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PubMed2001

Point mutations identify the glutamate binding pocket of the N-methyl-D-aspartate receptor as major site of conantokin-G inhibition.

Conantokin-G (Con-G), a gamma-carboxylglutamate (Gla) containing peptide derived from the venom of the marine cone snail Conus geographus, acts as a selective and potent inhibitor of N-methyl-D-aspartate (NMDA) receptors. Here, the effect of Con-G on recombinant NMDA receptors carrying point mutations within the glycine and glutamate binding pockets of the NR1 and NR2B subunits was studied using whole-cell voltage-clamp recording from cRNA injected Xenopus oocytes. At wild-type receptors, glutamate-induced currents were inhibited by Con-G in a dose-dependent manner at concentrations of 0.1-100 microM. Substitution of selected residues within the NR2B subunit reduced the inhibitory potency of Con-G, whereas similar mutations in the NR1 subunit had little effect. These results indicate a selective interaction of Con-G with the glutamate binding pocket of the NMDA receptor. Homology-based molecular modeling of the glutamate binding region based on the known structure of the glutamate binding site of the AMPA receptor protein GluR2 suggests how selected amino acid side chains of NR2B might interact with specific residues of Con-G.

Amino Acid Sequence↗

PubMed1997

A unique point mutation in the fibroblast growth factor receptor 3 gene (FGFR3) defines a new craniosynostosis syndrome.

The underlying basis of many forms of syndromic craniosynostosis has been defined on a molecular level. However, many patients with familial or sporadic craniosynostosis do not have the classical findings of those craniosynostosis syndromes. Here we present 61 individuals from 20 unrelated families where coronal synostosis is due to an amino acid substitution (Pro250Arg) that results from a single point mutation in the fibroblast growth factor receptor 3 gene on chromosome 4p. In this instance, a new clinical syndrome is being defined on the basis of the molecular finding. In addition to the skull findings, some patients had abnormalities on radiographs of hands and feet, including thimble-like middle phalanges, coned epiphyses, and carpal and tarsal fusions. Brachydactyly was seen in some cases; none had clinically significant syndactyly or deviation of the great toe. Sensorineural hearing loss was present in some, and developmental delay was seen in a minority. While the radiological findings of hands and feet can be very helpful in diagnosing this syndrome, it is not in all cases clearly distinguishable on a clinical basis from other craniosynostosis syndromes. Therefore, this mutation should be tested for in patients with coronal synostosis.

Adult↗

PubMed1993

A point mutation in the FMR-1 gene associated with fragile X mental retardation.

The vast majority of patients with fragile X syndrome show a folate-sensitive fragile site at Xq27.3 (FRAXA) at the cytogenetic level, and both amplification of the (CGG)n repeat and hypermethylation of the CpG island in the 5' fragile X gene (FMR-1) at the molecular level. We have studied the FMR-1 gene of a patient with the fragile X phenotype but without cytogenetic expression of FRAXA, a (CGG)n repeat of normal length and an unmethylated CpG island. We find a single point mutation in FMR-1 resulting in an lle367Asn substitution. This de novo mutation is absent in the patient's family and in 130 control X chromosomes, suggesting that the mutation causes the clinical abnormalities. Our results suggest that mutations in FMR-1 are directly responsible for fragile X syndrome, irrespective of possible secondary effects caused by FRAXA.

Adult↗

PubMed1992

Detection of topoisomerase I gene point mutation in CPT-11 resistant lung cancer cell line.

CPT-11, a recently developed topoisomerase I (Topo I) inhibitor, attracts the attention not only of basic researchers but also of clinicians because of its high antitumor activity. The CPT-11 resistant human lung cancer cell line, PC-7/CPT, showed 10-fold resistance compared to parental cell line, PC-7. The total activity of Topo I in the resistant cell line was one fourth that of the parental sensitive cell line. The Topo I from the resistant cells was also 5-fold more resistant to the inhibitory effect of CPT-11 than that of the parental cells. We speculated that the alteration of the Topo I gene may be responsible for the change in topoisomerase activity of the CPT-11 resistant cell line. Therefore, we analyzed the mutation of Topo I gene using the method of single strand conformation polymorphism of polymerase chain reaction and the reverse transcriptase. We divided Topo I cDNA into ten fragments which overlapped each other and covered whole coding sequences of the Topo I cDNA. We observed mobility shift of two fragments in the PC-7/CPT, suggesting the presence of some mutations in these fragments. We performed the direct-sequencing of these portions by the dideoxy chain termination method and observed an altered sequence having a G to A base change in PC-7/CPT. This base substitution results in replacement of the conserved threonine at 729 position with alanine. These results suggest that the point mutation of Topo I gene is related to the decreases of Topo I activity and the sensitivity to Topo I inhibitor in PC-7/CPT cells.

Antineoplastic Agents, Phytogenic↗

PubMed1999

Novel point mutation in the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor in a case of severe congenital neutropenia hyporesponsive to G-CSF treatment.

Severe congenital neutropenia (SCN) is a heterogeneous condition characterized by a drastic reduction in circulating neutrophils and a maturation arrest of myeloid progenitor cells in the bone marrow. Usually this condition can be successfully treated with granulocyte colony-stimulating factor (G-CSF). Here we describe the identification of a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSF-R) in an SCN patient who failed to respond to G-CSF treatment. When this mutant G-CSF-R was expressed in myeloid cells, it was defective in both proliferation and survival signaling. This correlated with diminished activation of the receptor complex as determined by signal transducer and activator of transcription (STAT) activation, although activation of STAT5 was more affected than STAT3. Interestingly, the mutant receptor showed normal affinity for ligand, but a reduced number of ligand binding sites compared with the wild-type receptor. This suggests that the mutation in the extracellular domain affects ligand-receptor complex formation with severe consequences for intracellular signal transduction. Together these data add to our understanding of the mechanisms of cytokine receptor signaling, emphasize the role of GCSFR mutations in the etiology of SCN, and implicate such mutations in G-CSF hyporesponsiveness.

Bone Marrow Cells↗

PubMed1997

A point mutation in the gamma2 subunit of gamma-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity.

Benzodiazepines allosterically modulate gamma-aminobutyric acid (GABA) evoked chloride currents of gamma-aminobutyric acid type A (GABAA) receptors. Coexpression of either rat gamma2 or gamma3, in combination with alpha1 and beta2 subunits, results both in receptors displaying high [3H]Ro 15-1788 affinity. However, receptors containing a gamma3 subunit display a 178-fold reduced affinity to zolpidem as compared with gamma2-containing receptors. Eight chimeras between gamma2 and gamma3 were constructed followed by nine different point mutations in gamma2, each to the homologous amino acid residue found in gamma3. Chimeric or mutant gamma subunits were coexpressed with alpha1 and beta2 in human embryonic kidney 293 cells to localize amino acid residues responsible for the reduced zolpidem affinity. Substitution of a methionine-to-leucine at position 130 of gamma2 (gamma2M130L) resulted in a 51-fold reduction in zolpidem affinity whereas the affinity to [3H]Ro 15-1788 remained unchanged. The affinity for diazepam was only decreased by about 2-fold. The same mutation resulted in a 9-fold increase in Cl 218872 affinity. A second mutation (gamma2M57I) was found to reduce zolpidem affinity by about 4-fold. Wild-type and gamma2M130L-containing receptors were functionally expressed in Xenopus oocytes. Upon mutation allosteric coupling between agonist and modulatory sites is preserved. Dose-response curves for zolpidem and for diazepam showed that the zolpidem but not the diazepam apparent affinity is drastically reduced. The apparent GABA affinity is not significantly affected by the gamma2M130L mutation. The identified amino acid residues may define part of the benzodiazepine binding pocket of GABAA receptors. As the modulatory site in the GABAA receptor is homologous to the GABA site, and to all agonist sites of related receptors, gamma2M130 may either point to a homologous region important for agonist binding in all receptors or define a new region not underlying this principle.

Animals↗

PubMed1995

Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport.

In the study of motor proteins, the molecular mechanism of mechanochemical coupling, as well as the cellular role of these proteins, is an important issue. To assess these questions we introduced cDNA of wild-type and site-directed mutant kinesin heavy chains into fibroblasts, and analyzed the behavior of the recombinant proteins and the mechanisms involved in organelle transports. Overexpression of wild-type kinesin significantly promoted elongation of cellular processes. Wild-type kinesin accumulated at the tips of the long processes, whereas the kinesin mutants, which contained either a T93N- or T93I mutation in the ATP-binding motif, tightly bound to microtubules in the center of the cells. These mutant kinesins could bind to microtubules in vitro, but could not dissociate from them even in the presence of ATP, and did not support microtubule motility in vitro, thereby indicating rigor-type mutations. Retrograde transport from the Golgi apparatus to the endoplasmic reticulum, as well as lysosome dispersion, was shown to be a microtubule-dependent, plus-end-directed movement. The latter was selectively blocked in the rigor-mutant cells, although the microtubule minus-end-directed motion of lysosomes was not affected. We found the point mutations that make kinesin motor in strong binding state with microtubules in vitro and showed that this mutant causes a dominant effect that selectively blocks anterograde lysosome membrane transports in vivo.

Adenosine Triphosphate↗

PubMed1999

A point mutation Thr(799)Met on the alpha(2) integrin leads to the formation of new human platelet alloantigen Sit(a) and affects collagen-induced aggregation.

A new platelet-specific alloantigen, termed Sit(a), was identified in a severe case of neonatal alloimmune thrombocytopenia. The Sit(a) alloantigen is of low frequency (1/400) in the German population. Immunochemical studies demonstrated that the Sit(a) epitopes reside on platelet glycoprotein (GP) Ia. Nucleotide sequence analysis of GPIa cDNA derived from Sit(a)-positive platelets showed C(2531)-->T(2531) point mutation, resulting in Thr(799)Met dimorphism. Analysis of genomic DNA from 22 Sit(a)-negative normal individuals showed that the Thr(799) is encoded by ACG(2532) (90.9%) or ACA(2532) (9.1%). To establish a DNA typing technique, we elucidated the organization of the GPIa gene adjacent to the polymorphic bases. The introns (421 bp and 1.2 kb) encompass a 142-bp exon with the 2 polymorphic bases 2531 and 2532. Polymerase chain reaction-restriction fragment length polymorphism analysis on DNA derived from 100 donors using the restriction enzyme Mae III showed that the Met(799) form of GPIa is restricted to Sit(a) (+) phenotype. Analysis of stable Chinese hamster ovary transfectants expressing allele-specific recombinant forms of GPIa showed that anti-Sit(a) exclusively reacted with the Glu(505)Met(799), but not with the Glu(505)Thr(799) and the Lys(505)Thr(799) isoforms. In contrast, anti-Br(a) (HPA-5b) only recognized the Lys(505)Thr(799) form, whereas anti-Br(b) (HPA-5a) reacted with both Glu(505)Thr(799) and Glu(505)Met(799) isoforms. These results demonstrated that the Met(799) is responsible for formation of the Sit(a) alloantigenic determinants, whereas amino acid 505 (Lys or Glu) specifically controls the expression of Br(a) and Br(b) epitopes, respectively. Platelet aggregation responses of Sit(a) (+) individuals were diminished in response to collagen, indicating that the Thr(799)Met mutation affects the function of the GPIa/IIa complex.

Animals↗

PubMed1999

Mitochondrial DNA point mutations detected in four cases of sudden infant death syndrome.

The aim of this study was to investigate the tRNA(Leu(UUR)) gene and the first part of the ND1 gene in mitochondrial DNA (mtDNA) in cases of sudden infant death syndrome (SIDS). A total of 158 cases of SIDS and 97 controls were included in the study, and the base pairs in the range 3230-3330 were investigated using polymerase chain reaction (PCR) and temporal temperature gradient electrophoresis (TTGE). If a band shift was detected by TTGE, the area investigated and the D-loop was sequenced. Three different point mutations (T3290C, T3308C and T3308G) were detected in four of the SIDS cases, while none of the controls were mutated. We also found a high D-loop substitution rate in these four cases. The findings indicate that mtDNA mutations may play a role in some cases of SIDS.

DNA, Mitochondrial↗

PubMed2005

A point mutation in the human melanin concentrating hormone receptor 1 reveals an important domain for cellular trafficking.

G protein-coupled receptors (GPCRs) are heptahelical integral membrane proteins that require cell surface expression to elicit their effects. The lack of appropriate expression of GPCRs may be the underlying cause of a number of inherited disorders. There is evidence that newly synthesized GPCRs must attain a specific conformation for their correct trafficking to the cell surface. In this study, we show that a single point mutation in human melanin-concentrating hormone receptor (hMCHR1) at position 255 (T255A), which is located at the junction of intracellular loop 3 and transmembrane domain 6, reduces the hMCHR1 cell surface expression level to 20% of that observed for the wild-type receptor. Most of these mutant receptors are located intracellularly, as opposed to the wild-type receptor, which is located primarily on the cell surface. Immunoprecipitation experiments show that hMCHR1-T255A has reduced glycosylation compared with the wild-type receptor and is associated with the chaperone protein, calnexin, and it colocalizes in the endoplasmic reticulum with KDEL-containing proteins. We also demonstrate that a cell-permeable small molecule antagonist of hMCHR1 can function as a pharmacological chaperone to restore cell surface expression of this and other MCHR1 mutants to wild-type levels. Once rescued, the T255A mutant couples to Gq proteins as efficiently as the wild-type receptor. These data suggest that this single mutation produces an hMCHR1 that folds incorrectly, resulting in its retention in the endoplasmic reticulum, but once rescued to the cell surface can still function normally.

Amino Acid Sequence↗

PubMed2003

Severe clinical forms of cytochrome b-negative chronic granulomatous disease (X91-) in 3 brothers with a point mutation in the promoter region of CYBB.

Chronic granulomatous disease (CGD) is a rare congenital syndrome that results in severe, recurrent bacterial and fungal infections. The most common form is caused by defects in the CYBB gene, leading to the absence of gp91phox associated with totally abolished NADPH oxidase activity (X91(0) CGD). We report 3 brothers with atypical cases of X-linked CGD, characterized by low levels of expression of gp91phox (X91(-) CGD). A point mutation (T-55C) identified in the CYBB gene's promoter region appears to prevent the full expression of this gene in neutrophils. This results in low levels of expression of gp91phox protein that are correlated with residual oxidase activity in the whole population of neutrophils. The total O(2)(-) production in these cells was approximately 5% of normal. Despite this oxidase activity, the patients experienced severe and life-threatening infections. It was concluded that the O(2)(-) production in the neutrophils of these patients was not sufficient to protect them against infections, and this X91(-) CGD phenotype must be considered to be a severe clinical form of CGD.

Base Sequence↗

PubMed1998

Single point mutations located outside the inter-monomer domains abolish trimerization of Schizosaccharomyces pombe PCNA.

We have generated proliferating cell nuclear antigen (PCNA) mutants by low fidelity PCR and screened for lethal mutations by testing for lack of complementation of a Schizosaccharomyces pombe strain disrupted for the pcn1 + gene. We thus identified eight lethal mutants out of the 50 cDNAs tested. Six were truncated in their C-terminal region due to the introduction of a stop codon within their coding sequences. Two were full-length with a single point mutation at amino acid 68 or 69. The two latter mutants were overexpressed in insect cells via a recombinant baculovirus and were purified. They were unable to stimulate DNA polymerase delta DNA replication activity on a poly(dA).oligo(dT) template. Cross-linking experiments showed that this was due to their inability to form trimers. Since these two mutations are adjacent and not located in a domain of the protein putatively involved in inter-monomer interactions, our results show that the beta-sheet betaF1 to which they belong must play an essential role in maintaining the 3-dimensional structure of S.pombe PCNA.

Animals↗

PubMed1996

Dejerine-Sottas disease with sensorineural hearing loss, nystagmus, and peripheral facial nerve weakness: de novo dominant point mutation of the PMP22 gene.

A 32 year old woman with Dejerine-Sottas disease and negative family history is reported. Clinical onset of her condition was with congenital weakness of her distal four extremities, accompanied by peripheral facial nerve weakness, deafness, and nystagmus. She has used a wheelchair all her life. Sural nerve biopsy showed proliferation of Schwann cells, extensive endoneural fibrosis, axon loss, and demyelination. MNCVs showed marked slowing. MRI of the brain was normal. Molecular genetic studies indicated a de novo dominant missense point mutation of exon 3 of the peripheral myelin protein 22 gene at nucleotide 264 causing replacement of serine with leucine.

Adult↗

PubMed1994

Adsorption of bacteriophage lambda on the LamB protein of Escherichia coli K-12: point mutations in gene J of lambda responsible for extended host range.

LamB is the cell surface receptor for bacteriophage lambda. LamB missense mutations yielding resistance to lambda group in two classes. Class I mutants block the growth of lambda with the wild-type host range (lambda h+) but support the growth of one-step host range mutants (lambda h). Class II mutants block lambda h but support the growth of two-step host range mutant (lambda hh*) phages. To identify amino acid residues in the J protein (the tail fiber of phage lambda) responsible for the extended host range phenotype of mutants of phage (lambda h+), we selected a series of one-step (lambda h) and two-step (lambda hh*) host range mutants and analyzed their corresponding J genes. Three different class I LamB missense mutants (mutations at sites 247, 245, and 148) were used to select 11 independent, new, one-step host range mutants (lambda h phages). DNA sequence analysis revealed a single-amino-acid change in each case. The 11 alterations affected only three residues in the distal part of J, corresponding to a Val-->Ala change at site 1077 in five cases, a Thr-->Met change at site 1040 in three cases, and a Leu-->Pro change at site 1127 in three cases. Recombination experiments confirmed that in the cases tested, the mutations identified were indeed responsible for the extended host range phenotype. The class II LamB mutant (Gly-->Asp at site 151) was used to select two-step extended host range mutants (lambda hh* phages) from three new lambda h phages, corresponding to different amino acid modifications in the J protein (at sites 1040, 1077, and 1127). The new lambda hh* phages analyzed corresponded to either double or triple point mutations located at the distal end of the J protein. In all, seven residues involved in the extended host range properties of lambda mutants were identified in the distal part of the J protein, suggesting that the last C-terminal portion of the J protein participates directly in the adsorption of the phage onto LamB. In agreement with the fact that the lambda h mutants (and the lambda hh* mutants) could grow on all of the lamB class I mutations tested, we found tha the nature of the J mutations did not depend on the LamB class I mutant used to select them. This is interpreted as meaning that the mutated residues in the J protein and in the LamB mutants are not involved in allele-specific protein-protein interactions. Rather, the LamB mutations would block a step in phage adsorption, and this block would be overcome by the mutations in the J protein.

Adsorption↗

PubMed1995

Point mutations in Staphylococcus aureus PBP 2 gene affect penicillin-binding kinetics and are associated with resistance.

In Staphylococcus aureus, penicillin-binding protein 2 (PBP 2) has been implicated in non-PBP 2a-mediated methicillin resistance. The PBP 2 gene (pbpB) was cloned from an expression library of a methicillin-susceptible strain of S. aureus (209P), and its entire sequence was compared with that of the pbpB gene from strains BB255, BB255R, and CDC6. Point mutations that resulted in amino acid substitutions near the conserved penicillin-binding motifs were detected in BB255R and CDC6, two low-level methicillin-resistant strains. Penicillin binding to PBP 2 in both BB255R and CDC6 is altered, and kinetic analysis indicated that altered binding of PBP 2 by penicillin was due to both lower binding affinity and more rapid release of bound drug. These structural and biochemical changes may contribute to the strains' resistance to beta-lactam antibiotics.

Amino Acid Sequence↗

PubMedDate not supplied

Overexpression of p53 protein and point mutation of K-ras genes in primary carcinoma of the small intestine.

Primary carcinoma of the small intestine is rare and represent about 0.5% of all gastrointestinal malignancies. The aim of this study was to examine the biological characteristics of primary carcinoma of the small intestine by immunohistochemical and nested polymerase chain reaction methods. Thirty-five primary carcinomas (12 in the duodenum and 23 in the jejunum or ileum) from 35 patients were studied clinicopathologically and examined for overexpression of p53 protein. In 22 of these 35 cases, point mutation at codon 12 of the K-ras gene was detected by the nested polymerase chain reaction and restriction fragment length polymorphism method. All the duodenal carcinomas were well-differentiated type and the rate of these carcinomas was significantly higher than that of jejunal or ileal carcinomas (100% vs. 65%). Fourteen cases showed overexpression of p53 (40%), and p53 tended to be expressed more frequently in poorly-differentiated type (71%) compared to well-differentiated type (30%). Only 2 out of 22 carcinoma cases showed K-ras gene mutation, and both were duodenal carcinomas. These findings suggest that p53 plays a major role in the progression of carcinoma of the small intestine, whereas the role of K-ras mutation is much less significant.

Adenocarcinoma↗

PubMed1992

Rare point mutation at codon 301 and 969 of FMS/M-CSF receptor in acute myelomonocytic and monocytic leukemia.

We have investigated whether point mutations occurred at codon 301 or 969 of FMS (M-CSF receptor) in 19 patients with acute myelomonocytic (M4) and monocytic leukemia (M5). Nineteen peripheral blood and bone marrow blood samples collected from M4 and M5 patients were examined by using polymerase chain reaction and hybridization to allele specific oligonucleotide probes. Mutations at codon 301 and 969 of FMS were not detected in any samples. FMS gene mutations at codon 301 and 969 were rarely involved in M4 and M5 patients in Japan.

Amino Acid Sequence↗

PubMed1982

Deletion loop mutagenesis: a novel method for the construction of point mutations using deletion mutants.

Deletion loop mutagenesis is a new, general method for site-directed mutagenesis that allows point mutations to the introduced within a sequence of DNA defined by a previously isolated deletion mutant. Wild type and deletion mutant DNA are cloned into a bacterial plasmid and each is cleaved with a different single cut restriction enzyme. Heteroduplexes are formed between the two DNAs to produce circular molecules containing a nick in each strand and a single-stranded deletion loop. The deletion loops are mutagenised using sodium bisulphite and the DNA transfected directly into a uracil repair deficient strain of Escherichia coli. Up to half of the resultant clones contain DNA produced by replication of the wild-type length strand and bear mutations exclusively within the target area. An example is given in which a deletion mutant lacking 21 nucleotides from the region coding for SV40 large-T was used. Eight of the possible nine target cytosine residues were mutagenised. The method described is specific, efficient and simple.

Base Sequence↗