Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “mutant identification”

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 163 records · Page 9Linked to original sources
PubMed1998

Characterization of glucose-specific catabolite repression-resistant mutants of Bacillus subtilis: identification of a novel hexose:H+ symporter.

Insertional mutagenesis was conducted on Bacillus subtilis cells to screen for mutants resistant to catabolite repression. Three classes of mutants that were resistant to glucose-promoted but not mannitol-promoted catabolite repression were identified. Cloning and sequencing of the mutated genes revealed that the mutations occurred in the structural genes for (i) enzyme II of the phosphoenolpyruvate-glucose phosphotransferase (PtsG), (ii) antiterminator GlcT, which controls PtsG synthesis, and (iii) a previously uncharacterized carrier of the major facilitator superfamily, which we have designated GlcP. The last protein exhibits greatest sequence similarity to the fucose:H+ symporter of Escherichia coli and the glucose/galactose:H+ symporter of Brucella abortus. In a wild-type B. subtilis genetic background, the glcP::Tn10 mutation (i) partially but specifically relieved glucose- and sucrose-promoted catabolite repression, (ii) reduced the growth rate in minimal glucose medium, and (iii) reduced rates of [14C]glucose and [14C]methyl alpha-glucoside uptake. In a delta pts genetic background no phenotype was observed, suggesting that expression of the glcP gene required a functional phosphotransferase system. When overproduced in a delta pts mutant of E. coli, GlcP could be shown to specifically transport glucose, mannose, 2-deoxyglucose and methyl alpha-glucoside with low micromolar affinities. Accumulation of the nonmetabolizable glucose analogs was demonstrated, and inhibitor studies suggested a dependency on the proton motive force. We conclude that B. subtilis possesses at least two distinct routes of glucose entry, both of which contribute to the phenomenon of catabolite repression.

Amino Acid Sequence↗
PubMed2004

Characterization of a Saccharomyces cerevisiae thermosensitive lytic mutant leads to the identification of a new allele of the NUD1 gene.

To improve our understanding of the factors involved in the osmotic stability of yeast cells, a search for novel conditional Saccharomyces cerevisiae cell lysis mutants was performed. Ten temperature-sensitive (ts) mutant strains of S. cerevisiae were isolated that lyse at the restrictive temperature on hypotonic, but not on osmotically supported medium. The ten mutants fell into four complementation groups: ts1 to ts4. To clone the wild-type gene corresponding to the ts4 mutation, a strategy aimed at complementing the thermosensitive phenotype-using low-copy and high-copy DNA libraries--was followed, but only two extragenic suppressors were identified. Another approach, in which classic genetic methods were combined with the use of yeast artificial chromosomes and traditional cloning procedures, allowed the identification of the NUD1 gene--which codes for a component of the spindle-pole body-as the wild-type gene corresponding to the ts4 mutation. Cloning and sequencing of the defective allele from the chromosome of the mutant cells resulted in the identification of a point mutation that produces a single amino acid change in the protein: a Gly-to-Glu change at position 585 (the nud1-G585E allele). Further analysis revealed that cells carrying this allele show a thermosensitive growth defect. At the restrictive temperature, the cells arrest with large buds, elongated spindles, and duplicated nuclei. In addition, with longer incubation times they are unable to maintain cellular integrity and lyse. Our results have allowed the identification of the first single amino acid mutation in NUD1, and suggest a link between cell cycle progression and cellular integrity.

Alleles↗
PubMed2001

[Creation of a collection of morphological insertional mutants of Arabidopsis thaliana].

A collection of transgenic Arabidopsis thaliana plants has been obtained by Agrobacterium-mediated transformation. The genomes of the transgenic plants contain insertions of T-DNA of the vector plasmids pLD3 or pPCVRN4. Genes bearing T-DNA insertions were shown to constitute 12-18% of the total number of A. thaliana genes. Seventy-five lines have been chosen from the collection and subjected to genetic and molecular-genetic analysis. Of these, 5 were dominant mutants, and 70, recessive insertion mutants with various morphological defects. Identification of mutant phenotypes and genetic characterization of the transgenic lines have been performed with the use of nutrient media supplemented with exogenous hormones, which revealed five recessive lethal mutants and one dominant sterile mutant.

Arabidopsis↗
PubMed1987

The defective proton-ATPase of uncD mutants of Escherichia coli. Identification by DNA sequencing of residues in the beta-subunit which are essential for catalysis or normal assembly.

Six mutant uncD alleles, affecting essential residues of the beta-subunit of Escherichia coli proton-ATPase, have been identified by intragenic complementation mapping, cloning, and DNA sequencing. Five of the mutations impair catalysis but do not cause structural perturbation of F1-ATPase. The amino acid substitutions found were as follows: uncD412, Gly-142----Ser; uncD430 and uncD431, both Arg-246----Cys; uncD478, Ser-174----Phe; and uncD484, Met-209----Ile. Kinetic characteristics of each corresponding mutant F1-ATPase are described or reviewed. In each case, the major determinant of impaired catalysis appears to be an attenuation of positive catalytic site cooperativity. Additionally, each mutation affects intrinsic properties of the catalytic site, including affinity for ATP, the ratio between unisite-bound substrate and products, and the rate of release of product inorganic phosphate under unisite ATP hydrolysis conditions. These effects are discussed in terms of a structural model of the catalytic nucleotide-binding domain of beta-subunit proposed recently (Duncan, T.M., Parsonage, D., and Senior, A.E. (1986) FEBS Lett. 208, 1-6). Each of the mutations lies within that domain. The uncD409 allele abolishes normal assembly of F1-ATPase. The amino acid substitution is Gly-214----Arg, which is suggested to affect a beta-turn connecting a beta-strand and an alpha-helix in the predicted nucleotide-binding domain of the beta-subunit.

Alleles↗
PubMed2003

Identification of Arabidopsis rat mutants.

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various "reverse genetic" approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.

Agrobacterium tumefaciens↗
PubMed1987

The defective proton-ATPase of uncA mutants of Escherichia coli. Identification by DNA sequencing of residues in the alpha-subunit which are essential for catalysis or normal assembly.

A group of mutant uncA alleles, affecting essential residues of the alpha-subunit of Escherichia coli proton-ATPase, have been identified by intragenic complementation mapping, cloning, and DNA sequencing. One of the mutations, uncA450, abolishes normal assembly of F1-ATPase. The amino acid substitution found was Glu-299----Lys, which is predicted to lie in an alpha-helix in alpha-subunit. The reversal of the charge at residue 299 is a likely cause of defective assembly. The uncA462 allele causes impairment of catalysis while allowing normal assembly of membrane-bound F1-ATPase. The amino acid substitution found was Ser-347----Phe. Three mutations which impair catalysis but do not cause structural perturbation of either membrane-bound or solubilized F1ATPase were characterized as follows: uncA401, Ser-373----Phe; uncA447, Gly-351----Asp; uncA453, Ser-375----Phe. We predict here that the nucleotide-binding domain of alpha-subunit is formed by the amino acids in the sequence from residue 160 to approximately residue 340. The mutations which cause impairment of catalysis lie in a short segment between residues 347-375 of alpha-subunit, at the C-terminal end of the predicted nucleotide-binding domain. This segment is suggested to be important for beta-alpha-beta intersubunit conformational interaction involved in positive catalytic cooperativity in F1-ATPase.

Alleles↗
PubMed2003

Classification and identification of Arabidopsis cell wall mutants using Fourier-Transform InfraRed (FT-IR) microspectroscopy.

We have developed a novel procedure for the rapid classification and identification of Arabidopsis mutants with altered cell wall architecture based on Fourier-Transform Infrared (FT-IR) microspectroscopy. FT-IR transmission spectra were sampled from native 4-day-old dark-grown hypocotyls of 46 mutants and the wild type treated with various drugs. The Mahalanobis distance between mutants, calculated from the spectral information after compression with the Discriminant Variables Selection procedure, was used for alpha hierarchical cluster analysis. Despite the completely unsupervised nature of the classification procedure, we show that all mutants with cellulose defects appeared in the same cluster. In addition, mutant alleles of similar strength for several unrelated loci were also clustered, which demonstrates the sensitivity of the method to detect a wide array of cell wall defects. Comparing the cellulose-deficient cluster with the cluster that contained wild-type controls led to the identification of wave numbers that were diagnostic for altered cellulose content in the context of an intact cell wall. The results show that FT-IR spectra can be used to identify different classes of mutants and to characterize cell wall changes at a microscopic level in unknown mutants. This procedure significantly accelerates the identification and classification of cell wall mutants, which makes cell wall polysaccharides more accessible to functional genomics approaches.

Alleles↗
PubMed2004

Selection of an antibiotic-hypersusceptible mutant of Pseudomonas aeruginosa: identification of the GlmR transcriptional regulator.

Tn501 random mutagenesis was applied to the Pseudomonas aeruginosa wild-type strain PAO1 to select for mutants hypersusceptible to aminoglycoside antimicrobial agents. One such mutant, called 19A, was found to be hypersusceptible to a wide range of antibiotics including aminoglycosides, beta-lactams, fluoroquinolones, colistin, erythromycin, rifampin, and glycopeptides. Light microscopy of the mutant strain revealed abnormal morphology characterized by large, filamentous cells. The drug supersusceptibility of 19A was accompanied by loss of motility, reduced resistance to osmotic and heat shock stress, and impaired growth at low temperatures. The insertion site of the Tn501 transposon in mutant 19A has occurred in an open reading frame (PA5550 according to the PAO1 genome project), whose gene product shows amino acid sequence similarity to the DeoR family of transcriptional repressors. The gene, which we called glmR, is located between the glmS (PA5549) and glmU (PA5552) homologues of E. coli, responsible for the synthesis of UDP-N-acetylglucosamine-1-P, a precursor of both lipopolysaccharide (LPS) and peptidoglycan. We showed that GlmR represses the transcription of the adjacent glmS homologue (PA5549) in P. aeruginosa, possibly affecting the pool of precursors for peptidoglycan and LPS synthesis. To our knowledge GlmR is the first regulator in P. aeruginosa that affects susceptibility to a large variety of antibiotics and is therefore a potential target for novel anti-infective agents.

Amino Sugars↗
PubMed2001

Large-scale mutagenesis directed at specific chromosomes in wheat.

A novel approach has been developed to allow for the efficient selection of loss-of-function wheat mutants in the M1 generation, following either physical or chemical mutagenesis. This has generated an order of magnitude increase in the efficiency of identification of mutants, and also greatly increases the likelihood that selected individuals reflect mutation events at the target locus, rather than at genes acting elsewhere in the disease resistance pathway. The approach relies only on prior knowledge of the chromosomal location of the target gene, and uses the polyploidy of wheat to construct populations for mutagenesis in which large numbers of individuals are hemizygous for the target gene. The idea is illustrated with the mass identification of mutants at three independent genes for race-specific resistance to yellow rust, and one gene for resistance to powdery mildew.

Chromosomes↗
PubMed2000

Misincorporation by wild-type and mutant T7 RNA polymerases: identification of interactions that reduce misincorporation rates by stabilizing the catalytically incompetent open conformation.

We have characterized the misincorporation properties of wild-type (wt) T7 RNAP and of 45 T7RNAP point mutants. The wt enzyme selects strongly against incorporation of an incorrect nucleotide. From the measured rates of misincorporation, an average error frequency of 1 in 2 x 10(4) is estimated. RNAs bearing 3'-mismatches are extended more slowly than correctly paired 3'-termini, and mismatches one or two bases away from the RNA 3'-end can also slow extension severely even when the 3'-base is correctly paired. Though it has been reported that T7RNAP has a 3' --> 5' nuclease activity, we were unable to detect any endogenous T7RNAP RNase activity in elongation complexes. Pyrophosphorolysis was detected but does not appear to contribute to proofreading. Therefore, unlike other RNAPs, T7RNAP fidelity appears to depend entirely on discrimination against incorporation of the incorrect nucleotide and not on post-misincorporation proofreading. Alanine substitution of the H784 side chain, which interacts with the 3' RNA.template base pair, increases both misincorporation and mismatch extension, while substitutions at G640, F644, and G645 increase misincorporation, but not mismatch extension. The latter three amino acids are in a part of the RNAP which interacts with the templating base and with the base immediately 5' to the templating base. Mutation of these amino acids not only increases misincorporation, but also eliminates pausing during promoter clearance. The effects of these mutations and the interactions observed in a crystal structure of a transcribing complex indicate that these mutations disrupt interactions which limit misincorporation rates by stabilizing the catalytically incompetent open conformation of the RNAP.

Alanine↗
PubMed1994

Yeast topoisomerase II mutants resistant to anti-topoisomerase agents: identification and characterization of new yeast topoisomerase II mutants selected for resistance to etoposide.

We describe a system that allows us to easily isolate and characterize mutants in yeast topoisomerase II that are resistant to antitumor agents that target this enzyme. The system uses yeast strains that are sensitive to those agents and that carry temperature-sensitive top2 mutations. The temperature-sensitive mutation allows the isolation of recessive drug-resistant mutations. The mutagenized TOP2 gene we have used is under the control of the yeast DED1 promoter; this overexpression of TOP2 is designed to avoid isolating mutants that are drug resistant solely because the mutated topoisomerase II has low enzymatic activity. We describe three mutants that we isolated using this system. Two of the three mutants show resistance to etoposide and amsacrine, while the third mutant is partially resistant to etoposide and fluoroquinolones but not to amsacrine. DNA sequence changes have been identified in all of these mutant TOP2 genes. The mutant with partial resistance to etoposide and fluoroquinolones has an amino acid change at position 738 of TOP2, which is three amino acids from the site homologous to Ser83 of E. coli gyrA, an amino acid which had previously been shown to be an important target for resistance to quinolones in bacteria. One of the alleles that confers resistance to both etoposide and amsacrine, top2-103, has changes in amino acid 824 and amino acid 1186 of TOP2. Reconstruction of the mutations by oligonucleotide-directed mutagenesis demonstrates that the change at amino acid 824 is responsible for the drug resistance of this allele.

Amino Acid Sequence↗
PubMed1989

Yeast mutants with enhanced ability to secrete human lysozyme: isolation and identification of a protease-deficient mutant.

Yeast mutant strains which secrete large amounts of human lysozyme were screened using an agar medium containing bacterial cells. Nine mutants secreted over 10 times more lysozyme than the wild-type parent strain. The mRNA levels for lysozyme in the mutants were not higher than that of the wild-type strain. Three of the mutant strains were deficient in carboxypeptidase Y activity. It was found that the protease deficiency was caused by a deficiency in conversion of proenzyme to mature enzyme in ssl1 mutant cells. The ssl1 gene was found to be closely linked to the centromere and determine both the efficiency of secretion of lysozyme and the processing of carboxypeptidase Y.

Blotting, Northern↗
PubMed2006

Inactive full-length p53 mutants lacking dominant wild-type p53 inhibition highlight loss of heterozygosity as an important aspect of p53 status in human cancers.

Over 1000 different mutants of the tumor suppressor protein p53 with one amino acid change in the core domain have been reported in human cancers. In mouse knock-in models, two frequent mutants displayed loss of wild-type (wt) p53 function, inhibition of wt p53 and wt p53-independent gain of function. The remaining mutants have been systematically characterized for loss of wt p53 function, but not other phenotypes. We report the concomitant assessment of loss of function and interference with wt p53 using URA3-based p53 yeast and confirmatory mammalian assays. We studied 76 mutants representing 54% of over 15 000 reported missense core domain mutations. The majority showed the expected complete loss of wt p53 function and dominant p53 inhibition. A few infrequent p53 mutants had wt p53-like activity. Remarkably, one-third showed no interference with wt p53 despite loss of wt p53 function at 37 degrees C. Half of this group consisted of temperature-sensitive p53 mutants, but the other half was surprisingly made up of mutants with complete loss of wt p53 function. Our findings illustrate the diverse behavior of p53 mutants and mechanisms of malignant transformation by p53 mutants. The identification of full-length p53 mutants without dominant inhibition of wt p53 highlights the importance of determining the status of the wt p53 allele in human cancers, in particular in the context of clinical studies. In the case of p53 mutants with no or weak dominant p53 inhibition, presence of the wt allele may indicate a good prognosis cancer, whereas loss of heterozygosity may spell an aggressive, therapy-resistant cancer.

Cell Line, Tumor↗
PubMed1994

Isolation and characterization of hypophosphite--resistant mutants of Escherichia coli: identification of the FocA protein, encoded by the pfl operon, as a putative formate transporter.

Hypophosphite was used as a toxic analogue to identify genes whose products have a putative function in the transport of formate. Two Tn10-derived insertion mutants were identified that exhibited increased resistance to high concentrations of hypophosphite in the culture medium. The transposon was located in the identical position in the focA (formate channel; previously termed orf) gene of the pfl operon in both mutants. A defined chromosomal focA nonsense mutant, which showed minimal polarity effects on pfl gene expression, had the same phenotype as the insertion mutants. Results obtained using a hycA-lacZ fusion to monitor changes in the intracellular formate concentration in a focA mutant indicated that the level of formate inside the cell was elevated compared with the wild type. Moreover, it could be shown that there was a corresponding reduction of approximately 50% in the amount of formate excreted by a focA mutant into the culture medium. Taken together, these results indicate that formate accumulates in anaerobic cells which do not have a functional focA gene product and that one function of FocA may be to export formate from the cell. A further significant result was that hypophosphite could substitute for formate in activating hycA gene expression. This hypophosphite-dependent activation of hycA gene expression was reduced 10-fold in a focA null mutant, suggesting that hypophosphite must first enter the cell before it can act as a signal to activate hycA expression. By analogy, these data suggest that focA may also be functional in the import of formate into anaerobic Escherichia coli cells. Site-specific mutagenesis identified the translation initiation codon of focA as a GUG. Therefore, the FocA polypeptide has a molecular weight of 30,958. FocA shows significant similarity at both the primary and secondary structural levels with the NirC protein of E. coli and the FdhC protein of Methanobacterium formicicum. All three proteins are predicted to be integral membrane proteins. A detailed in vivo TnphoA mutagenesis study predicted that FocA has six membrane-spanning segments.

Acetyltransferases↗
PubMed1995

Expression of a Drosophila GATA transcription factor in multiple tissues in the developing embryos. Identification of homozygous lethal mutants with P-element insertion at the promoter region.

GATA transcription factors are DNA-binding proteins that recognize the core consensus sequence, WGATAR. Previous studies indicated that GATA factors play ann important role in the development of tissue-specific functions in vertebrates. Here we report the identification of a new Drosophila melanogaster GATA factor, dGATAc, which displays a distinct expression pattern in embryos. The local concentration of dGATAc transcripts varies at different stages, being most prominent in the procephalic region at stages 6-10 and in the posterior spiracles, the gut, and the central nervous system at stages 11-13. On the basis of its predicted sequence, DNA-binding assays were performed to confirm that the dGATAc gene encodes a zinc finger protein that can bind the GATA consensus motif with predicted specificity. Two independent mutants carrying a P-element insertion at the dGATAc gene promoter region were identified that are homozygous lethal at the embryonic stage. Using a genetic scheme, it was demonstrated that the lack of dGATAc function can block normal embryonic development. Our results suggest that the dGATAc protein is a tissue-specific transcription factor that is vital to the development of multiple organ systems in D. melanogaster.

Amino Acid Sequence↗
PubMed1999

Mass spectrometric identification of a stable catalytic cysteinesulfinic acid residue in an enzymatically active chemically modified glucoamylase mutant.

Mass spectrometric identification of cysteinsulfinic acid resulting in restoration of activity of chemically modified Glu400 Cys catalytic-base glucoamylase (GA) mutants is described. This oxidation unexpectedly occurred during attempts to carboxyalkylate the Cys400 GA mutant using three different alkylation reagents. However, mass spectrometric peptide mapping did not show the presence of carboxyalkylation of the Cys400 residue but suggested an oxidation to cysteinsulfinic acid based on the observed mass increment. The presence of cysteinsulfinic acid was confirmed by employing matrix-assisted laser desorption/ionization mass spectrometry combined with post-source decay analysis. Furthermore, strong enhancement of metastable fragmentation was observed for peptides containing oxidized Cys in comparison with non-oxidized peptide.

Amino Acid Sequence↗
PubMed1988

Hemoglobin Pasadena: identification of the gene mutant by DNA analysis using synthetic DNA probes.

Hemoglobin Pasadena [beta 75(E19)Leu----Arg] was found in a boy who had an acute episode of anemia and rapid splenic enlargement. His father was the only other member of a large family with this hemoglobinopathy. We have used gene mapping techniques for direct identification of the beta-globin gene mutation. To correlate the DNA findings with the structural identification of this variant, we have also performed globin chain separation and analysis of the tryptic peptides using high performance liquid chromatography and secondary ion mass spectral analysis.

Adult↗
PubMed2000

Identification of an Arabidopsis thaliana mutant accumulating threonine resulting from mutation in a new dihydrodipicolinate synthase gene.

A novel Arabidopsis DHDPS gene named DHDPS2 was found through identification of a mutant by promoter trapping. The mutation promotes a reduction of growth resulting from combination of a defect in lysine biosynthesis and accumulation of a toxic level of threonine or derived products. The mutant also modifies the amino acid composition issuing from the pyruvate and aspartate pathways, affecting mainly the root compartment. These data are in accordance with the expression of DHDPS2 in the root apex as visualized by expression of the GUS reporter gene. This suggests that a large proportion of the amino acids derived from pyruvate and aspartate are synthesized in this organ.

Amino Acid Sequence↗