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At least 235 records · Page 13Linked to original sources
PubMed2006

A genetic dissection of Aip1p's interactions leads to a model for Aip1p-cofilin cooperative activities.

Actin interacting protein 1 (Aip1p) and cofilin cooperate to disassemble actin filaments in vitro and are thought to promote rapid turnover of actin networks in vivo. The precise method by which Aip1p participates in these activities has not been defined, although severing and barbed-end capping of actin filaments have been proposed. To better describe the mechanisms and biological consequences of Aip1p activities, we undertook an extensive mutagenesis of AIP1 aimed at disrupting and mapping Aip1p interactions. Site-directed mutagenesis suggested that Aip1p has two actin binding sites, the primary actin binding site lies on the edge of its N-terminal beta-propeller and a secondary actin binding site lies in a comparable location on its C-terminal beta-propeller. Random mutagenesis followed by screening for separation of function mutants led to the identification of several mutants specifically defective for interacting with cofilin but still able to interact with actin. These mutants suggested that cofilin binds across the cleft between the two propeller domains, leaving the actin binding sites exposed and flanking the cofilin binding site. Biochemical, genetic, and cell biological analyses confirmed that the actin binding- and cofilin binding-specific mutants are functionally defective, whereas the genetic analyses further suggested a role for Aip1p in an early, internalization step of endocytosis. A complementary, unbiased molecular modeling approach was used to derive putative structures for the Aip1p-cofilin complex, the most stable of which is completely consistent with the mutagenesis data. We theorize that Aip1p-severing activity may involve simultaneous binding to two actin subunits with cofilin wedged between the two actin binding sites of the N- and C-terminal propeller domains.

Actin Cytoskeleton↗
PubMed2005

Microarray analysis of zebrafish cloche mutant using amplified cDNA and identification of potential downstream target genes.

Zebrafish is an excellent model organism for studying vertebrate development and human disease. With the availability of increased numbers of zebrafish mutants and microarray chips, gene expression profiling has become a powerful tool for identification of downstream target genes perturbed by a specific mutation. One of the obstacles often encountered, however, is to isolate large numbers of zebrafish mutant embryos that are indistinguishable in morphology from the wild-type siblings for microarray analysis. Here, we report a method using amplified cDNA derived from five embryos for gene expression profiling of the 18-somite zebrafish cloche (clo) mutant, in which development of hematopoietic and endothelial lineages is severely impaired. In total, 31 differentially expressed target genes are identified, of which 13 have not been reported previously. We further determine that of these 13 new targets, 8 genes, including coproporphyrinogen oxidase (cpo), carbonic anhydrase (cahz), claudin g (cldn g), zinc-finger-like gene 2 (znfl2), neutrophil cytosol factor 1 (ncf1), matrix metalloproteinase 13 (mmp13), dual specificity phosphatase 5 (dusp5), and a novel gene referred as zebrafish vessel-specific gene 1 (zvsg1) are predominantly expressed in hematopoietic and endothelial cells. Comparative analysis demonstrates that this method is comparable and complementary to that of the conventional approach using unamplified sample. Our study provides valuable information for studying hematopoiesis and vessel formation. The method described here offers a powerful tool for gene expression profiling of zebrafish mutants in general.

Animals↗
PubMed1995

Methanol oxidation mutants in Methylobacterium extorquens AM1: identification of new genetic complementation groups.

Two-hundred-and-eight new Methylobacterium extorquens AM1 methanol oxidation (Mox) mutants were isolated and placed into complementation groups. Complementation analyses identified new Mox groups in the Mxb and Mxc loci and at a new locus, Mxd. Thirty-seven mutants at the Mxb locus were divided into MxbM and MxbD complementation groups on the basis of their complementation pattern. Twenty-nine mutants at the Mxc locus fell into three complementation groups, MxcB, MxcQ and MxcE. The direction of transcription for genes at this locus could be inferred from the subclones. Eighteen of the new mutants were not complemented by previously isolated M. extorquens AM1 clones but were complemented by two new overlapping clones. This locus was called Mxd and the mutants fell into two complementation groups, MxdR and MxdS. Immunoblots from all these mutant classes showed that all of the Mxb and Mxc strains had substantially reduced levels of MxaF (large subunit of methanol dehydrogenase) and cytochrome cL, compared to the wild-type. These mutants, particularly the Mxb mutants, also had elevated levels of cytochrome c-553. These results are consistent with a role for the MxbMD and MxcBQE complementation groups in the regulation of expression of mxaF. The MxdR and MxdS mutants had normal levels of MxaF and both c-type cytochromes.

Genes, Bacterial↗
PubMed1995

Saturation mutagenesis of His114 of EcoRI reveals relaxed-specificity mutants.

EcoRI recognizes and cleaves DNA at GAATTC sites and is one of the best characterized sequence-specific restriction endonucleases (ENases). In previous studies, an EcoRI mutant, which exhibited relaxed substrate specificity and cleaved both canonical and EcoRI star sites, was isolated. This mutant enzyme has Tyr instead of His114. Here, we subjected residue 114 of the EcoRI ENase to saturation mutagenesis. The resulting mutant enzymes were characterized both in vivo and in vitro, resulting in the identification of mutants with canonical (H114K, Q, D, I) or relaxed (H114Y, F, S, T) specificity, as well as one mutant with severely impaired activity (H114P). In the X-ray structure of an EcoRI-substrate complex, His114 is located between the catalytic and recognition regions of EcoRI and may directly contact the DNA phosphate backbone. Based on our genetic and biochemical findings and the X-ray structure, we propose that His114 participates in substrate recognition and catalysis, either directly, via protein-DNA interactions, or indirectly, by mediating conformational changes that trigger DNA cleavage in response to substrate recognition.

Amino Acid Sequence↗
PubMed1999

Identification of a novel surface mutant of hepatitis B virus in a seronegative chronic liver disease patient.

Hepatitis B virus (HBV) with mutations in the envelope proteins can emerge during natural infections, vaccinations or interferon therapy and appears occasionally to escape virus elimination or detection. The implications of such mutations at the molecular level are often obscure. We report the identification of a new surface mutant of HBV. This mutant was identified, and isolated from a chronic liver disease patient, negative for HBsAg as well as other serological markers but positive for HBV DNA. Several mutations were observed in the surface antigen gene out of which a Thr118-Ala118 change was predicted to have a destabilizing effect on the structural integrity of the 'a' determinant and also alter the antigenicity profile of the mutant HBsAg. Besides a RNA hairpin loop was predicted for the transcript generated by the small surface protein of this mutant, which could have an inhibitory effect at the translational level. These observations thus indicate that mutations in the surface gene could lead to a considerable decrease or complete absence of properly folded surface antigen which in turn could explain the absence of reactive HBsAg in the serum of the patient.

Amino Acid Substitution↗
PubMed1986

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis.

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

Adenoviruses, Human↗
PubMed1997

Screening of TnphoA mutants of Vibrio cholerae O139 for identification of antigens involved in colonisation.

A new serogroup of Vibrio cholerae non-O1, designated as O139, has emerged causing cholera-like disease among adults. Laboratory and field studies clearly show that there is no cross-protection between O1 and O139 pathogenic strains. Since colonisation of the intestine is a most important step in the pathogenesis of cholera caused by O1 strains and colonising antigens are known to be protective, investigation of the colonising antigens of O139 strain was initiated. By TnphoA mutagenesis, mutants were generated with insertions in the genome encoding membrane spanning or secretory proteins. Screening of the mutants for adherence to rabbit intestinal surface and colonisation in 5-day-old mice resulted in the identification of mutant clones, which were less adhesive than was the wild-type parent strain and which could not efficiently colonise the gut. Such non-colonising strains were attenuated in virulence. Analysis of the proteins by SDS-PAGE revealed that the non-colonising mutants did not express a 40-kDa outer-membrane protein.

Animals↗
PubMed1995

Characterisation of a gain-of-function mutant of poly(ADP-ribose) polymerase.

In order to examine the structure-function relationship of the poly (ADP-ribose) polymerase (PARP) catalytic domain, potential active-site residues in the catalytic domain have previously been described. Here, we have used mutagenesis with hydroxylamine to generate a random library of PARP mutants. The identification, overproduction in insect cells, purification and characterization of a gain-of-function mutant (L713F) is described. We show that the kcat of this mutant is increased over nine times compared to the wild-type enzyme; the Km for NAD+ is unchanged. The size and the branching structure of the ADP-ribose polymers are similar in both the wild-type and the mutant enzyme. This mutation may have an allosteric effect on the catalytic site and could be useful in analyzing the consequences of poly ADP-ribose overproduction in vivo on cell survival following DNA damage.

Allosteric Regulation↗
PubMed2003

Genetic identification of a female partial-sterile mutant in soybean.

We report here the genetic identification of a female partial-sterile mutant derived from soybean mutant L67-3483. L67-3483, which originated from the cultivar Clark after X-ray irradiation, is male and female fertile. All F1 plants in reciprocal pollinations of L67-3483 with 'Clark', 'Minsoy', or 'BSR 101' were female partial sterile. Partial sterility is expressed in the heterozygous condition at a single locus and upon self-pollination this locus exhibits a 1:1 segregation pattern. This locus is located on the terminus of the soybean molecular linkage group D1b+W, between simple sequence repeat (SSR) markers Satt157 and Satt266, and is linked to each by 5.3 and 1.2 cM, respectively. This gene is transmitted through both female and male gametes and there was no segregation distortion of SSR markers linked to this gene. We concluded that this female partial-sterile gene is a new mutation class, and differs from the previously reported mutation classes in soybean, i.e., sporophytic mutation, gametophytic female-specific mutation, and general gametophytic mutation. Restriction of recombination around the mutant gene suggested that this gene is located near or within (a) small inversion(s) or adjacent to (a) chromosomal deletion(s).

Chromosome Mapping↗
PubMed1996

Identification of naturally occurring dominant negative mutants of thyroid hormone alpha 1 and beta 1 receptors in a human hepatocellular carcinoma cell line.

To understand the function of thyroid hormone nuclear receptors (TRs) in human hepatocellular carcinoma cells (HCC), we characterized the hormone binding and transactivational activity of TRs in a HCC cell line, J7. TR alpha 1 (J7-TR alpha 1) and TR beta 1 (J7-TR beta 1) complementary DNAs were cloned from this cell line, and the binding activity to the hormone response elements (TREs) and to the thyroid hormone, 3,3',5-triiodo-L-thyronine (T3) of the expressed TR proteins were evaluated. J7-TR alpha 1 and J7-TR beta 1 bound to TREs similarly as the TRs isolated from other tissues. However, J7-TR alpha 1 did not bind to T3, and J7-TR beta 1 bound to T3 with only about 10% the affinity of the wild-type TR beta 1. Sequencing of the complementary DNAs shows a single Met259Ile mutation in J7-TR alpha 1 and Met334Val in J7-TR beta 1. Using reporters containing TREs, we found that J7-TR alpha 1 and J7-TR beta 1 had virtually lost their transactivational activity. Moreover, these two mutants inhibited the transactivational activity of the wild-type TRs by a dominant negative effect not only on the transfected TRs, but also on endogenous TRs in other two HCC cell lines, SK-Hep-1 and HepG2. The potency of the dominant negative effect of these two mutants inversely correlated with the expression level of endogenous TRs. The present studies identified two novel naturally occurring TR mutants that have potent dominant negative action. The identification of both the alpha and beta dominant negative mutants in J7 made this cell line a useful model system to further understand the molecular mechanism of the dominant negative action of TR mutants.

Carcinoma, Hepatocellular↗
PubMed1994

Isolation and characterization of DNA-binding mutants of a plasmid replication initiation protein utilizing an in vivo binding assay.

An in vivo screen was developed for the identification of mutants of the RK2 replication initiation protein, TrfA, that were altered in their binding to the iterons within the plasmid RK2 origin of replication. This assay is based on an antibiotic selection system originally described by Elledge, Sugiono, Guarente, and Davis (Proc. Natl. Acad. Sci. USA86, 3689-3693, 1989) for the isolation in vivo of genes encoding sequence-specific DNA-binding proteins. A TrfA-specific binding site consisting of two 17-bp iterons separated by a nonrandom 6-bp spacer was placed 3' to a strong constitutive promoter. This promoter-iteron fragment was then inserted into the assay vector convergent to the aadA gene such that an increased level of spectinomycin resistance by the Escherichia coli host was dependent on the binding of wild-type TrfA protein to the binding site. The in vivo system was used to specifically isolate TrfA mutants which were either defective in binding or capable of effecting increased levels of spectinomycin resistance as compared to wild-type TrfA. The defective TrfA mutants isolated by this screen were purified and found to be considerably less effective in DNA binding by in vitro gel mobility shift assays. The map location was determined for these six defective TrfA mutants. Each of the mutations consisted of a single base change and mapped within codons extending over a 162 amino acid sequence. All of the mutants which were capable of effecting increased levels of spectinomycin resistance in the in vivo DNA-binding assay also showed some alteration in RK2 replication in vivo with most of the mutants having a copy-up phenotype similar to previously isolated TrfA mutants able to maintain an eight-iteron RK2 origin plasmid at a higher copy number.

Amino Acid Sequence↗
PubMed2002

Rapid structural phenotyping of plant cell wall mutants by enzymatic oligosaccharide fingerprinting.

Various biochemical, chemical, and microspectroscopic methods have been developed throughout the years for the screening and identification of mutants with altered cell wall structure. However, these procedures fail to provide the insight into structural aspects of the cell wall polymers. In this paper, we present various methods for rapidly screening Arabidopsis cell wall mutants. The enzymatic fingerprinting procedures using high-performance anion-exchange-pulsed-amperometric detection liquid chromatography, fluorophore-assisted carbohydrate electrophoresis, and matrix-assisted laser-desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) were exemplified by the structural analysis of the hemicellulose xyloglucan. All three techniques are able to identify structural alterations of wall xyloglucans in mur1, mur2, and mur3, which in comparison with the wild type have side chain defects in their xyloglucan structure. The quickest analysis was provided by MALDI-TOF MS. Although MALDI-TOF MS per se is not quantitative, it is possible to reproducibly obtain relative abundance information of the various oligosaccharides present in the extract. The lack of absolute quantitation by MALDI-TOF MS was compensated for with a xyloglucan-specific endoglucanase and simple colorimetric assay. In view of the potential for mass screening using MALDI-TOF MS, a PERL-based program was developed to process the spectra obtained from MALDI-TOF MS automatically. Outliers can be identified very rapidly according to a set of defined parameters based on data collected from the wild-type plants. The methods presented here can easily be adopted for the analysis of other wall polysaccharides. MALDI-TOF MS offers a powerful tool to screen and identify cell wall mutants rapidly and efficiently and, more importantly, is able to give initial insights into the structural composition and/or modification that occurs in these mutants.

Arabidopsis↗
PubMed1972

Direct induction in wild-type Neurospora crassa of mutants (qa-1 c ) constitutive for the catabolism of quinate and shikimate.

A color test has been developed for the selection and identification of mutants in Neurospora crassa, constitutive for the three normally inducible enzymes which convert quinate to protocatechuate. By this means seven such mutants have been recovered after ultra violet irradiation of wild type and have been shown to be allelic (or very closely linked) to the qa-1(C) mutants previously obtained by other means. Thus, the regulation of the synthesis of these three catabolic enzymes is indicated to be under the control of a single gene, qa-1(+).

Alcohol Oxidoreductases↗
PubMed1995

Isolation of the polar and lateral flagellum-defective mutants in Vibrio alginolyticus and identification of their flagellar driving energy sources.

Vibrio alginolyticus has two types of flagella (polar and lateral) in one cell. We isolated mutants with only a polar flagellum (Pof+ Laf-) or only lateral flagella (Pof- Laf+). Using these mutants, we demonstrated that the energy sources of the lateral and polar flagellar motors in V. alginolyticus are H+ and Na+ motive forces, respectively, as in the related species V. parahaemolyticus.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed2006

Seeking completeness in bacterial mutant hunts.

The identification of most or all of the genetic functions that are required for a particular biological process could be achieved through phenotypic studies of high genome-coverage mutant collections. Technologies for creating such collections, in the form of mixed populations or individually arrayed sequence-defined mutants, are now available for numerous bacterial species. The analysis of mixed mutant collections using microarray-based detection procedures appears to be particularly effective in identifying functions required for complex processes such as virulence. The phenotypic analysis of sequence-defined mutant libraries provides a virtually complete identification of nonessential genes required for processes for which suitable screens can be devised. Such libraries also serve as a source of individual mutants for examining the biological relevance of gene associations revealed by transcriptional profiling or homology.

Bacteria↗
PubMed2001

Interruption of the cydB locus in Brucella abortus attenuates intracellular survival and virulence in the mouse model of infection.

Brucellosis is characterized by abortion in ruminants and a protracted undulant fever in humans, which often results in severe pathological manifestations. Scant information exists about the molecular mechanisms employed by Brucella abortus to combat host defenses or to persist and replicate within host cells. Transposon (Tn5) mutagenesis of B. abortus and the subsequent screening of mutants for sensitivity to killing in murine macrophages and in the mouse model led to the identification of mutants which were severely attenuated for intracellular survival. One group of mutants was interrupted in cydB, a gene that is part of the cydAB operon encoding cytochrome bd oxidase, which catalyzes an alternate terminal electron transport step in bacterial respiration. The elevated affinity for molecular oxygen of this enzyme in Escherichia coli has suggested that it is involved in the protection of sensitive enzymatic activities such as those of hydrogenases and nitrogenases from damage. B. abortus cydB::Tn5 strains exhibited heightened sensitivity to the respiratory inhibitors zinc and azide, highly reactive oxygen species such as hydrogen peroxide, low pH, and attenuated virulence in the mouse model of infection. Virulence was restored by an intact copy of cydAB or by B. abortus genes encoding the oxidative radical-scavenging enzyme Cu/Zn superoxide dismutase or catalase. These results suggest a bifunctional role for the products of the cydAB operon, both in preventing the buildup of oxidative free radicals and in detoxifying the intracellular compartment, thus indicating the importance of these products in preventing intracellular destruction. Intracellular conditions that favor expression of the cydAB operon are under investigation and may be linked to the acid sensitivity also observed in this strain.

Animals↗
PubMed2002

[Construction of Saccharopolyspora erythraea M synthesizing a novel ketolide 3-deoxy-3-oxo-erythronolide B].

Genetic engineering on macrolide antibiotics was a new field in recent years and more than 100 novel polyketides had been produced until then. Using genomic DNA of S. erythraea A226 as a template, about 3.2 kb DNA fragment without KR6 domain was amplified by overlapping PCR technique and cloned into pWHM3 carrier, which resulted in the construction of homologous recombinant plasmid pWHM2201. Plasmid pWHM2201 was introduced into protoplasts of S. erythraea A226 by PEG-mediated transformation and integrated into the gene locus for erythromycin biosynthesis. After integrants grew for two generations on R3M media without Tsr, they were protoplasted and grown on R3M plates. By PCR identification, 8 mutants without KR6 domain were selected out and named S. erythraea M(1-8). With the identification of mass spectrometry, it was proved that S. erythraea M1 synthesized a novel ketolide compound 3-deoxy-3-oxo-erythronolide B.

Anti-Bacterial Agents↗
PubMed1971

Phenotypic reversion in some early blocked sporulation mutants of Bacillus subtilis: isolation and phenotype identification of partial revertants.

Single-step mutants of Bacillus subtilis blocked at stage zero of sporulation (spoO mutants) are pleiotropic. They are impaired in several traits, including increased sensitivity to polymyxin B, when compared to the wild type. Revertants for one or another of the traits associated in this pleiotropy have been isolated and classified according to their phenotypes. Attention was centered on partial revertants (spoO still), of which at least three classes could be recognized. Class I partial revertants recovered a high level of resistance to polymyxin and, in a constant order, a variable number of the other traits; some kind of hierarchy is thus revealed to exist among the traits affected in the original pleiotropy. Partial revertants in the other two classes are still hypersensitive to polymyxin. In class II revertants, ability to excrete exoenzymes and antibiotic(s) is recovered to various extents. Only inducibility of nitrate reductase by nitrate is recovered in class III mutants. The possible significance of these observations is discussed.

Anti-Bacterial Agents↗