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PubMed2005

Chromosome segment duplications in Neurospora crassa and their effects on repeat-induced point mutation and meiotic silencing by unpaired DNA.

The size and extent of four Neurospora crassa duplications, Dp(AR17), Dp(IBj5), Dp(OY329), and Dp(B362i), was determined by testing the coverage of RFLP markers. The first three duplications were all > approximately 350 kb and have been shown in earlier studies to act as dominant suppressors of repeat-induced point mutation (RIP) in gene-sized duplications, possibly via titration of the RIP machinery. Dp(B362i), which is only approximately 117 kb long, failed to suppress RIP. RIP suppression in gene-sized duplications by large duplications was demonstrated using another test gene, dow, and supposedly applies generally. Crosses homozygous for Dp(AR17) or Dp(IBj5) were as barren as heterozygous crosses. Barrenness of the heterozygous but not the homozygous crosses was suppressible by Sad-1, a semidominant suppressor of RNAi-dependent meiotic silencing by unpaired DNA. A model is proposed in which large duplications recessively suppress semidominant Sad-1 mutations. The wild-isolated Sugartown strain is hypothesized to contain a duplication that confers not only dominant suppression of RIP but also a barren phenotype, which is linked (9%) to supercontig 7.118 in LG VII.

Chromosome Mapping↗
PubMed1998

Detection of point mutations associated with resistance of Helicobacter pylori to clarithromycin by hybridization in liquid phase.

When the standard procedure for determining antibiotic susceptibility of bacteria is used, the results are delayed, especially for bacteria that grow slowly, such as Helicobacter pylori. Treatment for this bacterium may involve clarithromycin, a compound for which resistance has been associated with point mutations on the 23S rRNA gene. This resistance is currently found in organisms isolated from 0 to 15% of patients and jeopardizes the success of the treatment. We have designed a test involving amplification and colorimetric hybridization in the liquid phase to detect the mutation at the molecular level. First, four reference strains, including the wild type and three strains with the mutations A2143C, A2143G, and A2144G, were used to optimize the method. Amplification was carried out with primers previously published. The amplified products were added to probe-coated microtiter wells. A DNA enzyme immunoassay was used to detect the hybrids. The optimal conditions of the hybridization were defined for each probe. Nineteen H. pylori strains resistant to clarithromycin and 22 susceptible according to phenotypic data were submitted to restriction with BsaI and BbsI, and part of the 23S rRNA gene was sequenced in order to determine the mutation involved for the resistant strains. The new assay showed a complete correlation with the reference methods, except for one strain. Cross-hybridizations as well as application of the reaction to other bacteria did not lead to optical densities higher than the cutoff values chosen with the receiving operating characteristic curve. This method can be easily standardized and gives a result within a day. Its application directly to the biopsy specimens or infected gastric juice is planned in the future.

Anti-Bacterial Agents↗
PubMed2004

Alteration of sugar donor specificities of plant glycosyltransferases by a single point mutation.

In comparison with the amino acid sequences of seven species of glucosyltransferases and six species of galactosyltransferases, glutamine and histidine are highly conserved as the last amino acid residue of a glycosyltransferase-specific conserved region (UDPGT) in glucosyltransferases and galactosyltransferases, respectively. Consequently, the sugar donor specificities of glycosyltransferases are successfully altered by a single amino acid point mutation. UDP-galactose:anthocyanin galactosyltransferase (ACGaT), isolated from Aralia cordata, acquired glucosyltransferase activity in addition to the inherent galactosyltransferase activity by replacing histidine with glutamine. In contrast, UDP-glucose:flavonoid glucosyltransferase (UBGT), isolated from Scutellaria baicalensis, did not acquire galactosyltransferase activity by replacing glutamine with histidine, and exhibited a remarkable decrease in glucosyltransferase activity.

Amino Acid Sequence↗
PubMed2006

Dual actions of enflurane on postsynaptic currents abolished by the gamma-aminobutyric acid type A receptor beta3(N265M) point mutation.

BACKGROUND: At concentrations close to 1 minimum alveolar concentration (MAC)-immobility, volatile anesthetics display blocking and prolonging effects on gamma-aminobutyric acid type A receptor-mediated postsynaptic currents. It has been proposed that distinct molecular mechanisms underlie these dual actions. The authors investigated whether the blocking or the prolonging effect of enflurane is altered by a point mutation (N265M) in the beta3 subunit of the gamma-aminobutyric acid type A receptor. Furthermore, the role of the beta3 subunit in producing the depressant actions of enflurane on neocortical neurons was elucidated. METHODS: Spontaneous inhibitory postsynaptic currents were sampled from neocortical neurons in cultured slices derived from wild-type and beta3(N265M) mutant mice. The effects of 0.3 and 0.6 mm enflurane on decay kinetics, peak amplitude, and charge transfer were quantified. Furthermore, the impact of enflurane-induced changes in spontaneous action potential firing was evaluated by extracellular recordings in slices from wild-type and mutant mice. RESULTS: In slices derived from wild-type mice, enflurane prolonged inhibitory postsynaptic current decays and decreased peak amplitudes. Both effects were almost absent in slices from beta3(N265M) mutant mice. At clinically relevant concentrations between MAC-awake and MAC-immobility, the anesthetic was less effective in depressing spontaneous action potential firing in slices from beta3(N265M) mutant mice compared with wild-type mice. CONCLUSION: At concentrations between MAC-awake and MAC-immobility, beta3-containing gamma-aminobutyric acid type A receptors contribute to the depressant actions of enflurane in the neocortex. The beta3(N265M) mutation affects both the prolonging and blocking effects of enflurane on gamma-aminobutyric acid type A receptor-mediated inhibitory postsynaptic currents in neocortical neurons.

Action Potentials↗
PubMed2001

Nonenzymatic autoligation in direct three-color detection of RNA and DNA point mutations.

Enzymatic ligation methods are useful in diagnostic detection of DNA sequences. Here we describe the investigation of nonenzymatic phosphorothioate-iodide DNA autoligation chemistry as a method for detection and identification of both RNA and DNA sequences. Combining ligation specificity with the hybridization specificity of the ligated product is shown to yield discrimination of a point mutation as high as >10(4)-fold. Unlike enzymatic ligations, this reaction is found to be equally efficient on RNA or DNA templates. The reaction is also shown to exhibit a significant level of self-amplification, with the template acting in catalytic fashion to ligate multiple pairs of probes. A strategy for fluorescence labeling of three autoligating energy transfer (ALET) probes and directly competing them for autoligation on a target sequence is described. The method is tested in several formats, including solution phase, gel, and blot assays. The ALET probe design offers direct RNA detection, combining high sequence specificity with an easily detectable color change by fluorescence resonance energy transfer (FRET).

Base Sequence↗
PubMed2005

Detection of point mutations at codon 12 of KI-ras in ophthalmic pterygia.

AIMS: Ophthalmic pterygium is a potentially vision-threatening lesion of unknown etiology, related to an exposure to solar light. Mutations to the ras genes are frequently observed in lesions related to an exposure to solar light. The present study aims at screening pterygia for mutations at codons 12 and 13 of the ras genes. METHODS: In all, 50 pterygia were examined, together with respective blood samples and specimens of normal conjunctiva. A PCR reaction was performed to amplify sequences containing codons 12 and 13 of Ki-ras, H-ras, and N-ras. An RFLP analysis was then performed to detect point mutations at codon 12. The mutational status at codons 12 and 13 was further explored with sequencing of PCR products. RESULTS: RFLP analysis revealed Ki-ras mutations at codon 12 in five (10%) of pterygia, whereas H-ras or N-ras mutations were not observed. Sequencing confirmed Ki-ras mutations at codon 12 and revealed absence of mutations at codon 13. The presence of Ki-ras mutations was significantly correlated with postoperative recurrence (P=0.02) and young age (P=0.04). Mutations were not observed in specimens of blood or normal conjunctiva for any of the genes examined. CONCLUSIONS: The absence of N-ras mutations is in agreement with previous reports concerning mucosal lesions. The detection of Ki-ras mutations and the association with postoperative recurrence implies a possible role of Ki-ras in the clinical profile of pterygium. The mechanism of Ki-ras mutations is unclear and could be independent of the action of UV light.

Adult↗
PubMed2004

A case of complicated spastic paraplegia 2 due to a point mutation in the proteolipid protein 1 gene.

Pelizaeus-Merzbacher disease (PMD) is a rare X-linked dysmyelinating disorder resulting from mutation of the proteolipid protein gene (PLP1). Clinical features of PMD include progressive psychomotor developmental delay, nystagmus, spastic quadriplegia, dystonia, and cerebellar ataxia. PMD is clinically classified into three subtypes according to the severity of the disease: connatal, transitional, and classic forms. Patients with PMD have been identified with duplication, point mutations, and deletion of PLP1. In addition, spastic paraplegia 2 (SPG2) is allelic to PMD and typically caused by missense mutations in the second extracellular domain of PLP1 or in the PLP1-specific region that is spliced out during formation of the DM20 isoform. The authors describe a Korean boy diagnosed with SPG2 caused by a mutation that results in a Pro215Leu substitution in the second extracellular domain. Analysis of phenotypes resulting from mutations affecting PLP1 has been valuable in identifying functional domains of this still incompletely understood major myelin protein. Null mutations and mutations affecting the PLP1-specific domain cause peripheral neuropathy. The PLP1-specific domain also is important in the long-term maintenance of axonal integrity. This patient's phenotype was relatively mild, in contrast with other mutations at position 215 of PLP1 that cause severe PMD. One of these severe mutations is also a missense mutation substituting an aliphatic residue, alanine, for proline. The distinct severity difference between the Pro215Leu and Pro215Ala substitutions suggests that this region of the protein is very sensitive to subtle structural changes and likely plays a critical role in PLP1 function.

Brain↗
PubMed2006

Point mutations in two EVI1 Zn fingers abolish EVI1-GATA1 interaction and allow erythroid differentiation of murine bone marrow cells.

EVI1 is an aggressive nuclear oncoprotein deregulated by recurring chromosomal abnormalities in myelodysplastic syndrome (MDS). The expression of the corresponding gene is a very poor prognostic marker for MDS patients and is associated with severe defects of the erythroid lineage. We have recently shown that the constitutive expression of EVI1 in murine bone marrow results in a fatal disease with features characteristic of MDS, including anemia, dyserythropoiesis, and dysmegakaryopoiesis. These lineages are regulated by the DNA-binding transcription factor GATA1. EVI1 has two zinc finger domains containing seven motifs at the N terminus and three motifs at the C terminus. Supported by results of assays utilizing synthetic DNA promoters, it was earlier proposed that erythroid-lineage repression by EVI1 is based on the ability of this protein to compete with GATA1 for DNA-binding sites, resulting in repression of gene activation by GATA1. Here, however, we show that EVI1 is unable to bind to classic GATA1 sites. To understand the mechanism utilized by EVI1 to repress erythropoiesis, we used a combination of biochemical assays, mutation analyses, and in vitro bone marrow differentiation. The results indicate that EVI1 interacts directly with the GATA1 protein rather than the DNA sequence. We further show that this protein-protein interaction blocks efficient recognition or binding to DNA by GATA1. Point mutations that disrupt the geometry of two zinc fingers of EVI1 abolish the protein-protein interaction, leading to normal erythroid differentiation of normal murine bone marrow in vitro.

Animals↗
PubMed1996

A point mutation in exon 3 (His 107-->Tyr) in two unrelated Japanese patients with carbonic anhydrase II deficiency with central nervous system involvement.

We have analyzed two unrelated Japanese patients with carbonic anhydrase II deficiency born to consanguineous parents. We have identified the same mutation as that reported to be homozygous in a Belgian family and compound heterozygous in an American family. It comprises to C-to-T transition that results in the amino acid substitution of Tyr (TAT) for His (CAT) at position 107. This point mutation creates an AccI site that can be conveniently screened by the polymerase chain reaction/restriction fragment length polymorphism method using a restriction enzyme for gene tracking. Our patients exhibit severe mental retardation, not seen in the Belgian and American patients.

Carbonic Anhydrases↗
PubMed2002

Point mutations in HpuB enable gonococcal HpuA deletion mutants to grow on hemoglobin.

Neisseria gonorrhoeae ordinarily requires both HpuA and HpuB to use hemoglobin (Hb) as a source of iron for growth. Deletion of HpuA resulted in reduced Hb binding and failure of growth on Hb. We identified rare Hb-utilizing colonies (Hb(+)) from an hpuA deletion mutant of FA1090, which fell into two phenotypic classes. One class of the Hb(+) revertants required expression of both TonB and HpuB for growth on Hb, while the other class required neither TonB nor HpuB. All TonB/HpuB-dependent mutants had single amino acid alterations in HpuB, which occurred in clusters, particularly near the C terminus. The point mutations in HpuB did not restore normal Hb binding. Human serum albumin inhibited Hb-dependent growth of HpuB point mutants lacking HpuA but did not inhibit growth when expression of HpuA was restored. Thus, HpuB point mutants internalized heme in the absence of HpuA despite reduced binding of Hb. HpuA facilitated Hb binding and was important in allowing use of heme from Hb for growth.

Bacterial Outer Membrane Proteins↗
PubMed1995

Functional analysis of linker insertions and point mutations in the alpha-Amy2/54 GA-regulated promoter.

Functional analysis of a gibberellin-regulated wheat alpha-amylase promoter, alpha-Amy2/54, has indicated that three regions were essential for expression. By studying the ability of mutant promoters, containing a randomly inserted 22 bp excision linker, to direct expression in oat aleurone protoplasts we have refined the positions and extents of these three cis elements and also demonstrated the presence of two additional elements. By converting the linker insertions to either single base point mutations or deletions using the class IIS restriction endonuclease BsmI we have shown that nucleotides -119 and -109 within the GARE -121GTAACAGAGTCTGG-108 and nucleotide -152 within the proposed element -156GATTGACTTGACC-144 are essential for high level expression from this promoter.

Base Sequence↗
PubMed1993

Direct diagnosis of carriers of point mutations in Duchenne muscular dystrophy.

In about one-third of patients with Duchenne/Becker muscular dystrophy, the causative mutation does not involve gross alterations in the structure of the dystrophin gene. Prenatal diagnosis and carrier detection for such families is therefore dependent on the indirect method of linkage analysis with polymorphic DNA markers, a method that is subject to error. The identification of point mutations in the dystrophin gene in six affected males enabled us to develop direct assays, based on the polymerase chain reaction, that are specific for each of the mutations. In all six cases, the assays allowed us to offer families accurate carrier testing and prenatal diagnosis.

Base Sequence↗
PubMed2001

Absence of point mutations within the AML-1 gene in patients with MDS/AML and loss of chromosome 5q or 7.

There is increasing evidence that the acute myeloid leukemia 1 (AML-1) gene plays a versatile role in hematopoiesis, and its inactivation has been described in various hematopoetic disorders, e.g., leukemia or familial thrombocytopenia. AML-1 can be affected by various mechanisms, such as chromosomal translocations or point mutations. On the other hand, the specific underlying molecular lesions in myelodysplastic syndromes (MDS) or leukemias with aberrations of chromosomes 5q or 7, respectively, are largely unknown. Despite extraordinary scientific effort no specific genes on chromosome 5q or 7, which act as tumor suppressors, have definitely been identified. Therefore, it has recently been speculated that the AML-1 gene, even if distantly located on chromosome 21q22, may be involved in leukemogenesis in patients with aberrations at chromosome 5q or monosomy 7 [2]. Therefore, we sequenced all exons of the AML-1 gene in 15 patients with MDS/AML and deleted chromosome 5q or 7q, respectively. None of the patients analyzed had any AML-1 mutation.

Adolescent↗
PubMed1994

Double P53 point mutation in extramedullary blast crisis of chronic myelogenous leukemia.

A patient with Philadelphia-positive chronic myelogenous leukemia (CML) evolved in extramedullary blast crisis, was studied for the presence of alterations of the P53 tumor suppressor gene in the different stages of disease progression. No P53 gene aberrations were detected during the chronic and accelerated phases. Two identical missense point mutations, involving codons 249 and 281 and leading to the amino acid substitutions Arg-Ser and Thy-Asp, were identified in cells of an extramedullary mass and then in peripheral blood blast crisis cells. The data indicate that the medullary and extramedullary blast cells belong to the same cellular clone. They also strongly suggest that in this case, the alteration of P53 gene is strictly related to the progression of the disease, although this mechanism is certainly neither the only nor the most frequent molecular event leading to the acute phase.

Adult↗
PubMed1996

A single point mutation in CTP synthetase of Chlamydia trachomatis confers resistance to cyclopentenyl cytosine.

A Chlamydia trachomatis strain (L2/CPEC) resistant to the cytotoxic effects of cyclopentenyl cytosine (CPEC) was isolated by a stepwise selection procedure. This strain showed an approximate 350-fold increase in resistance to CPEC. Sequencing of the gene encoding CTP synthetase from this resistant strain revealed a single point mutation, resulting in a change of amino acid 149 from Asp to Glu. This appeared to be the only mutation in L2/CPEC, because no changes in CTP transport, CTP synthetase expression, or incorporation of CPEC into DNA or RNA could be detected. The mutation in the chlamydial CTP synthetase resulted in a loss of CTP feedback inhibition. This was demonstrated both in vivo using Escherichia coli cells carrying the cloned gene, and an in vitro assay using partially purified preparations of CTP synthetase. As a result of the loss of feedback inhibition, E. coli cells carrying the CPECR CTP synthetase showed a 22-fold increase in their CTP pools. However, examination of the CTP pools of L2/CPEC revealed no change in CTP levels when compared with wild type C. trachomatis.

Amino Acid Sequence↗
PubMed1993

Dense nonsymmetrical DNA methylation resulting from repeat-induced point mutation in Neurospora.

Cytosine methylation has been implicated in epigenetic control of gene expression in animals, plants, and fungi. It has been assumed that all methylation in eukaryotes is at symmetrical sequences such as CpG/GpC, because this can explain perpetuation of methylation states. Here the bisulfite genomic sequencing method was used to examine methylation in DNA from a Neurospora gene exposed to repeat-induced point mutation. 5-Methylcytosine was not limited to symmetrical sites and individual molecules showed different patterns and amounts of modification. The methylation extended beyond the mutated region and even beyond the edge of the duplicated segment.

5-Methylcytosine↗
PubMed2005

First description in Tunisia of a point mutation at codon 119 (CCT-->TCT) in the alpha1-globin gene: Hb Groene Hart in association with the -alpha3.7 deletion.

Herein we describe the case of a Tunisian girl who presented with 3% Hb Bart's (gamma4) at birth. At the age of 3 years, she showed microcytosis and hypochromia in the absence of iron deficiency. The first step of molecular analysis was to test for the common Mediterranean mutations and the classical -alpha3.7 deletion was found in the heterozygous state. Since this finding could not explain the level of Hb Bart's at birth, or the hypochromia and microcytosis, all the alpha-globin genes were sequenced. This revealed a rare point mutation at codon 119 (CCT-->TCT) in the alpha1-globin gene, identified for the first time in Tunisia, and which has previously been described as an unstable hemoglobin (Hb) variant named Hb Groene Hart [alpha119(H2)Pro-->Ser (alpha1)]. Here the -alpha3.7/alpha(alpha)119(CCT-->TCT) genotype is responsible for the alpha-thalassemia (thal) trait phenotype.

Child, Preschool↗
PubMed1998

Structure-function analysis of Qk1: a lethal point mutation in mouse quaking prevents homodimerization.

Qk1 is a member of the KH domain family of proteins that includes Sam68, GRP33, GLD-1, SF1, and Who/How. These family members are RNA binding proteins that contain an extended KH domain embedded in a larger domain called the GSG (for GRP33-Sam68-GLD-1) domain. An ethylnitrosourea-induced point mutation in the Qk1 GSG domain alters glutamic acid 48 to a glycine and is known to be embryonically lethal in mice. The function of Qk1 and the GSG domain as well as the reason for the lethality are unknown. Here we demonstrate that the Qk1 GSG domain mediates RNA binding and Qk1 self-association. By using in situ chemical cross-linking studies, we showed that the Qk1 proteins exist as homodimers in vivo. The Qk1 self-association region was mapped to amino acids 18 to 57, a region predicted to form coiled coils. Alteration of glutamic acid 48 to glycine (EG) in the Qk1 GSG domain (producing protein Qk1:EG) abolishes self-association but has no effect on the RNA binding activity. The expression of Qk1 or Qk1:EG in NIH 3T3 cells induces cell death by apoptosis. Approximately 90% of the remaining transfected cells are apoptotic 48 h after transfection. Qk1:EG was consistently more potent at inducing apoptosis than was wild-type Qk1. These results suggest that the mouse quaking lethality (EG) occurs due to the absence of Qk1 self-association mediated by the GSG domain.

3T3 Cells↗