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At least 523 records · Page 29Linked to original sources
PubMed1996

Isolation and characterization of two new negative regulatory mutants for nitrate assimilation in Chlamydomonas reinhardtii obtained by insertional mutagenesis.

Plasmid DNA carrying either the nitrate reductase (NR) gene or the argininosuccinate lyase gene as selectable markers and the corresponding Chlamydomonas reinhardtii mutants as recipient strains have been used to isolate regulatory mutants for nitrate assimilation by insertional mutagenesis. Identification of putative regulatory mutants was based on their chlorate sensitivity in the presence of ammonium. Among 8975 transformants, two mutants, N1 and T1, were obtained. Genetic characterization of these mutants indicated that they carry recessive mutations at two different loci, named Nrg1 and Nrg2. The mutation in N1 was shown to be linked to the plasmid insertion. Two copies of the nitrate reductase plasmid, one of them truncated, were inserted in the N1 genome in inverse orientation. In addition to the chlorate sensitivity phenotype in the presence of ammonium, these mutants expressed NR, nitrite reductase and nitrate transport activities in ammonium-nitrate media. Kinetic constants for ammonium (I4C-methylammonium) transport, as well as enzymatic activities related to the ammonium-regulated metabolic pathway for xanthine utilization, were not affected in these strains. The data strongly suggest that Nrg1 and Nrg2 are regulatory genes which specifically mediate the negative control exerted by ammonium on the nitrate assimilation pathway in C. reinhardtii.

Animals↗
PubMed1996

Mutations in the amino-terminal domain of the human poly(ADP-ribose) polymerase that affect its catalytic activity but not its DNA binding capacity.

Poly-ADP ribosylation of nuclear proteins is activated when poly(ADP-ribose) polymerase (PARP), a nuclear zinc-finger enzyme, binds to single-strand DNA breaks. To understand how the signal emerging from its DNA-binding domain (DBD) bound to such breaks is transduced to its catalytic domain, the structure-function relationship of the DBD was investigated. We have used mutagenesis by the polymerase chain reaction (PCR) to generate a random library of PARP mutants. In this work, we describe the identification of catalytically inactive mutants bearing single point mutations, located outside the two zinc fingers in the DBD, that have conserved their full capacity to bind DNA. The results obtained demonstrate that the DNA-dependent activation of PARP requires not only a capacity to bind DNA but also a number of crucial residues to maintain a conformation of the domain necessary to transfer an 'activation signal' to the catalytic domain.

Catalysis↗
PubMed1996

Optimized BlaM-transposon shuttle mutagenesis of Helicobacter pylori allows the identification of novel genetic loci involved in bacterial virulence.

Helicobacter pylori is an important etiologic agent of gastroduodenal disease in humans. In this report, we describe a general genetic approach for the identification of genes encoding exported proteins in H. pylori. The novel TnMax9 mini-blaM transposon was used for insertion mutagenesis of a H. pylori gene library established in Escherichia coli. A total of 192 E. coli clones expressing active beta-lactamase fusion proteins (BlaM+) were obtained, indicating that the corresponding target plasmids carry H. pylori genes encoding putative extracytoplasmic proteins. Natural transformation of H. pylori P1 or P12 using the 192 mutant plasmids resulted in 135 distinct H. pylori mutant strains (70%). Screening of the H. pylori collection of mutant strains allowed the identification of mutant strains impaired in motility, in natural transformation competence and in adherence to gastric epithelial cell lines. Motility mutants could be grouped into distinct classes: (i) mutant strains lacking the major flagellin subunit FlaA and intact flagella (class I); (ii) mutant strains with apparently normal flagella, but reduced motility (class II), and (iii) mutant strains with obviously normal flagella, but completely abolished motility (class III). Two independent mutations that exhibited defects in natural competence for genetic transformation mapped to different genetic loci. In addition, two independent mutant strains were isolated by their failure to bind to the human gastric carcinoma cell line KatoIII. Both mutant strains carried a transposon in the same gene, 0.8 kb apart, and showed decreased autoagglutination when compared to the wild-type strain.

Amino Acid Sequence↗
PubMed2005

Proteomic analysis of different mutant genotypes of Arabidopsis led to the identification of 11 proteins correlating with adventitious root development.

A lack of competence to form adventitious roots by cuttings or explants in vitro occurs routinely and is an obstacle for the clonal propagation and rapid fixation of elite genotypes. Adventitious rooting is known to be a quantitative genetic trait. We performed a proteomic analysis of Arabidopsis (Arabidopsis thaliana) mutants affected in their ability to develop adventitious roots in order to identify associated molecular markers that could be used to select genotypes for their rooting ability and/or to get further insight into the molecular mechanisms controlling adventitious rooting. Comparison of two-dimensional gel electrophoresis protein profiles resulted in the identification of 11 proteins whose abundance could be either positively or negatively correlated with endogenous auxin content, the number of adventitious root primordia, and/or the number of mature adventitious roots. One protein was negatively correlated only to the number of root primordia and two were negatively correlated to the number of mature adventitious roots. Two putative chaperone proteins were positively correlated only to the number of primordia, and, interestingly, three auxin-inducible GH3-like proteins were positively correlated with the number of mature adventitious roots. The others were correlated with more than one parameter. The 11 proteins are predicted to be involved in different biological processes, including the regulation of auxin homeostasis and light-associated metabolic pathways. The results identify regulatory pathways associated with adventitious root formation and represent valuable markers that might be used for the future identification of genotypes with better rooting abilities.

Adaptation, Physiological↗
PubMed1994

On the hsp26 of Saccharomyces cerevisiae.

The function of the small size hsps in Saccharomyces cerevisiae has yet to be convincingly established. In this paper we present some aspects of the physiology of hsp26. Several mutant strains were analyzed with respect to the expression of the HSP26 gene using anti-hsp26 antibody for identification. The bcy1 mutant which lacks the regulating subunit of protein kinase A failed to produce full expression of HSP26 under heat shock whereas a ras2 mutation which lowers significantly the level of cAMP, produced no detectable effect. During normal growth hsp26 protein is induced during diauxie and its synthesis continues during the second exponential phase. Both BCY1 and CYR1 genes seen to be required for induction during the transition phase albeit not directly but rather interacting with some other regulatory component. The structure of hsp26 is discussed by homology with other small hsps.

Amino Acid Sequence↗
PubMed2002

In vivo imaging of embryonic vascular development using transgenic zebrafish.

In this study we describe a model system that allows continuous in vivo observation of the vertebrate embryonic vasculature. We find that the zebrafish fli1 promoter is able to drive expression of enhanced green fluorescent protein (EGFP) in all blood vessels throughout embryogenesis. We demonstrate the utility of vascular-specific transgenic zebrafish in conjunction with time-lapse multiphoton laser scanning microscopy by directly observing angiogenesis within the brain of developing embryos. Our images reveal that blood vessels undergoing active angiogenic growth display extensive filopodial activity and pathfinding behavior similar to that of neuronal growth cones. We further show, using the zebrafish mindbomb mutant as an example, that the expression of EGFP within developing blood vessels permits detailed analysis of vascular defects associated with genetic mutations. Thus, these transgenic lines allow detailed analysis of both wild type and mutant embryonic vasculature and, together with the ability to perform large scale forward-genetic screens in zebrafish, will facilitate identification of new mutants affecting vascular development.

Animals↗
PubMed1998

Characterization of the lipid-carrier involved in the synthesis of enterobacterial common antigen (ECA) and identification of a novel phosphoglyceride in a mutant of Salmonella typhimurium defective in ECA synthesis.

The polysaccharide chains of enterobacterial common antigen (ECA) consist of linear trisaccharide repeat units with the structure -->3)-alpha-d-Fuc4NAc-(1-->4)-beta-d-ManNAcA-(1--> 4)-alpha-d-GlcNAc-(1-->, where Fuc4NAc is 4-acetamido-4, 6-dideoxy-d-galactose, ManNAcA is N -acetyl-d- mannosaminuronic acid, and GlcNAc is N -acetyl-d-glucosamine. The major form of ECA (ECAPG) consists of polysaccharide chains that are believed to be covalently linked to diacylglycerol through phosphodiester linkage; the phospholipid moiety functions to anchor molecules in the outer membrane. The ECA trisaccharide repeat unit is assembled as a polyisoprenyl-linked intermediate which has been tentatively identified as Fuc4NAc-ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid III). Subsequent chain-elongation presumably occurs by a block-polymerization mechanism. However, the identity of the polyisoprenoid carrier-lipid has not been established. Accordingly, the current studies were conducted in an effort to structurally characterize the polyisoprenyl lipid-carrier involved in ECA synthesis. Isolation and characterization of the lipid carrier was facilitated by the accumulation of a ManNAcA-GlcNAc-pyrophosphorylpolyisoprenyl lipid (lipid II) in mutants of Salmonella typhimurium defective in the synthesis of TDP-Fuc4NAc, the donor of Fuc4NAc residues for ECA synthesis. Analyses of lipid II preparations by fast atom bombardment tandem mass spectroscopy (FAB-MS/MS) resulted in the identification of the lipid-carrier as the 55-carbon polyisoprenyl alcohol, undecaprenol. These analyses also resulted in the identification of a novel glycolipid which copurified with lipid II. FAB-MS/MS analyses of this glycolipid revealed its structure to be 1,2-diacyl- sn -glycero-3-pryophosphoryl-GlcNAc-ManNAcA (DGP-disaccharide). An examination of purified ECAPGby phosphorus-31 nuclear magnetic resonance spectroscopy confirmed that the polysaccharide chains are linked to diacylglycerol through phosphodiester linkage. Thus, DGP-disaccharide does not appear to be an intermediate in ECAPGsynthesis. Nevertheless, although the available evidence clearly indicate that lipid II is a precursor of DGP-disaccharide, the function of this novel glycolipid is not yet known, and it may be an intermediate in the biosynthesis of a molecule other than ECAPG.

Antigens, Bacterial↗
PubMed1988

Identification of group specific component/vitamin D-binding protein (GC/DBP) mutants by isoelectric focusing in immobilized pH gradients.

The six common genetic types of the group specific component/vitamin D-binding protein (GC/DBP) system are usually classified by isoelectric focusing in carrier ampholytes, followed by visualization of the GC proteins by immunoprinting with monospecific antiserum. In addition, more than 120 mutant GC types have been discovered. For their identification additional methods were necessary, including polyacrylamide gel electrophoresis, isoelectric focusing in the presence of 3 M urea as well as isoelectric focusing in immobilized pH gradients. The application of the last method is described in detail and several examples of GC/DBP mutants identified thereby are presented.

Gels↗
PubMed2005

Screening for Bacillus subtilis mutants deficient in pressure induced spore germination: identification of ykvU as a novel germination gene.

Exposure to high pressure induces germination in spores of Bacillus subtilis. To investigate the mechanisms of this process and to compare the pressure and nutrient induced germination pathways, a random transposon knock-out library of B. subtilis was constructed and screened for clones with a compromised pressure induced germination at 100 MPa. Two mutants were isolated and their transposon insertion was mapped to gerAC and ykvU respectively. While GerAC is required for production of the l-alanine receptor which has been implicated in pressure-induced germination before, YkvU is shown here to be a novel germination determinant in B. subtilis, affecting germination by high (100 MPa) and very high (600 MPa) pressure, by nutrients and by dodecylamine, but not by Ca(2+)-dipicolinic acid.

Amines↗
PubMed2006

Temperature-sensitive paralytic mutants: insights into the synaptic vesicle cycle.

Forward genetic screens have identified numerous proteins with critical roles in neurotransmission. One particularly fruitful screening target in Drosophila has been TS (temperature-sensitive) paralytic mutants, which have revealed proteins acutely required in neuronal signalling. In the present paper, we review recent insights and current questions from one recently cloned TS paralytic mutant, rbo (rolling blackout). The rbo mutant identifies a putative integral lipase of the pre-synaptic plasma membrane that is required for the SV (synaptic vesicle) cycle. Identification of this mutant adds to a growing body of evidence that lipid-modifying enzymes locally control specialized lipid microenvironments and lipid signalling pathways with key functions regulating neurotransmission strength. The RBO protein is absolutely required for phospholipase C signalling in phototransduction. We posit that RBO might be required to regulate the availability of fusogenic lipids such as phosphatidylinositol 4,5-bisphosphate and diacylglycerol that may directly modify membrane properties and/or activate lipid-binding fusogenic proteins mediating SV exocytosis.

Animals↗
PubMed2002

P53 gene mutations: case study of a clinical marker for solid tumors.

P53 is a tumor-suppressor gene that codes for a multifunctional DNA-binding protein involved in cell cycle arrest, DNA repair, differentiation, and apoptosis. The P53 gene is mutated in approximately 50% of human cancers and in germline DNA of families with inherited cancer syndromes. The role of P53 mutations in the program of carcinogenic genetic alterations differs among tumor sites ranging from the earliest mutations that can be detected in premalignant cells to mutations that trigger malignant transformation of a benign neoplasm. P53 mutations can cause expression of abnormal proteins or result in complete absence of P53 expression. For these reasons the role of P53 genetic disruption has different implications in different tumor types and may vary depending on the effect of the mutation on P53 protein function. Immunohistochemical detection of P53, commonly used as a surrogate for identification of a mutant gene, has imperfect sensitivity and specificity, further complicating correlations between P53 gene status and clinical outcomes. The presence of P53 mutations has been shown to affect prognosis of some cancers. The identity of P53 mutations can be used to determine tumor clonality. The detection of P53 mutations suggests the severity of premalignant lesions. Evolving technology for more accurate identification of P53 mutations, better understanding of the function of mutant P53 protein, and more detailed analysis of individual tumor types may expand the relevance of P53 gene analysis for clinical outcomes and therapeutic response.

Apoptosis↗
PubMed2004

An interactive gene network for securin-separase, condensin, cohesin, Dis1/Mtc1 and histones constructed by mass transformation.

The small genome of fission yeast Schizosaccharomyces pombe contains 4824 predicted genes and gene disruption suggests that approximately 850 are essential for viability. To obtain information on interactions among genes required for chromosome segregation, an approach called Strategy B was taken using mass transformation of the 1015 temperature-sensitive (ts) mutants that were made by random mutagenesis and transformed by plasmids carrying the genes for securin, separase, condensin, cohesin, kinetochore microtubule-binding proteins Dis1/Mtc1 or histones. Mutant strains whose phenotypes were either suppressed or inhibited by plasmids were selected. Each plasmid interacted positively or negatively with the average 14 strains. Identification of the mutant gene products by cloning revealed many hitherto unknown interactions. The interactive networks of segregation therefore may consist of genes with a variety of functions. For example, separase/Cut1 interacts with Cdc48/p97/VCP, which stabilizes securin and separase. Surprisingly, S. pombe cdc48 mutants displayed the mitotic phenotype highly similar to separase/cut1 mutants. This approach also provides a novel way of mutant isolation, resulting in two histone H2B strains and a cohesion mutant with a new phenotype.

Adenosine Triphosphatases↗
PubMed1989

Identification of additional virulence determinants on the pYV plasmid of Yersinia enterocolitica W227.

This paper describes the mutagenesis of the pYV plasmid from Yersinia enterocolitica W22703 (serotype O:9) with Tn2507, a new element generating operon fusions. Analysis of the mutants allowed the identification of an additional Yop protein called Yop20 and the mapping of yop20, yop44, yop48, and lcrV, the gene encoding the V antigen. The last gene appeared to be part of an operon that also may contain yop37 and yop44. At 37 degrees C, mutants affected in this operon grew poorly, irrespective of the presence of Ca2+, or they even died in the presence of Ca2+. This operon is thus involved in the regulation by Ca2+, and we called it car, for Ca2+ regulation. It is presumably the Y. enterocolitica counterpart of the lcrGVH operon of Yersinia pestis. Transcription of yop20 and of the car operon was strongly regulated by temperature and only slightly by calcium. Hence, these genes behaved like the other genes of the yop regulon. Mutants affected in yop20 or in yop48 were markedly less virulent for the desferrioxamine-treated mouse than was the parental strain. Yop20 and Yop48 thus probably are Yersinia virulence factors.

Animals↗
PubMed1989

The Drosophila EGF receptor homolog (DER) gene is allelic to faint little ball, a locus essential for embryonic development.

Recessive lethal mutations in the genetic locus of the Drosophila EGF receptor homolog (DER) were isolated. Identification of mutations in the gene is based on assays of DER protein autophosphorylation activity. Most DER alleles show little or no in vivo autophosphorylation. The ability to monitor these activities in vivo and in vitro offers a preliminary insight into the functional defects in the different mutant proteins. The identification of the DER locus was also confirmed by partial rescue of the mutant phenotype with a DER P-element construct. Homozygous DER mutants display a complex embryonic phenotype. Most notably, the anterior structures deteriorate, ventral denticle bands are missing, the germ band does not retract, and the central nervous system shows a collapse of commissure and midline pattern. Mutations in DER were shown to be allelic to the previously described locus faint little ball.

Alleles↗
PubMed2000

A series of novel mutants of Arabidopsis thaliana that are defective in the formation of continuous vascular network: calling the auxin signal flow canalization hypothesis into question.

For the genetic analysis of molecular mechanisms underlying temporal and spatial regulation of vascular pattern formation, we isolated mutants of Arabidopsis thaliana that are impaired in vascular patterning. Microscopic examination of the cotyledonary venation of 3,400 M(3) lines led to the identification of 12 mutant lines. Genetic analysis of 8 of these mutant lines indicated that vein pattern formation in these lines resulted from monogenic recessive mutations in 7 different genes, designated VAN1 through VAN7. Mutations in VAN1 through VAN6 genes caused fragmentation (disconnection or partial loss) of lateral veins of the cotyledon and tertiary veins of the rosette leaf whereas they were less injurious to the formation of major veins. Detailed characterization of the van3 mutant using pAthb8::GUS and pTED3::GUS, as molecular markers for the early stage of vascular tissue formation showed that the provascular tissue of the cotyledonary lateral veins was differentiated in fragments during late embryogenesis. These phenotypes of the van mutants are discussed in relation to the auxin signal flow canalization hypothesis and the diffusion-reaction prepattern hypothesis, with the fragility of the continuity in the minor vein formation favoring the latter hypothesis.

Arabidopsis↗
PubMed1979

[Separation of the biosynthesis products of a mutant strain of Actinomyces chrysomallus var. carotenoides and the identification of actinomycin antibiotics].

An orange antibiotically active substance isolated from the mycelium of a mutant strain of Actinomyces chrysomallus var. carotenoides was identified as a mixture of actinomycins according to its light absorption spectra, circular dichroism spectra, IR spectra and chromatographic comparison with the standard samples. A scheme for successive extraction of the biologically active substances from the mycelium resulting in isolation of a fraction enriched with antibiotic substances and a fraction enriched with pigments is presented. A method for separation and purification of 3 groups of biologically active substances from the mycelium enriched extract was developed.

Antibiotics, Antineoplastic↗
PubMedDate not supplied

[Noncapsular mutants of Vibrio cholerae serogroup O139: their isolation, identification and use for the preparation of an O139 diagnostic antiserum].

On the basis of V. cholerae strain P16064, serogroup O139, spontaneous and transposon mutants with the stable lose of the capacity for producing the polysaccharide capsule, but retaining antigen O139, have been obtained. As revealed in this study, capsular and noncapsular strains differ in their sensitivity to cholera phages 20 and Inaba, as well as in agglutination with O139-antiserum. These data make it possible to use of bacteriophages for the differentiation of capsular and noncapsular strains. The use of noncapsular mutants ensure obtaining rabbit O139-antisera with higher antibody titer.

Animals↗
PubMed2005

Design and evaluation of 5'-modified nucleoside analogs as prodrugs for an E. coli purine nucleoside phosphorylase mutant.

Our studies have led to the identification of an E. coli PNP mutant (M64V) that is able to cleave numerous 5'-modified nucleoside analogs with much greater efficiency than the wild-type enzyme. The biological activity of the three best substrates of this mutant (9-[6-deoxy-alpha-L-talofuranosyl]-6-methylpurine (methyl(talo)-MeP-R), 9-[6-deoxy-alpha-L-talofuranosyl]-2-F-adenine, and 9-[alpha-L-lyxofuranosyl]-2-F-adenine) were evaluated so that we can optimally utilize these compounds. Our results indicated that the mechanism of toxicity of methyl(talo)-MeP-R to mice was due to its cleavage to MeP by a bacterial enzyme, and that the toxicity of the two F-Ade analogs was due to their cleavage to F-Ade by mammalian methylthioadenosine phosphorylase.

Animals↗