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 559 records · Page 31Linked to original sources
PubMed2005

Identification of a potent inverse agonist at a constitutively active mutant of human P2Y12 receptor.

Human platelets express two P2Y receptors: G(q)-coupled P2Y(1), and G(i)-coupled P2Y(12). Both P2Y(1) and P2Y(12) are ADP receptors on human platelets and are essential for ADP-induced platelet aggregation that plays pivotal roles in thrombosis and hemostasis. Numerous constitutively active G protein-coupled receptors have been described in natural or recombinant systems, but in the P2Y receptors, to date, no constitutive activity has been reported. In our effort to identify G protein coupling domains of the human platelet ADP receptor, we constructed a chimeric hemagglutinin-tagged human P2Y(12) receptor with its C terminus replaced by the corresponding part of human P2Y(1) receptor and stably expressed it in Chinese hamster ovary-K1 cells. It is interesting that the chimeric P2Y(12) mutant exhibited a high level of constitutive activity, as evidenced by decreased cAMP levels in the absence of agonists. The constitutive activation of the chimeric P2Y(12) mutant was dramatically inhibited by pertussis toxin, a G(i) inhibitor. The constitutively active P2Y(12) mutant retained normal responses to 2-methylthio-ADP, with an EC(50) of 0.15 +/- 0.04 nM. The constitutively active P2Y(12) mutant caused Akt phosphorylation that was abolished by the addition of pertussis toxin. Pharmacological evaluation of several P2Y(12) antagonists revealed (E)-N-[1-[7-(hexylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]-pyrimidin-3-yl]-1,5,6-trideoxy-beta-d-ribo-hept-5-enofuranuronoyl]-l-aspartic acid (AR-C78511) as a potent P2Y(12) inverse agonist and 5'-adenylic acid, N-[2-(methylthio)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-, monoanhydride with (dichloromethylene)bis[phosphonic acid] (AR-C69931MX) as a neutral antagonist. In conclusion, this is the first report of a cell line stably expressing a constitutively active mutant of human platelet P2Y(12) receptor and the identification of potent inverse agonist.

Animals↗
PubMed1986

Galactose metabolism in Rhizobium meliloti L5-30.

Data from previous studies of Rhizobium meliloti mutants have been consistent with the catabolism of hexoses via the Entner-Doudoroff pathway. However, galactose metabolism was not impaired in those mutants. We show here by enzymatic assay and by identification of a galactose mutant lacking 2-keto-3-deoxy-6-phosphogalactonate aldolase that the De Ley-Doudoroff pathway is used for galactose metabolism. Mutants in this pathway have not been previously reported for any organism.

Aldehyde-Lyases↗
PubMed1995

Identification of a chemotaxis operon with two cheY genes in Rhodobacter sphaeroides.

A large chemotaxis operon was identified in Rhodobacter sphaeroides WS8-N using a probe based on the 3' terminal portion of the Rhizobium meliloti cheA gene. Two genes homologous to the enteric cheY were identified in an operon also containing cheA, cheW, and cheR homologues. The deduced protein sequences of che gene products were aligned with those from Escherichia coli and shown to be highly conserved. A mutant with an interrupted copy of cheA showed normal patterns of swimming, unlike the equivalent mutants in E. coli which are smooth swimming. Tethered cheA mutant cells showed normal responses to changes in organic acids, but increased, inverted responses to sugars. The unusual behaviour of the cheA mutant and the identification of two homologues of cheY suggests that R. sphaeroides has at least two pathways controlling motor activity. To identify functional similarity between the newly identified R. sphaeroides Che pathway and the methyl-accepting chemotaxis protein (MCP)-dependent pathway in enteric bacteria, the R. sphaeroides cheW gene was expressed in a cheW mutant strain of E. coli and found to complement, causing a partial return to a swarming phenotype. In addition, expression of the R. sphaeroides gene in wild-type E. coli resulted in the same increased tumbling and reduced swarming as seen when the native gene is overexpressed in E. coli. The identification of che homologues in R. sphaeroides and complementation by cheW suggests the presence of MCPs in an organism previously considered to use only MCP-independent sensing. The MCP-dependent pathway, appears conserved.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗
PubMed2004

Generation of Yersinia pestis attenuated strains by signature-tagged mutagenesis in search of novel vaccine candidates.

In a search for novel attenuated vaccine candidates for use against Yersinia pestis, the causative agent of plague, a signature-tagged mutagenesis strategy was used and optimized for a subcutaneously infected mouse model. A library of tagged mutants of the virulent Y. pestis Kimberley53 strain was generated. Screening of 300 mutants through two consecutive cycles resulted in selection of 16 mutant strains that were undetectable in spleens 48 h postinfection. Each of these mutants was evaluated in vivo by assays for competition against the wild-type strain and for virulence following inoculation of 100 CFU (equivalent to 100 50% lethal doses [LD50] of the wild type). A wide spectrum of attenuation was obtained, ranging from avirulent mutants exhibiting competition indices of 10(-5) to 10(-7) to virulent mutants exhibiting a delay in the mean time to death or mutants indistinguishable from the wild type in the two assays. Characterization of the phenotypes and genotypes of the selected mutants led to identification of virulence-associated genes coding for factors involved in global bacterial physiology (e.g., purH, purK, dnaE, and greA) or for hypothetical polypeptides, as well as for the virulence regulator gene lcrF. One of the avirulent mutant strains (LD50, >10(7) CFU) was found to be disrupted in the pcm locus, which is presumably involved in the bacterial response to environmental stress. This Kimberley53pcm mutant was superior to the EV76 live vaccine strain because it induced 10- to 100-fold-higher antibody titers to the protective V and F1 antigens and because it conferred efficacious protective immunity.

Animals↗
PubMed1997

Identification and characterization of hydrogen peroxide-sensitive mutants of Escherichia coli: genes that require OxyR for expression.

Escherichia coli produces an inducible set of proteins that protect the cell from exogenous peroxide stress. A subset of these genes is induced by hydrogen peroxide and is controlled at the transcriptional level by the OxyR protein. To identify additional genes involved in protection from hydrogen peroxide, a library of random transcriptional fusions of lambda(plac)Mu53 was screened for hydrogen peroxide sensitivity and 27 such mutants were identified. These fusions were transduced into nonlysogenic strains to ensure that the phenotypes observed were the result of a single mutation. The mutants were grouped into three classes based on the expression of the lacZ fusion during growth in oxyR+ and deltaoxyR backgrounds. The expression of the lacZ fusion in 8 mutants was independent of OxyR, 10 mutants required OxyR for expression, and 6 mutants showed reduced levels of expression in the presence of OxyR. OxyR dependence varied from 2- to 50-fold in these mutants. The OxyR-dependent phenotype was complemented by a plasmid-borne copy of oxyR gene in all mutants. Three mutants exhibited dual regulation by OxyR and RpoS. We sequenced the fusion junctions of several of these mutants and identified the genetic loci responsible for the hydrogen peroxide-sensitive (hps) phenotype. In this study, we report the identification of several genes that require OxyR for expression, including hemF (encoding coproporphyrinogen III oxidase), rcsC (encoding a sensor-regulator protein of capsular polysaccharide synthesis genes), and an open reading frame, f497, that is similar to arylsulfatase-encoding genes.

Bacterial Proteins↗
PubMed2004

The poliovirus replication machinery can escape inhibition by an antiviral drug that targets a host cell protein.

Viral replication depends on specific interactions with host factors. For example, poliovirus RNA replication requires association with intracellular membranes. Brefeldin A (BFA), which induces a major rearrangement of the cellular secretory apparatus, is a potent inhibitor of poliovirus RNA replication. Most aspects governing the relationship between viral replication complex and the host membranes remain poorly defined. To explore these interactions, we used a genetic approach and isolated BFA-resistant poliovirus variants. Mutations within viral proteins 2C and 3A render poliovirus resistant to BFA. In the absence of BFA, viruses containing either or both of these mutations replicated similarly to wild type. In the presence of BFA, viruses carrying a single mutation in 2C or 3A exhibited an intermediate-growth phenotype, while the double mutant was fully resistant. The viral proteins 2C and 3A have critical roles in both RNA replication and vesicle formation. The identification of BFA resistant mutants may facilitate the identification of cellular membrane-associated proteins necessary for induction of vesicle formation and RNA replication. Importantly, our data underscore the dramatic plasticity of the host-virus interactions required for successful viral replication.

Antiviral Agents↗
PubMed2003

Development of an efficient "substrate-trapping" mutant of Src homology phosphotyrosine phosphatase 2 and identification of the epidermal growth factor receptor, Gab1, and three other proteins as target substrates.

Src homology containing phosphotyrosine phosphatase 2 (SHP2) is a positive effector of growth factor, cytokine, and integrin signaling. However, neither its physiological substrate nor its mechanism of action in tyrosine kinase signaling has been demonstrated. We reasoned that the identification of physiological substrates of SHP2 would be a stepping stone in elucidating its mechanism of action, and, thus, we constructed a potent trapping mutant of SHP2. Surprisingly, the frequently used Asp to Ala substitution did not give rise to a trapping mutant. However, we were able to develop an efficient trapping mutant of SHP2 by introducing Asp to Ala and Cys to Ser double mutations. The double mutant (DM) protein identified the epidermal growth factor receptor (EGFR), the Grb2 binder 1, and three other, as yet unidentified, phosphotyrosyl proteins as candidate physiological substrates. Given that substrate trapping occurred in intact cells and that the interaction was very specific, it is highly likely that EGFR and Gab1 represent physiological SHP2 substrates. Therefore, the DM protein would serve as an important tool in future SHP2 studies, including identification of p190, p150, and p90.

3T3 Cells↗
PubMed1996

Identification of a class of Saccharomyces cerevisiae mutants defective in fatty acid repression of gene transcription and analysis of the frm2 gene.

Exogenous fatty acids transcriptionally control the expression of a wide variety of eukaryotic genes, many of which encode proteins involved in lipid metabolism. To identify gene products involved in the lipid signalling pathway, a reporter plasmid containing the 5'-upstream region of a gene demonstrated to be repressed by unsaturated fatty acids (OLE1) was fused in frame to the Escherichia coli gene lacZ encoding beta-galactosidase. Saccharomyces cerevisiae mutants defective in transcriptional control by lipids were identified and this class of mutants has been named frm (fatty acid repression mutant). The mutants were organized into six complementation groups designated frm1-6. Mutants from two of the complementation groups, frm1 and frm3, were also defective in their ability to activate a reporter construct containing the 5'-upstream region of POX1. POX1 has been shown to be transcriptionally activated in the presence of unsaturated fatty acids. frm2 was rescued by a region of DNA localized to chromosome III. This region contained an open reading frame of 579 nucleotides predicted to encode a M(r) 21 116 polypeptide. The upstream region of FRM2 contained a number of potential response elements which have previously been identified as important in regulating gene expression in response to glucose and certain fatty acids. Consistent with this observation, lacZ activity driven by FRM2 or frm2 promoters was induced two- to three-fold dependent upon the carbon and fatty acid source utilized. The properties of FRM2 suggest that it functions in the fatty acid signalling pathway and that it is itself regulated by fatty acids.

Base Sequence↗
PubMed2000

Identification and molecular characterization of a natural mutant of the p50.2/KIR2DS2 activating NK receptor that fails to mediate NK cell triggering.

P50/KIR2DS molecules represent the activating form of the HLA-C-specific inhibitory NK receptors. They are characterized, in the transmembrane portion, by a charged amino acid that is involved in coupling with signal-transducing adaptor polypeptides. In this study we identified a novel p50.2/KIR2DS2 surface molecule, isolated from NK cell clones derived from an otherwise normal donor, that was unable to transduce activating signals. Sequence analysis of the cDNA encoding this molecule revealed six non-conservative codon mutations in the exon coding for the putative transmembrane portion. Notably, one of such mutations involved the charged residue lysine thought to be important for the association with signal-transducing polypeptides. Indeed, co-transfection experiments revealed that this naturally occurring p50.2/KIR2DS2 mutant, termed Mp50.2, displayed a sharply reduced ability to associate with DAP12 polypeptides. These data provide the first in vivo demonstration of the crucial role played by the transmembrane region of p50.2 receptor molecules in the functional association with DAP12 adaptor molecules and in the process of activation of NK-mediated cytotoxicity.

Amino Acid Sequence↗
PubMed2005

Identification and functional characterization of five novel mutant alleles in 58 Italian patients with Gaucher disease type 1.

Gaucher disease (GD) is the most frequent lysosomal glycolipid storage disorder due to an autosomal recessive deficiency of acid beta-glucosidase characterized by the accumulation of glucocerebroside. In this work we carried out the molecular analysis of the glucocerebrosidase gene (GBA) in 58 unrelated patients with GD type 1. We identified five novel genetic alterations: three missense changes c.187G>A (p.D63N), c.473T>G (p.I158S), c.689T>A (p.V230E), a gene-pseudogene recombinant allele and a non-pseudogene-derived complex allele [c.1379G>A;c.1469A>G] encoding [p.G460D;p.H490R]. All mutant alleles were present as compound heterozygotes in association with c.1226A>G (p.N409S), the most common mutation in GD1. The missense mutant proteins were expressed in vitro in COS-1 cells and analyzed by enzyme activity, protein processing and intracellular localization. Functional studies also included the c.662C>T (p.P221L) mutation recently reported in the Spanish GD population (Montfort et al., 2004). The missense mutant alleles retained an extremely low residual enzyme activity with respect to wild type; the complex allele expressed no activity. Processing of the mutant proteins was unaltered except for c.473T>G which was differently glycosylated due to the exposition of an additional glycosylation site. Immunofluorescence studies showed that protein trafficking into the lysosomes was unaffected in all cases. Finally, the characterization of the novel recombinant allele identified a crossover involving the GBA gene and pseudogene between intron 5 and exon 7.

Adult↗
PubMed1998

Identification of a novel Lymantria dispar nucleopolyhedrovirus mutant that exhibits abnormal polyhedron formation and virion occlusion.

In previous studies on the formation of Lymantria dispar nuclear polyhedrosis virus (LdMNPV) few polyhedra (FP) mutants, several polyhedron formation mutants (PFM) were identified that appeared to be unique. These viral mutants are being characterized to investigate the processes of polyhedron formation and virion occlusion. LdMNPV isolate PFM-1 is one of these mutants, and is described in this report. Genetic techniques were used to determine if isolate PFM-1 contained a mutation in the polyhedrin or 25K FP gene. Wild-type viruses were recovered after coinfection of Ld652Y cells with isolate PFM-1 and a FP mutant, and with isolates PFM-1 and PFM-C (isolate PFM-C contains a mutation in the polyhedrin gene). These viruses were analyzed by genomic restriction endonuclease digestion and found to be chimeras of the original PFMs used in the coinfections. Marker rescue studies mapped the mutation in isolate PFM-1 to a genomic region that does not include the polyhedrin or 25K FP genes. Isolate PFM-1 produced approximately 14-fold fewer polyhedra than LdMNPV isolate A21-MPV, an isolate that produces wild-type levels of polyhedra, and approximately 2-fold more polyhedra compared to the FP isolate 122-2. Polyhedra generated by isolate PFM-1 were normal in size and shape but contained very few viral nucleocapsids. The same amount of budded virus (BV) was released from cells infected with isolates PFM-1 and A21-MPV. In contrast, isolate 122-2 yielded significantly more BV than isolates PFM-1 and A21-MPV.

Animals↗
PubMed1993

Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencing, complementation and co-suppression.

A tomato phytoene synthase gene, Psy1, has recently been isolated as the clone GTOM5 and shown by sequence identity to be the gene from which the major fruit-ripening cDNA clone TOM5 was derived. Sequence analysis of transcripts from two allelic yellow-fruited tomato mutants, mapped to chromosome 3, has shown the lack of carotenoids in fruit of these mutants to be due to the production of aberrant TOM5 transcripts which are unlikely to encode a functional phytoene synthase enzyme. In one mutant (yellow flesh) the aberrant transcript contained a sequence that, by its strong hybridization to a wide size range of genomic fragments, appeared to be repeated many times within the genome. Southern and PCR analysis of the phytoene synthase genes in the mutant revealed restriction fragment length polymorphisms, suggesting that the production of altered mRNAs was associated with specific genomic rearrangements. Constitutive over-expression of a TOM5 cDNA clone in transgenic mutant plants restored synthesis of the carotenoid lycopene in ripening fruit and also led to unscheduled pigment production in other cell types. In some mutant plants transformed with the TOM5 cDNA construct, inhibition of carotenoid production in immature green fruit, leaves and flowers was observed, due to the phenomenon of co-suppression, indicating that different insertion events with the same gene construct can lead to overexpression or co-suppression in transgenic plants. Green organs of these plants were susceptible to photobleaching, due to the lack of carotenoids. These results suggest the existence of separate Psy genes for carotenoid synthesis in green organs.

Alkyl and Aryl Transferases↗
PubMed1997

Identification of an assimilatory nitrate reductase in mutants of Paracoccus denitrificans GB17 deficient in nitrate respiration

A Paracoccus denitrificans strain (M6Omega) unable to use nitrate as a terminal electron acceptor was constructed by insertional inactivation of the periplasmic and membrane-bound nitrate reductases. The mutant strain was able to grow aerobically with nitrate as the sole nitrogen source. It also grew anaerobically with nitrate as sole nitrogen source when nitrous oxide was provided as a respiratory electron acceptor. These growth characteristics are attributed to the presence of a third, assimilatory nitrate reductase. Nitrate reductase activity was detectable in intact cells and soluble fractions using nonphysiological electron donors. The enzyme activity was not detectable when ammonium was included in the growth medium. The results provide an unequivocal demonstration that P. denitrificans can express an assimilatory nitrate reductase in addition to the well-characterised periplasmic and membrane-bound nitrate reductases.

Journal Article↗
PubMed1999

Identification of T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium.

We have identified T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium tumefaciens (rat mutants). These mutants are highly recalcitrant to the induction of both crown gall tumors and phosphinothricin-resistant calli. The results of transient GUS (beta-glucuronidase) assays suggest that some of these mutants are blocked at an early step in the Agrobacterium-mediated transformation process, whereas others are blocked at a step subsequent to translocation of T-DNA into the nucleus. Attachment of Agrobacterium to roots of the mutants rat1 and rat3 was decreased under various incubation conditions. In most mutants, the transformation-deficient phenotype co-segregated with the kanamycin resistance encoded by the mutagenizing T-DNA. In crosses with susceptible wild-type plants, the resistance phenotype of many of these mutants segregated either as a semi-dominant or dominant trait.

Agrobacterium tumefaciens↗
PubMed1989

Identification and characterisation of an antithrombin III mutant (AT Dublin 2) with marginally decreased heparin reactivity.

The antithrombin III (ATIII) isoform patterns of plasma and serum samples from cancer patients and controls were analysed by isoelectric focusing and immunoblotting. A novel ATIII banding pattern was identified in two individuals: a patient with breast carcinoma who developed deep venous thrombosis and a blood donor. Family studies in the patient showed the abnormal pattern to be due to a mutant form of ATIII (AT Dublin 2). The coagulation properties of AT Dublin 2 heterozygotes were normal. Immunologic and activity levels of ATIII, measured by standard techniques, were normal. Mutant plasma ATIII showed reduced thrombin reactivity at low concentrations of thrombin and demonstrated decreased reactivity with heparin over a range of heparin concentrations. This was confirmed using a modified ATIII heparin cofactor activity assay with varying heparin concentrations. The abnormal ATIII was also found to elute from heparin agarose at a lower ionic strength than normal ATIII. Two dimensional gel electrophoresis showed the abnormal ATIII to have similar molecular size distribution to normal ATIII. Neuraminidase treatment of normal and mutant plasma reduced the ATIII isoforms to one in both samples. The possible role of AT Dublin 2 in predisposing to hypercoagulation is discussed.

Adult↗
PubMed2004

Identification and complementation of abnormal hyphal branch mutants ahbA1 and ahbB1 in Aspergillus nidulans.

Branching generates new axes of polar growth in filamentous fungi and is critical for development, reproduction, and pathogenicity. To investigate branching we screened an Aspergillus nidulans temperature-sensitive mutant collection for abnormal hyphal branch (ahb) mutants. We identified two mutants, ahbA1, which showed reduced branching relative to wild type at restrictive temperature, and ahbB1, which showed increased branching relative to wild type at restrictive temperature. Both mutants also showed abnormal conidiophore development at restrictive temperature. The ahbA1 hypobranching mutant showed defects in nuclear division and hydroxyurea resistance. Complementation and sequencing showed that ahbA1 is a previously identified allele of the cell cycle regulator nimX. The ahbB1 hyperbranching mutant had an increased number of nuclei, was osmotically remedial and Calcofluor resistant. The ahbB gene is predicted to encode a novel protein that has homologues exclusively in filamentous fungi. The C-terminal domain of the predicted AhbB protein showed homology with the heme-binding domain of a cytochrome P450 protein and sequencing of the ahbB1 mutant allele showed that the lesion lies just before this putative heme-binding domain. The ahbB1 mutant showed increased sensitivity to the ergosterol biosynthesis inhibitor imidazole. Our results suggest a link between nuclear division and branching and a possible role for membrane synthesis in branching.

Amino Acid Sequence↗
PubMed2003

Identification and characterization of p100HB, a new mutant form of p100/NF-kappa B2.

P100, which is encoded by NF-kappa B2, inhibits Rel dimers. It can also be processed into p52, one of the DNA binding sub-units of NF-kappa B/Rel factors. Several p100 C-terminal truncations that result from gene rearrangements are associated with lymphomagenesis. Here, we characterized a new p100 mutant that we termed p100HB. It originates from a point-mutation that generates a premature stop-codon, and thus the protein lacks the last 125 amino acids. We have detected p100HB in several human tumor cell lines. The truncated protein is mainly unprocessed, and although it still binds Rel dimers, it has reduced inhibitory potency compared to p100 and translocates into the nucleus. Thus, p100HB may be associated with deregulated NF-kappa B/Rel functions.

Active Transport, Cell Nucleus↗