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At least 217 records · Page 12Linked to original sources
PubMed1992

Evidence for proteolytic cleavage of the 120-kilodalton outer membrane protein of rickettsiae: identification of an avirulent mutant deficient in processing.

The 120-kDa rickettsial outer membrane protein (rOmpB) is encoded by a gene with the capacity to encode a protein of approximately 168 kDa. The carboxy-terminal end of the molecule is apparently cleaved to yield 120- and 32-kDa products. Both polypeptides are surface exposed and remain associated with the outer membrane of intact rickettsiae. All species of rickettsiae examined display similar cleavage of rOmpB. Comparison of diverse species of rickettsiae demonstrate a conserved N terminus of the 32-kDa fragment, with a predicted procaryotic secretory signal peptide immediately upstream of the proposed cleavage site. Coprecipitation of the 120-kDa rOmpB protein and the 32-kDa peptide by monoclonal antibodies specific for the 120-kDa portion of the molecule suggests that the two fragments remain noncovalently associated on the surface of rickettsiae. Analysis of an avirulent mutant of Rickettsia rickettsii revealed reduced amounts of the 120- and 32-kDa fragments, but with a correspondingly larger rOmpB protein that displayed properties expected of the putative precursor. This avirulent mutant grows intracellularly but fails to cause the lysis of infected cells that is typical of R. rickettsii. DNA sequence analysis of the region of the gene encoding the cleavage site of the avirulent strain revealed no difference from the sequence obtained from virulent R. rickettsii. The 168-kDa putative precursor of the avirulent strain of R. rickettsii was not extracted from the surface by dilute buffers, as is the 120-kDa protein of virulent R. rickettsii or R. prowazekii. These latter results suggest that the 32-kDa C-terminal region of the molecule may serve as a membrane anchor domain.

Amino Acid Sequence↗
PubMed1996

Identification of Legionella pneumophila mutants that have aberrant intracellular fates.

After uptake by macrophages, Legionella pneumiophila evades phagosome-lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum. A collection of bacterial mutants defective for growth in macrophages were isolated, and the intracellular fate of each mutant strain was analyzed by fluorescence microscopy. To measure intracellular replication, bacteria inside macrophages were stained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI). Evasion of the endocytic pathway was quantified by immunofluorescence localization of lp120 [correction of IgpI20] (LAMP-1), a membrane protein of late endosomes and lysosomes, or by measuring colocalization of bacteria with a fluorescent tracer, Texas red-ovalbumin, preloaded into lysosomes. Replication vacuoles were quantified by immunofluorescence localization of BiP, an endoplasmic reticulum protein. By these approaches, four phenotypic groups of mutants were classified. One class formed replication vacuoles less efficiently than the wild type did; another formed replication vacuoles, but replication was abortive; in another class, most phagosomes containing bacteria acquired markers of the endocytic pathway but a minority formed replication vacuoles and the bacteria replicated; finally, a fourth class, the one most defective for intracellular growth, occupied vacuoles that acquired markers of the endocytic pathway.

Animals↗
PubMed2004

Identification of a compensatory mutant (lpg2-REV) of Leishmania major able to survive as amastigotes within macrophages without LPG2-dependent glycoconjugates and its significance to virulence and immunization strategies.

Different Leishmania species rely to different extents on abundant glycoconjugates, such as lipophosphoglycan (LPG) and related molecules, in mammalian infections. Previously, we showed that Leishmania major deletion mutants lacking the Golgi GDP-mannose transporter LPG2, which is required for assembly of the dominant phosphoglycan (PG) repeats of LPG, were unable to survive in macrophages. These lpg2- mutants, however, retained the ability to generate asymptomatic, persistent infections in mice. In contrast, Ilg and colleagues showed that Leishmania mexicana LPG2 mutants retained virulence for mice. Here we identified a partial revertant population of the L. major lpg2- mutants (designated lpg2(-)REV) that had regained the ability to replicate in macrophages and induce disease pathology through a compensatory change. Like the lpg2 parent, the lpg2(-)REV revertant was unable to synthesize LPG2-dependent PGs in the promastigote stage and thus remained highly attenuated in the ability to induce infection. However, after considerable delay lpg2(-)REV revertant-infected mice exhibited lesions, and amastigotes isolated from these lesions were able to replicate within macrophages despite the fact that they were unable to synthesize PGs. Thus, in some respects, the lpg2(-)REV amastigotes resemble L. mexicana amastigotes. Future studies of the gene(s) responsible may shed light on the mechanisms employed by L. major to survive in the absence of LPG2-dependent glycoconjugates and may also improve the potential of the lpg2- L. major line to serve as a live parasite vaccine by overcoming its tendency to revert toward virulence.

Animals↗
PubMed1998

Identification of highly attenuated mutants of simian immunodeficiency virus.

Deletion mutants of the pathogenic clone of simian immunodeficiency virus isolate 239 (SIVmac239) were derived that are missing nef, vpr, and upstream sequences (US) in the U3 region of the LTR (SIVmac239 delta3), nef, vpx, and US (SIVmac239 delta3x), and nef, vpr, vpx, and US (SIVmac239 delta4). These multiply deleted derivatives replicated well in the continuously growing CEMx174 cell line and were infectious for rhesus monkeys. However, on the basis of virus load measurements, strength of antibody responses, and lack of disease progression, these mutants were highly attenuated. Measurements of cell-associated viral load agreed well with assays of plasma viral RNA load and with the strengths of the antibody responses; thus, these measurements likely reflected the extent of viral replication in vivo. A derivative of SIVmac239 lacking vif sequences (SIVmac239 delta vif) could be consistently grown only in a vif-complementing cell line. This delta vif virus appeared to be very weakly infectious for rhesus monkeys on the basis of sensitive antibody tests only. The weak antibody responses elicited by SIVmac239 delta vif were apparently in response to low levels of replicating virus since they were not elicited by heat-inactivated virus and the anti-SIV antibody responses persisted for greater than 1 year. These results, and the results of previous studies, allow a rank ordering of the relative virulence of nine mutant strains of SIVmac according to the following order: delta vpr > delta vpx > delta vpr delta vpx approximately delta nef > delta3 > delta3x > or = delta4 > delta vif > delta5. The results also demonstrate that almost any desired level of attenuation can be achieved, ranging from still pathogenic in a significant proportion of animals (delta vpr and delta vpx) to not detectably infectious (delta5), simply by varying the number and location of deletions in these five loci.

Animals↗
PubMed2005

Identification of zebrafish insertional mutants with defects in visual system development and function.

Genetic analysis in zebrafish has been instrumental in identifying genes necessary for visual system development and function. Recently, a large-scale retroviral insertional mutagenesis screen, in which 315 different genes were mutated, that resulted in obvious phenotypic defects by 5 days postfertilization was completed. That the disrupted gene has been identified in each of these mutants provides unique resource through which the formation, function, or physiology of individual organ systems can be studied. To that end, a screen for visual system mutants was performed on 250 of the mutants in this collection, examining each of them histologically for morphological defects in the eye and behaviorally for overall visual system function. Forty loci whose disruption resulted in defects in eye development and/or visual function were identified. The mutants have been divided into the following phenotypic classes that show defects in: (1) morphogenesis, (2) growth and central retinal development, (3) the peripheral marginal zone, (4) retinal lamination, (5) the photoreceptor cell layer, (6) the retinal pigment epithelium, (7) the lens, (8) retinal containment, and (9) behavior. The affected genes in these mutants highlight a diverse set of proteins necessary for the development, maintenance, and function of the vertebrate visual system.

Animals↗
PubMed1984

Identification of Saccharomyces cerevisiae mutants deficient in DNA topoisomerase I activity.

Mutants of the yeast, Saccharomyces cerevisiae, deficient in DNA topoisomerase I activity have been identified. One mutant has normal topoisomerase I activity when assayed at 25 degrees C and about 20% of normal activity when assayed at 36 degrees C. Strains with this mutation grow normally at all temperatures tested. The mutation has been mapped to MAK1, a gene required for maintenance of killer RNA. Three previously isolated mak1 mutants exhibit less than 1% of normal topoisomerase I activity in our assay, but yet they grow normally. The implications of these results for the role of DNA topoisomerase I in the cell are discussed.

DNA Topoisomerases, Type I↗
PubMed1996

Mutation analysis of very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency: identification and characterization of mutant VLCAD cDNAs from four patients.

Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency is a newly identified disease. A 105-bp deletion in the VLCAD cDNA in two patients has been reported, and detailed molecular characterization of this disease has remained to be done. We report here five mutations identified in four patients: a 135-bp deletion encompassing bases 343-477, a C-1837-to-T transition (R613W), 3-bp deletions at the nucleotide positions 388-390 (E130del) and 895-897 (K299del), and an A-1144-to-C transversion (K382Q). Sequencing of genomic DNA amplified by PCR revealed a 135-bp deletion caused by exon skipping due to a 1-bp deletion in a 3' splice site of an intron. In cDNA expression experiments using Chinese hamster ovary (CHO) cells, we found that each of the mRNAs derived from E130del and K299del clones were unstable and that translation products from R613W, E130del, K299del, and K382Q clones were labile. Each of R613W, E130del, K299del, and K382Q proteins expressed in CHO cells appeared abnormal in dimer assembly, as shown in gel-filtration analysis. VLCAD activity was not detected in mutants' transfectants. Thus, we verified that all five mutations identified in these four patients were disease-causing alterations.

Acyl-CoA Dehydrogenase, Long-Chain↗
PubMed1994

[Screening and rapid identification of Bacillus thuringiensis mutants].

Mutants of Bacillus thuringiensis subsp. kurstaki NTU 9 and Bt 158, which were isolated previously for using the diamondback moth as a target insect in Taiwan, were screening by either protein electrophoresis of intracellular proteins or enzyme-linked immunosorbent assay (ELISA). The optimal conditions of effective protein electrophoresis were (1) 24-hour cells harvested from nutrient broth were crashed by petite glass beads followed by centrifugation. And (2) the supernatant pretreated by heating at 60 degrees C for 2 minutes was electrophoresed with 7.5% native PAGE at 110 voltages. On ELISA, the antiserum used was obtained from rabbits immunized with Bt 158 crystal protein. Optimal antigen coating concentration of ELISA, attained by chequer-board titration method, was 10 micrograms/ml. Antigens (crystal protein) in samples were detected by competitive inhibition method with antiserum diluted to 10(4) fold. By using protein electrophoresis and ELISA methods, two isolates A 71 and BN 11, were denoted respectively as qualitative and quantitative mutants of Bacillus thuringiensis.

Animals↗
PubMed2002

A novel extinction screen in Arabidopsis thaliana identifies mutant plants defective in early microsporangial development.

Few Arabidopsis mutants defective in early male or female germline development have been reported. A novel extinction screen has been devised which permits the identification of mutants deficient in the earliest stages of anther development. Using mutagenized plants carrying GUS reporter constructs driven by tapetal-specific promoters originally derived from Brassica genes, a wide spectrum of mutants have been identified in Arabidopsis, ranging from those defective in archesporial cell differentiation to others expressed later in development. Crosses between these lines and known anther development mutants have enabled the identification of lines carrying mutations in genes expressed during very early anther formation. Initial characterization reveals these early mutants fall into two classes, gne (GUS-negative) 1-like, and gne2-like. Members of the gne1 mutant class initiate all four layers of the anther wall and an appropriate number of sporogenous cells; however, as development proceeds the tapetal and middle-layer cells enlarge, eventually crushing the sporogenous cells. The gne2 class anthers are disrupted at an earlier stage, with the middle and tapetal layers failing to form, and an excess of sporogenous cells developing until the germline aborts late in meiosis II. Analysis of these mutants has already raised questions about the accuracy of current models of angiosperm anther development.

Arabidopsis↗
PubMed1979

Structure of the lipopolysaccharide from an Escherichia coli heptose-less mutant. I. Chemical degradations and identification of products.

The structure of lipopolysaccharide from a heptose-less mutant of Escherichia coli K-12 has been investigated. Lipopolysaccharide isolated from 32P-labeled cells was treated with mild alkali to yield two separable components: [OH-LPS]-I (approximately 70%) and [OH-LPS]-II (approximately 30%). Mild acidic treatment of [OH-LPS]-I gave mainly a product which was identified as (4-O-phosphoryl-N-beta-hydroxymyristyl-D-glucosaminyl)-beta(1 leads to 6)-N-beta-hydroxymyristyl-D-glucosamine 1-phosphate (Compound I). Further acidic hydrolysis of both [OH-LPS]-I and [OH-LPS]-II yielded as the main product (4-O-phosphoryl-N-beta-hydroxymyristyl-D-glucosaminyl)-beta(1 leads to 6)-N-beta-hydroxymyristyl-D-glucosamine (Compound II). The structures of the above products were deduced by a combination of compositional analyses, sensitivity to phosphomonoesterase, rates of hydrolysis of the phosphate groups and alkali-catalyzed beta elimination of the phosphate residues following appropriate oxidation of hydroxyl groups. These studies together with work reported in the accompanying papers have led to the identification of two species of lipopolysaccharide in the E. coli strain both of which contain a single glucosamine dissacharide unit but differ in having monosubstituted phosphate or pyrophosphate groups at the glycosidic position. Each species of lipopolysaccharide also appeared to be heterogeneous with respect to the number of esterified fatty acyl groups.

Alkaline Phosphatase↗
PubMed1965

CONDITIONAL-LETHAL MUTANTS OF AN ANIMAL VIRUS: IDENTIFICATION OF TWO CISTRONS.

Two different temperature-sensitive conditional-lethal mutants of Sindbis virus, an animal virus that contains RNA, have been isolated. When cultured in chick fibroblast monolayers at 42 degrees C, these mutants yield less than 0.05 percent as much virus as does the wild type, whereas at 27 degrees C they grow normally. One mutant appears to be altered in the synthesis of a protein that is produced early in the infection and is required for viral RNA synthesis. The other mutant produces as much infectious RNA as the wild type at 42 degrees C and appears to be altered in the synthesis of a protein produced late in the infection.

Animals↗
PubMed1988

Selection and immunochemical analysis of lipooligosaccharide mutants of Neisseria gonorrhoeae.

The identification of enterobacterial mutants that contain alterations in the lipopolysaccharide (LPS) oligosaccharide core structure facilitated the development of the model of the physicochemical and immunochemical structures of enteric LPS. Results of recent immunochemical studies have suggested that the structural model of the lipooligosaccharides (LOSs) of Neisseria gonorrhoeae may differ from the enteric LPS model. The difficulties in the analysis of the wild-type gonococcal LOS have precluded understanding of the precise nature of the LOS structure. This study was undertaken to isolate a series of mutants of N. gonorrhoeae 1291 that had sequential saccharide deletions in the LOS. Results of preliminary studies suggested that the pyocin, designated pyocin C, allowed selection of gonococci with such mutant LOS structures. Results also indicated that the receptor for pyocin C binding was an LOS component. Pyocin C selection led to the isolation of five strains with LOS patterns on sodium dodecyl sulfate-polyacrylamide gels which differed from the LOS of parent strain 1291. In this system, the Mr of the parent LOS was 4,715, while the LOSs from the mutant strains demonstrated progressive saccharide deletions, with Mrs of 4,230, 4,089, 3,627, 3,262, and 3,197. Protein patterns of these mutants on sodium dodecyl sulfate-polyacrylamide gels were qualitatively similar to those of the parent strains. Results of studies with five monoclonal antibodies specific for neisserial LOS indicated that shared as well as unique epitopes were present on the mutant LOSs. Results of ketodeoxyoctonate analysis of the mutant LOSs indicated that the majority of the ketodeoxyoctonate residues may be substituted on C-4 or C-5. Chemical and immunological analysis of such LOS mutants should expedite the development of the model for the structure of gonococcal LOS.

Antibodies, Monoclonal↗
PubMed1988

A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants.

We describe an in vitro selection procedure for oligodeoxynucleotide-directed mutagenesis, which produces mutants at frequencies of greater than 90%, facilitating the identification of mutants directly by nucleotide sequencing. The method is based on the selective methylation of the mutant strand by the incorporation of 5-methyl-dCTP. Restriction endonuclease digestion of the resulting hemimethylated DNA with MspI results in the nicking of only the nonmethylated-parental strand. The parental strand is removed by treatment with exonuclease III. The mutants are recovered by transformation of a mcrAB strain of Escherichia coli with the nascent strand.

Base Sequence↗
PubMed2003

Genetic approaches to understanding sugar-response pathways.

Plants as photoautotrophic organisms are able to produce the carbohydrates they require and have developed mechanisms to co-ordinate carbohydrate production and its metabolism. Carbohydrate-derived signals regulate the expression of genes involved in both photosynthesis and metabolism, and control carbohydrate partitioning. A number of genetic approaches have been initiated to understand sugar-response pathways in plants and identify the components involved. Screening strategies to date have been based on the effects of high sugar media on early seedling development or on changes in the enzyme activity or expression of sugar-responsive genes. These screens have established roles for plant hormones in sugar-response pathways, in particular for abscisic acid. The present emphasis on the role of plant hormones in sugar responses is due to the fact that mutants could be readily identified as belonging to these established pathways, but also results from the nature of the mutant screens in use. Progress is being made on the identification of mutants and genes that may be specific to sugar-signalling pathways. It is also expected that the modification of existing screens may target sugar-signalling pathways more directly. Genetic approaches may be especially useful in identifying components of novel signalling pathways unique to plants, and their combination with genomic and molecular approaches will guide future research.

Abscisic Acid↗
PubMed1996

Pseudomonas fluorescens adhesion and transport through porous media are affected by lipopolysaccharide composition.

The objectives of this work were (i) to use transposon mutagenesis to produce mutants of Pseudomonas fluorescens that were altered in adhesion ability and transport through porous media and (ii) to identify the alterations in surface characteristics that were responsible for the changes in attachment. Mutants of P. fluorescens were generated with TnphoA, which enabled identification of mutants that were altered in surface proteins. Transposon mutants were screened for alterations in adhesion ability by attachment assays on hydrophobic polystyrene and water-wettable polystyrene. Four TnphoA mutants with increased adhesion to the hydrophobic surface and decreased adhesion to the water-wettable surface were obtained. Transport of the strains through porous media was evaluated by passing suspensions of each mutant and the parent through columns containing quartz sand and determining the number of cells retained in the columns. The mutants all demonstrated increased adhesion and retention in the columns. Southern analysis demonstrated two types of mutants with separate transposon insertion sites. Polyacrylamide gel electrophoresis of the strains demonstrated that the O antigen on the lipopolysaccharide was either attenuated or absent. Lack of this polysaccharide, and the consequent increased exposure of the lipid moiety of the lipopolysaccharide, is probably responsible for the increase in adhesion to the hydrophobic substrata and retention in the sand column. This work combined with previous studies of attachment of P. fluorescens demonstrates that more than one type of polymer can mediate the adhesion of this organism to nonbiological surfaces.

Bacterial Adhesion↗
PubMedDate not supplied

Global regulation of expression of antifungal factors by a Pseudomonas fluorescens biological control strain.

The root-colonizing bacterium Pseudomonas fluorescens BL915 protects a variety of seedlings from damping-off disease caused by the fungal pathogen Rhizoctonia solani. Spontaneous pleiotropic mutants of P. fluorescens strain BL915 which fail to synthesize antifungal factors such as chitinase, cyanide, and pyrrolnitrin and exhibit altered colony morphology were isolated. Such mutants fail to inhibit the growth of R. solani in vitro, and their biological control capability is sharply reduced. We characterized a genomic DNA fragment from strain BL915 which, when introduced into these pleiotropic mutants, restored the lost functions, the wild-type colony morphology, and bio-control activity. DNA sequence analysis of the genomic fragment revealed the presence of genes homologous to those of numerous bacterial global regulatory systems and identified a cluster of genes identical in organization to the Escherichia coli gene cluster consisting of uvrY, uvrC, pgsA, and glyW. Coordinate biosynthesis of multiple antifungal products in some heterologous Pseudomonas strains in response to the introduction of the strain BL915 genomic fragment confirmed the regulatory nature of sequences contained on this fragment. Further genetic analysis indicated a gene homologous to response regulators of bacterial two-component systems was sufficient to complement the pleiotropic mutants and to activate antifungal genes in heterologous strains. Marker exchange of a truncated version of this gene into the P. fluorescens BL915 chromosome generated pleiotropic mutants indistinguishable from the original spontaneous mutants. Cloning and sequencing of the response regulator gene from several spontaneous mutants allowed identification of various nucleotide changes associated with the gene in such mutants.

Amino Acid Sequence↗
PubMed1984

Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichia coli K12: identification of a new mutation (recQ1) that blocks the RecF recombination pathway.

An Escherichia coli K12 mutant resistant to thymineless death (TLD) was isolated, and its genetic analysis led us to identify a new mutation (recQ1) located between corA and metE on the standard linkage map. The mutation was found to result in increased sensitivity to ultraviolet light and deficiency in conjugational recombination when placed in the recBC sbcB background, indicating that it blocked the RecF pathway of recombination. It seemed likely that this mutation is also capable of causing partial resistance to TLD, but we reserve the possibility of a separate mutation closely linked to recQ1 giving rise to this phenotype. The original mutant was shown to carry an additional mutation probably in the vicinity of the uhp locus, which was also required for the full TLD resistance of the mutant to be expressed.

Chromosome Mapping↗