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 739 records · Page 41Linked to original sources
PubMed2000

Annexin XII E105K crystal structure: identification of a pH-dependent switch for mutant hexamerization.

Annexins are a family of calcium- and phospholipid-binding proteins involved with numerous cellular processes including membrane fusion, ion channel activity, and heterocomplex formation with other proteins. The annexin XII (ANXB12) crystal structure presented evidence that calcium mediates the formation of a hexamer through a novel intermolecular calcium-binding site [Luecke et al. (1995) Nature 378, 512-515]. In an attempt to disrupt hexamerization, we mutated a conserved key ligand in the intermolecular calcium-binding site, Glu105, to lysine. Despite its occurrence in a new spacegroup, the 1.93 A resolution structure reveals a hexamer with the Lys105 epsilon-amino group nearly superimposable with the original intermolecular calcium position. Our analysis shows that the mutation is directly involved in stabilizing the hexamer. The local residues are reoriented to retain affinity between the two trimers via a pH-dependent switch residue, Glu76, which is now protonated, allowing it to form tandem hydrogen bonds with the backbone carbonyl and nitrogen atoms of Thr103 located across the trimer interface. The loss of the intermolecular calcium-binding site is recuperated by extensive hydrogen bonding favoring hexamer stabilization. The presence of this mutant structure provides further evidence for hexameric annexin XII, and possible in vivo roles are discussed.

Animals↗
PubMed1998

Trehalose-6-phosphate phosphatases from Arabidopsis thaliana: identification by functional complementation of the yeast tps2 mutant.

It is currently thought that most flowering plants lack the capacity to synthesize trehalose, a common disaccharide of bacteria, fungi and invertebrates that appears to play a major role in desiccation tolerance. Attempts have therefore been made to render plants more drought-resistant by the expression of microbial genes for trehalose synthesis. It is demonstrated here that Arabidopsis thaliana itself possesses genes for at least one of the enzymes required for trehalose synthesis, trehalose-6-phosphate phosphatase. The yeast tps2 mutant, which lacks this enzyme, is heat-sensitive, and Arabidopsis cDNA able to complement this effect has been screened for. Half of the yeast transformants that grew at 38.6 degrees C were also able to produce trehalose. All of these expressed one of two Arabidopsis cDNA, either AtTPPA or AtTPPB, which are both homologous to the C-terminal part of the yeast TPS2 gene and other microbial trehalose-6-phosphate phosphatases. Yeast tps2 mutants expressing AtTPPA or AtTPPB contained trehalose-6-phosphate phosphatase activity that could be measured both in vivo and in vitro. The enzyme dephosphorylated trehalose-6-phosphate but not glucose-6-phosphate or sucrose-6-phosphate. Both genes are expressed in flowers and young developing tissue of Arabidopsis. The finding of these novel Arabidopsis genes for trehalose-6-phosphate phosphatase strongly indicates that a pathway for trehalose biosynthesis exists in plants.

Amino Acid Sequence↗
PubMed2001

Identification of new JNK substrate using ATP pocket mutant JNK and a corresponding ATP analogue.

Modification of the ATP pocket on protein kinases allows selective use of an ATP analogue that exhibits high affinity for the altered kinases. Using this approach, we altered the ATP-binding site on JNK and identified N(6)-(2-phenythyl)-ATP, a modified form of ATP that exhibits high specificity and affinity for the modified, but not the wild type form, of JNK. Using modified JNK and its ATP analogue enables the detection of novel JNK substrates. Among substrates identified using this approach is heterogeneous nuclear ribonucleoprotein K, which is involved in transcription and post-transcriptional mRNA metabolism. The newly identified substrate can be phosphorylated by JNK on amino acids 216 and 353, which contribute to heterogeneous nuclear ribonucleoprotein K mediated transcriptional activities.

Adenosine Triphosphate↗
PubMed2000

Identification of ascorbic acid-deficient Arabidopsis thaliana mutants.

Vitamin C (l-ascorbic acid) is a potent antioxidant and cellular reductant present at millimolar concentrations in plants. This small molecule has roles in the reduction of prosthetic metal ions, cell wall expansion, cell division, and in the detoxification of reactive oxygen generated by photosynthesis and adverse environmental conditions. However, unlike in animals, the biosynthesis of ascorbic acid (AsA) in plants is only beginning to be unraveled. The previously described AsA-deficient Arabidopsis mutant vtc1 (vitamin c-1) was recently shown to have a defect in GDP-mannose pyrophosphorylase, providing strong evidence for the recently proposed role of GDP-mannose in AsA biosynthesis. To genetically define other AsA biosynthetic loci, we have used a novel AsA assay to isolate four vtc mutants that define three additional VTC loci. We have also isolated a second mutant allele of VTC1. The four loci represented by the vtc mutant collection have been genetically characterized and mapped onto the Arabidopsis genome. The vtc mutants have differing ozone sensitivities. In addition, two of the mutants, vtc2-1 and vtc2-2, have unusually low levels of AsA in the leaf tissue of mature plants.

Arabidopsis↗
PubMed2003

Identification of genes showing differential expression in anorexia mutant mouse.

Differential display (DD) PCR was applied to identify genes regulated by anorexia (anx) in mouse brain. Of the four apparently differentially expressed genes, three (H2-H4) had high homology with known genes such as apoptotic protease activating factor 1 (Apaf 1), adenylate cyclase 6 (Adcy 6), and myelin proteolipid protein (PLP). H1, designated as HIPA1, had a homology with unknown function gene fragments. Northern blot analysis confirmed HIPA1 expression induced by anorexia. A 1579 bp full-length cDNA of HIPA1 was isolated from a mouse brain cDNA library using a probe from the differentially displayed H1 fragment. Sequence analysis showed that HIPA1 had 98.7% homology to mouse hippocampus cDNA. In situ hybridization demonstrated that the HIPA1 mRNA was highly up-regulated in the hippocampus of anx/anx mouse brain.

Amino Acid Sequence↗
PubMed1994

Identification of a gene that complements an Arabidopsis mutant deficient in chloroplast omega 6 desaturase activity.

Membrane lipids of the fad6 (formerly fadC) mutant of Arabidopsis, which is deficient in chloroplast omega 6 desaturase activity, have increased levels of monounsaturated fatty acids and are deficient in trienoic fatty acids. A putative fad6 cDNA clone was isolated by probing a cDNA library with a degenerate oligonucleotide based on a conserved region within known omega 3 desaturase genes. Expression of the cDNA in transgenic plants of a fad6 mutant restored normal levels of all fatty acids. When used as a hybridization probe, the cDNA identified a restriction fragment-length polymorphism that co-segregated with the fad6 mutation. Thus, on the basis of a genetic complementation test and genetic map position, the fad6 gene is encoded by the cDNA. The cDNA encoded a 418-amino acid polypeptide of 47,727 D that displayed a high degree of sequence similarity to a delta 12 desaturase from the cyanobacterium Synechocystis. The fad6 gene exhibited less sequence homology to any known higher plant desaturase, including an endoplasmic reticulum-localized omega 6 desaturase corresponding to the Arabidopsis fad2 gene.

Amino Acid Sequence↗
PubMed1980

Morphogenesis of the bacterial division septum: identification of potential sites of division in lkyD mutants of Salmonella typhimurium.

It previously has been shown that lkyD mutants of Salmonella typhimurium form large blebs of outer membrane over the septal and polar regions of dividing cells. To determine whether the outer membrane blebs are formed over potential sites of division even in the absence of septal ingrowth, lkyD strains were studied under conditions in which ingrowth of inner membrane and murein was prevented by inactivation of the envA gene product. In aseptate filaments of the LkyD EnvA strain, outer membrane blebs occurred with the usual frequency and were preferentially located over regions where new septa were formed when cell division was subsequently permitted to resume. The results indicate that the outer membrane blebs of the LkyD strain are markers for potential sites of cell division, implying that an alteration in association of outer membrane and murein exists in these sites before the initiation of septal ingrowth. This localized change in cell envelope organization is independent of the septation-inducing effects of the envA gene product.

Cell Division↗
PubMed1985

Phasmid vectors for identification of genes by complementation of Escherichia coli mutants.

A bacteriophage lambda cloning vector was designed to facilitate the isolation of genes from procaryotic organisms by complementation of Escherichia coli mutants. This vector, lambda SE4, was constructed by attaching a very-low-copy-number replication system (from the plasmid NR1) and a spectinomycin resistance gene to the left arm of lambda 1059 (Karn et al., Proc. Natl. Acad. Sci. U.S.A. 77:5172-5176, 1980). This phasmid cloning vector is capable of growing lytically as a phage in a nonimmune host or lysogenically as a phasmid in an immune host. This phasmid utilizes the Spi- selection for insertions of DNA into the vector and has the ability to accept 2- to 19-kilobase Sau3A1, BamHI, BglII, BclI, or XhoII fragments; recombinants lysogenize immune hosts as single-copy-number selectable plasmids at 100% frequency. An E. coli library was constructed by using the initial vector lambda SE4, and clones of a number of representative genes were identified. A typical clone, lambda ant+, was shown to be readily mutagenized by a mini-Tn10 transposon. A general method for transferring cloned DNA segments onto bacteriophage lambda was developed. The method involves the use of in vivo recombination with a selection and was used to construct two derivatives of lambda SE4. Possible uses of these vectors and of the method for transferring cloned DNA onto phage lambda are discussed.

Bacteriophage lambda↗
PubMed1979

More sensitive test agar for detection of dextranase-producing oral streptococci and identification of two glucan synthesis-defective dextranase mutants of Streptococcus mutans 6715.

A more sensitive test agar was developed to detect oral microbes with relatively low dextranase activity and to identify dextranase-negative mutants. Several oral streptococci that had previously been scored as dextranase negative readily decolorized the new, blue dextran-containing medium. To assess whether dextranase plays a role in glucan synthesis by oral streptococci, various glucan synthesis-defective mutants were tested for dextranase activity on the new medium. Mutants 4 and 27, which do not cause smooth-surface caries and which synthesize more soluble glucan than their parent, Streptococcus mutans 6715-13, were markedly deficient in these dextranase activity tests.

Culture Media↗
PubMed1995

Identification and characterization of a temperature-sensitive mutant of human immunodeficiency virus type 1 by alanine scanning mutagenesis of the integrase gene.

We have created a temperature-sensitive (ts) mutant of human immunodeficiency virus type 1, using the technique of charge-cluster-to-alanine scanning mutagenesis to introduce specific changes into the integrase coding region. In the ts mutant virus, the lysine at amino acid 136 and the glutamic acid at amino acid 138 of integrase have been replaced with alanines (K136A/E138A). When K136A/E138A is synthesized at 35 degrees C, it replicates to a similar degree as wild-type virus during infection of CEM cells at 35 degrees C on the basis of syncytium formation, levels of core antigen, and reverse transcriptase activity. However, during infection at the nonpermissive temperature of 39.5 degrees C, K136A/E138A is capable of only one round of integration. Mutant virions formed at 39.5 degrees C do not integrate but are indistinguishable from wild-type virions when scored for activity of reverse transcriptase and correct expression and processing of Gag and Pol proteins. We demonstrate that the defect responsible for the ts phenotype of K136A/E138A is localized to a step after proviral formation and integrase protein synthesis but prior to particle maturation. It is the temperature at which the K136A/E138A virion is synthesized, not the temperature at which infection occurs, which determines the ability of the virus to integrate.

Alanine↗
PubMed1998

gag, vif, and nef genes contribute to the homologous viral interference induced by a nonproducer human immunodeficiency virus type 1 (HIV-1) variant: identification of novel HIV-1-inhibiting viral protein mutants.

We previously demonstrated that expression of the nonproducer F12-human immunodeficiency virus type 1 (HIV-1) variant induces a block in the replication of superinfecting HIV that does not depend on the down-regulation of CD4 HIV receptors. In order to individuate the gene(s) involved in F12-HIV-induced interference, vectors expressing each of the nine F12-HIV proteins were transfected in HIV-susceptible HeLa CD4 cells. Pools of cell clones stably producing each viral protein were infected with HIV-1, and virus release was measured in terms of reverse transcriptase activity in supernatants. We hereby demonstrate that HeLa CD4 cells expressing the F12-HIV gag, vif, or nef gene were resistant, to different degrees, to infection with T-cell-line-adapted HIV-1 strains. Conversely, expression of either the tat, rev, or vpu F12-HIV gene increased the rate of HIV release, and no apparent effects on HIV replication were observed in cells expressing either the F12-HIV vpr, pol, or env gene. No variation of CD4 exposure was detected in any of the uninfected HeLa CD4 pools. These data indicate that F12-HIV homologous viral interference is the consequence of the synergistic anti-HIV effects of Gag, Vif, and Nef proteins. Retrovirus vectors expressing F12-HIV vif or nef allowed us to further establish that the expression of each mutated protein (i) inhibits the replication of clinical HIV-1 isolates as well, (ii) impairs the infectivity of the virus released by cells chronically infected with HIV-1, and (iii) limitedly to F12-HIV Vif protein, induces HIV resistance in both vif-permissive and vif-nonpermissive cells. The levels of action of F12-HIV vif and nef anti-HIV effects were also determined. We observed that HIV virions emerging from the first viral cycle on F12-HIV vif-expressing cells, although released in unaltered amounts, had a strongly reduced ability to initiate the retrotranscription process when they reinfected parental HeLa CD4 cells. Differently, we observed that expression of F12-HIV Nef protein affects the HIV life cycle at the level of viral assembling and/or release. For the first time, an inhibitory effect on the HIV life cycle in both acutely and chronically infected cells induced by mutated Vif and Nef HIV-1 proteins is described. These genes could thus be proposed as new useful reagents for anti-HIV gene therapy.

Amino Acid Sequence↗
PubMed1998

Identification and characterization of a constitutively active STAT5 mutant that promotes cell proliferation.

STAT (signal transducers and activators of transcription) proteins are transcription factors which are activated by phosphorylation on tyrosine residues upon stimulation by cytokines. Seven members of the STAT family are known, including the closely related STAT5A and STAT5B, which are activated by various cytokines. Except for prolactin-dependent beta-casein production in mammary gland cells, the biological consequences of STAT5 activation in various systems are not clear. We applied PCR-driven random mutagenesis and a retrovirus-mediated expression screening system to identify constitutively active forms of STAT5. By this strategy, we have identified a constitutively active STAT5 mutant which has two amino acid substitutions; one is located upstream of the putative DNA binding domain (H299R), and the other is located in the transactivation domain (S711F). The mutant STAT5 was constitutively phosphorylated on tyrosine residues, localized in the nucleus, and was transcriptionally active. Expression of the mutant STAT5 partially dispenses with interleukin 3 (IL-3) as a growth stimulant of IL-3-dependent cell lines. Further analyses of the mutant STAT5 have demonstrated that both of the mutations are required for nuclear localization, efficient transcriptional activation, and induction of IL-3-independent growth of an IL-3-dependent cell line, Ba/F3, and have indicated that a molecular basis for the constitutive activation is the stability of the phosphorylated form of the mutant STAT5.

Animals↗
PubMed1989

Identification of the haplotype pattern associated with the mutant PKU allele in the Gypsy population of Wales.

Using the full length cDNA probe, the RFLP haplotype patterns at the phenylalanine hydroxylase locus have been studied in the extensive and highly consanguineous Welsh Gypsy population. The pattern associated with the mutant PKU allele is identical to haplotype 4 in the northern European population. Two children with classical PKU are homozygous for this haplotype. We have tracked the mutant allele through four generations to a great grandfather who died 22 years ago. Both affected children almost certainly have inherited a double dose of the same mutant PKU allele from one common ancestor. It should be possible to identify the specific mutation that is associated with haplotype 4 which results in the more serious form of PKU.

Alleles↗
PubMed1980

Identification and partial characterization of an Escherichia coli mutant with altered hydrogenase activity.

An Escherichia coli mutant strain with altered hydrogenase activity was isolated using a filter paper assay. This assay depends on the ability of hydrogenase-containing microorganisms to reduce methyl viologen impregnated in filter paper, producing purple-colored colonies in the presence of hydrogen. Membrane-bound and cytoplasmic hydrogenase activities of wild-type and mutant strains were compared by amperometric measurement of hydrogen production. The cytoplasmic activities of mutant and wild type were comparable. The membrane-bound activity was lower in the mutant than in the wild type. Upon addition of detergent to the membrane fraction the specific activity of the enzyme from the mutant strain increased so that it equalled that of the wild type. The mutant requires an exogenous electron acceptor for anaerobic growth providing evidence for the function of the hydrogenase in anaerobic growth.

Escherichia coli↗
PubMed2006

Identification of a novel germ line variant hotspot mutant p53-R175L in pediatric adrenal cortical carcinoma.

Hotspot mutations in the p53 tumor suppressor gene result in the disruption of DNA contact points or alter the overall structure of the protein to prevent DNA binding. When inherited, hotspot mutants are associated with Li-Fraumeni syndrome (LFS), a familial cancer predisposition. One of the most common hotspot mutations occurs at codon 175, resulting in an arginine to histidine substitution. We have identified a novel germ line variant of the 175 mutant (Arg to Leu; R175L) in a pediatric patient who developed adrenal cortical carcinoma. Surprisingly, the family is not tumor prone or associated with LFS. In vitro, the R175L mutant displayed an attenuated tumor suppressor activity in the regulation of transcription, colony formation, and apoptosis when compared with wild-type p53 and the R175H mutant. These findings suggest that p53-R175L retains sufficient activity to suppress LFS, but not adrenal cortical carcinoma. Therefore, not all hotspot mutants are functionally equivalent and the biochemical nature of the mutant may significantly influence clinical outcome. The implications of these results for genetic counseling are discussed.

Adrenal Cortex Neoplasms↗
PubMed2002

Isolation and characterization of new Saccharomyces cerevisiae mutants perturbed in nuclear pore complex assembly.

BACKGROUND: Nuclear pore complexes (NPCs) are essential for facilitated, directional nuclear transport; however, the mechanism by which ~30 different nucleoporins (nups) are assembled into NPCs is unknown. We combined a genetic strategy in Saccharomyces cerevisiae with Green Fluorescence Protein (GFP) technology to identify mutants in NPC structure, assembly, and localization. To identify such mutants, a bank of temperature sensitive strains was generated and examined by fluorescence microscopy for mislocalization of GFP-tagged nups at the non-permissive temperature. RESULTS: A total of 121 mutant strains were isolated, with most showing GFP-Nic96 and Nup170-GFP mislocalized to discrete, cytoplasmic foci. By electron microscopy, several mutants also displayed an expansion of the endoplasmic reticulum (ER). Complementation analysis identified several mutant groups with defects in components required for ER/Golgi trafficking (sec13, sec23, sec27, and bet3). By directed testing, we found that mutant alleles of all COPII components resulted in altered GFP-Nup localization. Finally, at least nine unknown complementation groups were identified that lack secretion defects. CONCLUSION: The isolation of sec mutants in the screen could reflect a direct role for vesicle fusion or the COPII coat during NPC assembly; however, only those sec mutants that altered ER structure affected Nup localization. This suggests that the GFP-Nup mislocalization phenotypes observed in these mutants were the indirect result of overproliferation of the ER and connected outer nuclear envelope. The identification of potentially novel mutants with no secretory defects suggests the distinct GFP-Nup localization defects in other mutants in the collection will provide insights into NPC structure and assembly.

Genetic Complementation Test↗
PubMed2006

[Identification of a common deletion in different curly mutants in Drosophila melanogaster].

Curly is a easily distinguishable dominant mutant wing character. The Cy mutation is the most commonly used dominant marker for the second chromosome balancers in Drosophila melanogaster, but little is known about the Cy gene. Based on known genomic and cytogenetic information, a 102 bp deletion which is located between the Genes synaptotagmin (syt) and Activin Like Protein at 23B(Alp23B) on the Drosophila melanogaster genome (release 4) had been found to be commonly contained on Cy chromosome in three different curly strains. Meanwhile, when using the deletion as a DNA marker, the result suggested that Cy homozygote be lethal in embryo period. These results will provide some helpful information to investigate molecular mechanism of curly wings in the further study.

Animals↗