insensitive acetylcholinesterase, in the olive fruit fly Bactrocera oleae.

A 2.2-kb full length cDNA containing an ORF encoding a putative acetylcholinesterase (AChE) precursor of 673 amino acid residues was obtained by a combined degenerate PCR and RACE strategy from an organophosphate-susceptible Bactrocera oleae strain. A comparison of cDNA sequences of individual insects from susceptible and resistant strains, coupled with an enzyme inhibition assay with omethoate, indicated a novel glycine-serine substitution (G488S), at an amino acid residue which is highly conserved across species (G396 of Torpedocalifornica AChE), as a likely cause of AChE insensitivity. This mutation was also associated with a 35-40% reduction in AChE catalytic efficiency. The I199V substitution, which confers low levels of resistance in Drosophila, was also present in B. oleae (I214V) and in combination with G488S produced up to a 16-fold decrease in insecticide sensitivity. This is the first agricultural pest where resistance has been associated with an alteration in AChE, which arises from point mutations located within the active site gorge of the enzyme.

Acetylcholinesterase↗