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 379 records · Page 21Linked to original sources
PubMed1999

Identification and characterization of Arabidopsis mutants with reduced quenching of chlorophyll fluorescence.

Regulation of nonradiative dissipation of absorbed light energy in PSII is an indispensable process to avoid photoinhibition in plants. To dissect molecular mechanisms of the regulation, we identified Arabidopsis mutants with reduced quenching of Chl fluorescence using a fluorescence imaging system. By analyses of Chl fluorescence induction pattern in the light and quantum yield of both photosystems, 37 mutants were classified into three groups. The first group was characterized by an extremely high level of minimum Chl fluorescence at the open PSII center possibly due to a defect in PSII. Mutants with significant reduction in the nonphotochemical quenching formation but not in quantum yield of both photosystems were classified into the second group. Mutants in the third group showed reduction in quantum yield of both photosystems possibly due to a defect in the electron transport activity. Mutants in the second and third groups were further characterized by light intensity dependence of Chl fluorescence parameters and steady state redox level of P700.

Arabidopsis↗
PubMed2004

Directed enzyme evolution guided by multidimensional analysis of substrate-activity space.

The directed evolution of protein function frequently involves identification of mutants with improved properties from a population of variants obtained by mutagenesis. The selection of clones to parent the subsequent generation is crucial to the continued creation of superior progeny. In the present study, multivariate analysis guided the evolution of human glutathione transferase (GST) T1-1 to 65-fold enhanced alkyltransferase activity. Six alternative substrates monitored the substrate-activity space that characterized a mutant library of enzymes, obtained by recombination of DNA and heterologous expression in Escherichia coli. A subset of mutants was identified by their proximity in the targeted region of six-dimensional factor space. DNA from these mutants was recombined to create a new generation of GST variants from which an improved enzyme was isolated. The multidimensional cluster analysis is applicable to quantitative properties in any population of molecules undergoing evolution and can guide the tailoring of proteins, nucleic acids and other chemical structures to novel and improved functions.

Alkyl and Aryl Transferases↗
PubMed2005

Elevated intraocular pressure and transgenic applications in the mouse.

Exploitation of the mouse as a model for glaucoma has been advanced by the development of methods to measure mouse intraocular pressure (IOP), identification of mutant mouse strains in which IOP spontaneously increases, and the development of treatments to induce elevated IOP. These developments enable investigations that directly test the influence of specific gene product alterations on the progression of glaucoma. Moreover, new transgenic mouse models have been produced with genetic mutations that parallel human gene mutations that have been linked to the onset of glaucoma. These new mouse models and technologies have potential for uncovering the biological basis of glaucoma as well as for evaluating new treatments.

Animals↗
PubMed1993

Identification of a human cytomegalovirus mutant in the pp150 matrix phosphoprotein gene with a growth-defective phenotype.

Following amplification by PCR of a portion of the matrix phosphoprotein pp150 gene, electrophoretic analysis revealed the simultaneous presence of two viral variants of human cytomegalovirus in the blood of a heart transplant recipient. Repeated denaturation-annealing cycles during the amplification reaction led to the formation of heteroduplex molecules with altered electrophoretic mobility. Sequence analysis of the amplification products showed the presence of a viral variant carrying an in-frame three nucleotide deletion, which caused the absence of an aspartic acid in the corresponding protein. Attempts to plaque-purify the deletion mutant were unsuccessful, suggesting that the variant was growth-defective.

Base Sequence↗
PubMed1996

Mutational analysis of the influenza virus A/Victoria/3/75 PA protein: studies of interaction with PB1 protein and identification of a dominant negative mutant.

The RNA polymerase activity and PB1 binding of influenza virus PA mutants were studied using an in vivo-reconstituted polymerase assay and a two hybrid system. Deletions covering the whole PA protein abolished polymerase activity, but the deletion of the 154 N-terminal amino acids allowed PB1 binding, indicating that the PA protein N terminus is not absolutely required for this interaction. Further internal or C-terminal deletions abolished PB1 interaction, suggesting that most of the protein is involved in this association. As a novel finding we showed that a single amino acid insertion mutant, PAI672, was responsible for a temperature-sensitive phenotype. Mutant PAS509, which had a serine insertion at position 509, bound to PB1 like wild-type PA but did not show any polymerase activity. Over-expression of PAS509 interfered with the polymerase activity of wild-type PA, identifying PAS509 as a dominant negative mutant.

Animals↗
PubMed2002

Identification and biochemical characterization of mutants in the proanthocyanidin pathway in Arabidopsis.

Proanthocyanidin (PA), or condensed tannin, is a polymeric flavanol that accumulates in a number of tissues in a wide variety of plants. In Arabidopsis, we found that PA precursors (detected histochemically using OsO(4)) accumulate in the endothelial cell layer of the seed coat from the two-terminal cell stage of embryo development onwards. To understand how PA is made, we screened mature seed pools of T-DNA-tagged Arabidopsis lines to identify mutants defective in the synthesis of PA and found six tds (tannin-deficient seed) complementation groups defective in PA synthesis. Mutations in these loci disrupt the amount (tds1, tds2, tds3, tds5, and tds6) or location and amount of PA (tds4) in the endothelial cell layer. The PA intermediate epicatechin has been identified in wild type and mutants tds1, tds2, tds3, and tds5 (which do not produce PA) and tds6 (6% of wild-type PA), whereas tds4 (2% of wild-type PA) produces an unidentified dimethylaminocinnamaldehyde-reacting compound, indicating that the mutations may be acting on genes beyond leucoanthocyanidin reductase, the first enzymatic reduction step dedicated to PA synthesis. Two other mutants were identified, an allele of tt7, which has a spotted pattern of PA deposition and produces only 8% of the wild-type level of type PA as propelargonidin, and an allele of tt8 producing no PA. Spotted patterns of PA deposition observed in seed of mutants tds4 and tt7-3 result from altered PA composition and distribution in the cell. Our mutant screen, which was not exhaustive, suggests that the cooperation of many genes is required for successful PA accumulation.

Alleles↗
PubMed1988

Identification and characterization of TnphoA mutants of Salmonella that are unable to pass through a polarized MDCK epithelial cell monolayer.

Surface protein mutants of the invasive Salmonella species, S. choleraesuis, were generated using the transposon TnphoA. 626 alkaline phosphatase (PhoA+) fusion mutants were identified and screened for their ability to pass through (transcytose) polarized epithelial monolayers of Madin Darby canine kidney (MDCK) cells grown on membrane filters. Forty two mutants were unable to pass through this barrier. All of these transcytosis mutants were unable to adhere to or invade MDCK monolayers, yet these mutations were not in the genes encoding type 1 pili or mannose-resistant haemagglutination (MRHA). These transcytosis mutants could be grouped into six classes. Class 1 mutants had altered lipopolysaccharide (LPS) O side-chain structures while Class 2 mutants had defects in their LPS core. Mutants belonging to Classes 5 and 6 did not decrease the transepithelial electrical resistance of polarized MDCK cell monolayers, in contrast to the parental strain and the other mutants (Classes 1, 2, 3 and 4). Mutants belonging to Class 1 were less virulent in mice, while Class 2 (defective core) and Classes 4 and 5 (normal LPS) mutant strains were avirulent in mice. Mutants from Classes 3 and 6 were as virulent in mice as S. choleraesuis. These results suggest that the ability to pass through epithelial barriers may be an important virulence characteristic of Salmonella. These data indicate that bacterial adherence, internalization and monolayer transcytosis are closely linked events. It was also demonstrated that a mutant with decreased rates of intracellular replication still passed through the monolayer at rates similar to wild-type S. choleraesuis.

Alkaline Phosphatase↗
PubMed1993

Organization of the genes necessary for hydrogenase expression in Rhodobacter capsulatus. Sequence analysis and identification of two hyp regulatory mutants.

A 25 kbp DNA fragment from the chromosome of Rhodobacter capsulatus B10 carrying hydrogenase (hup) determinants was completely sequenced. Coding regions corresponding to 20 open reading frames were identified. The R. capsulatus hydrogenase-specific gene (hup and hyp) products bear significant structural identity to hydrogenase gene products from Escherichia coli (13), from Rhizobium leguminosarum (16), from Azotobacter vinelandii (10) and from Alcaligenes eutrophus (11). The sequential arrangement of the R. capsulatus genes is: hupR2-hupU-hypF-hupS-hupL-hupM-hu pD-hupF-hupG-hupH-hupJ-hupK-hypA- hypB-hupR1- hypC-hypD-hypE-ORF19-ORF20, all contiguous and transcribed from the same DNA strand. The last two potential genes do not encode products that are related to identified hydrogenase-specific gene products in other species. The sequence of the 12 R. capsulatus genes underlined above is presented. The mutation site in two of the Hup- mutants used in this study, RS13 and RCC12, was identified in the hypF gene (deletion of one G) and in the hypD gene (deletion of 54 bp), respectively. The hypF gene product shares 45% identity with the product of hydA from E. coli and the product of hypF from R. leguminosarum. Those products present at their N-terminus a Cys arrangement typical of zinc-finger proteins. The G deletion in the C-terminal region of hypF in the RS13 mutant prevented the expression of a hupS::lacZ translational fusion from being stimulated by H2 as it is observed in the wild-type strain B10. It is inferred that the HypF protein is a factor involved in H2 stimulation of hydrogenase expression.

Amino Acid Sequence↗
PubMed2004

Screening for synthetic lethal mutants in Escherichia coli and identification of EnvC (YibP) as a periplasmic septal ring factor with murein hydrolase activity.

Bacterial cytokinesis is driven by the septal ring apparatus, the assembly of which in Escherichia coli is directed to mid-cell by the Min system. Despite suffering aberrant divisions at the poles, cells lacking the minCDE operon (Min(-)) have an almost normal growth rate. We developed a generally applicable screening method for synthetic lethality in E. coli, and used it to select for transposon mutations (slm) that are synthetically lethal (or sick) in combination with DeltaminCDE. One of the slm insertions mapped to envC (yibP), proposed to encode a lysostaphin-like, metallo-endopeptidase that is exported to the periplasm by the general secretory (Sec) pathway. Min(-) EnvC(-) cells showed a severe division defect, supporting a role for EnvC in septal ring function. Accordingly, we show that an EnvC-green fluorescent protein fusion, when directed to the periplasm via the twin-arginine export system, is both functional and part of the septal ring apparatus. Using an in-gel assay, we also present evidence that EnvC possesses murein hydrolytic activity. Our results suggest that EnvC plays a direct role in septal murein cleavage to allow outer membrane constriction and daughter cell separation. By uncovering genetic interactions, the synthetic lethal screen described here provides an attractive new tool for studying gene function in E. coli.

Cell Division↗
PubMed2004

Identification of novel dominant INO2c mutants with an Opi- phenotype.

The INO2 gene of Saccharomyces cerevisiae is required for derepression of the phospholipid biosynthetic genes in response to inositol depletion. Conversely, the OPI1 gene is required for repression in response to inositol supplementation. Results of an in vitro assay have led to a model in which Opi1p interacts with Ino2p. However, there is no in vivo evidence to support this model. Additionally, most of the previously isolated ino2 mutants offer little insight into this model. Here, we report the isolation of a new class of dominant mutations in the INO2 gene, which yield constitutive expression of a target gene (i.e. an Opi(-) mutant phenotype). Two mutations reside in a region of the Ino2p required for interaction with Opi1p in vitro. Three other mutations are at the amino-terminus in a transcriptional activation domain.

Basic Helix-Loop-Helix Proteins↗
PubMed1997

Frequency and clinico-pathological associations of ras mutations in colorectal cancer in the Victorian population.

BACKGROUND: Mutations in the oncogene ras occur in 20-50% of colorectal cancers. The presence of these mutations allows screening tests to be developed based on the identification of mutant DNA in cells derived from cancers. A study of the prevalence and clinicopathological associations of ras mutations was undertaken. METHODS: The frequency of mutations in codons 12 and 13 of the K-ras gene was investigated in 103 colorectal carcinomas using restriction fragment length polymorphism. RESULTS: Mutations were detected in 32% (33/103) of the tumours, predominantly in codon 12 (25/33). No mutations were detected in normal-appearing mucosa from the same patients. CONCLUSIONS: Analysis of the frequency of ras mutations compared with various independent clinical variables revealed a sex-linked relationship between the presence of a ras mutation and nodal status but no correlation with any other clinical parameter was found. The findings suggest that screening tests based on ras mutation detection may lack sensitivity because of the presence of mutations in only 32% of tumours.

Aged↗
PubMed1994

Identification of genetic defect of an epilepsy: strategies for therapeutic advances.

Advances in molecular genetics and molecular biology are transforming the biology of human disease. Cures for diseases previously refractory to all treatments have become the reality for some disorders and the legitimate promise for others. In the case of the epilepsies, identification of mutant genes underlying familial epilepsies may lead to a new pharmacology, through the development of in vitro expression systems permitting rapid search for novel drugs, creation of highly specific animal models based on expression of the precise mutation, and correction of disease phenotype by introducing novel and highly specific genetic information into the person with epilepsy.

Adult↗
PubMed2001

Construction of mini-Tn5cyaA' and its utilization for the identification of genes encoding surface-exposed and secreted proteins in Bordetella bronchiseptica.

A mini-Tn5 transposon derivative, mini-Tn5cyaA', has been constructed. It contains a promoter-less and ribosome binding site-deficient reporter gene, encoding the catalytic domain of Bordetella pertussis adenylate cyclase toxin (CyaA'). We used this system to mutagenize B. bronchiseptica and we developed a screen for identification of mutants containing cyaA' translational fusions. This system was used to identify B. bronchiseptica genes that encode surface-exposed and secreted proteins.

Adenylate Cyclase Toxin↗
PubMed1975

Genetic regulation: the Lac control region.

The nucleotide sequence of the lac promoter-operator region has been determined. The 122 base pairs comprising this region include the recognition sites for RNA polymerase, the positive regulatory protein, CAP, and the negative regulatory protein, the repressor. Identification of mutant variants of the sequence combined with the in vitro biochemical studies of others has allowed us to tentatively identify the recognition site for each of these proteins, and to suggest how CAP might act at a distance to affect the interaction of RNA polymerase with the promoter.

Base Sequence↗
PubMed2000

The osteoblast: a sophisticated fibroblast under central surveillance.

The study of the biology of osteoblasts, or bone-forming cells, illustrates how mammalian genetics has profoundly modified our understanding of cell differentiation and physiologic processes. Indeed, genetic-based studies over the past 5 years have revealed how osteoblast differentiation is controlled through growth and transcription factors. Likewise, the recent identification, using mutant mouse models, of a central component in the regulation of bone formation expands our understanding of the control of bone remodeling. This regulatory loop, which involves the hormone leptin, may help to explain the protective effect of obesity on bone mass in humans. In addition, it provides a novel physiologic concept that may shed light on the etiology of osteoporosis and help to identify new therapeutic targets.

Animals↗
PubMed1992

Utilization of 3-chloro-2-methylbenzoic acid by Pseudomonas cepacia MB2 through the meta fission pathway.

Pseudomonas cepacia MB2 grew on 3-chloro-2-methylbenzoate as a sole carbon source by metabolism through the meta fission pathway with the subsequent liberation of chloride. meta pyrocatechase activity in cell extracts was induced strongly by 3-chloro-2-methylbenzoate, but not by nongrowth analogs 4- or 5-chloro-2-methylbenzoate. Although rapid turnover of metabolites precluded direct identification, a mutant strain MB2-G5 lacking meta pyrocatechase activity produced 4-chloro-3-methylcatechol when incubated with 3-chloro-2-methylbenzoate. The catecholic product, confirmed by nuclear magnetic resonance and mass spectral analyses, produced a transient meta fission product (lambda max = 391 nm) from cell extracts of the wild-type MB2 strain. Further confirmation of meta pyrocatechase activity was noted by conversion of 4-chlorocatechol to 2-hydroxy-5-chloromuconic semialdehyde, which was not further metabolized. In contrast to 3-chlorocatechol, which was not metabolized and is known to generate suicidal products, 4-chlorocatechols do not generate acyl halides. Thus, further metabolism of the ring fission products is governed in strain MB2 by their suitability as substrates for the hydrolase.

Burkholderia cepacia↗
PubMed1973

Temperature-sensitive nonsense mutations in essential genes of Escherichia coli.

Cells containing nonsense mutations in essential genes have been isolated in a strain of Escherichia coli that carried the su4(ts) gene which specifies a temperature-sensitive tyrosine transfer ribonucleic acid. Such cells are unable to form colonies at temperatures which inactivate this suppressor transfer ribonucleic acid. A screening procedure for the identification of mutants that carry temperature-sensitive nonsense mutations in essential genes is described, and certain properties of two such mutants are reported.

Chromosome Mapping↗