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Genetic Transformation Of The Lyme Disease Agent Borrelia Burgdorferi With Coumarin-Resistant Gyrb., D. Scott Samuels, Kathleen E. Mach, Claude F. Garon
Genetic Transformation Of The Lyme Disease Agent Borrelia Burgdorferi With Coumarin-Resistant Gyrb., D. Scott Samuels, Kathleen E. Mach, Claude F. Garon
Biological Sciences Faculty Publications
No useful method to genetically manipulate Borrelia burgdorferi, the causative agent of Lyme disease, has been developed previously. We have used resistance to the coumarin antibiotic coumermycin A1, an inhibitor of DNA gyrase, as a genetic marker to monitor the transformation of B. burgdorferi by electroporation. Introduction of site-directed mutations into the gyrB gene demonstrated that transformation was successful, provided evidence that homologous recombination occurs on the chromosome, and established that mutations at Arg-133 of DNA gyrase B confer coumermycin A1 resistance in B. burgdorferi. The coumermycin A1-resistant gyrB marker and genetic transformation can now be applied toward dissecting the …
Mechanism Of Gtp Hydrolysis By G-Protein Α Subunits, Christiane Kleuss, André S. Raw, Ethan Lee, Stephen R. Sprang, Alfred G. Gilman
Mechanism Of Gtp Hydrolysis By G-Protein Α Subunits, Christiane Kleuss, André S. Raw, Ethan Lee, Stephen R. Sprang, Alfred G. Gilman
Biological Sciences Faculty Publications
Hydrolysis of GTP by a variety of guanine nucleotide-binding proteins is a crucial step for regulation of these biological switches. Mutations that impair the GTPase activity of certain heterotrimeric signal-transducing G proteins or of p21(ras) cause tumors in man. A conserved glutamic residue in the α subunit of G proteins has been hypothesized to serve as a general base, thereby activating a water molecule for nucleophilic attack on GTP. The results of mutagenesis of this residue (Glu-207) in G(iα1) refute this hypothesis. Based on the structure of the complex of G(iα1) with GDP, Mg2+, and AlF4/ …
Use Of An Oriented Transmembrane Protein To Probe The Assembly Of A Supported Phospholipid Bilayer, Paul Brian Contino, Carol A. Hasselbacher, J. B. Alexander Ross, Yale Nemerson
Use Of An Oriented Transmembrane Protein To Probe The Assembly Of A Supported Phospholipid Bilayer, Paul Brian Contino, Carol A. Hasselbacher, J. B. Alexander Ross, Yale Nemerson
Chemistry and Biochemistry Faculty Publications
Planar-supported phopholipid bilayers formed by te adsorpton of vesicles are increasingly used in the investigation of lipK-ependent reactis. We have studied the way in which these bilayers are forned with phopholipid vesicles coaining the btranembrane protein Tssue Factor (TF). TF complexed with te senne protease, factor Vlla, is the primary initiator of bklod coagulation by way of activation of the zymogen factor X. TF has been shown to orient randomly on the inner and outer leaflets of vesicles. We used proteolytic digestion to produce vesicles in which the exracellular domain of TF is located on the inner leaflet These vesicles …
Analysis Of The Distribution And Molecular Heterogeneity Of The Ospd Gene Among The Lyme Disease Spirochetes: Evidence For Lateral Gene Exchange, Richard T. Marconi, D. Scott Samuels, Robert K. Landry, Claude F. Garon
Analysis Of The Distribution And Molecular Heterogeneity Of The Ospd Gene Among The Lyme Disease Spirochetes: Evidence For Lateral Gene Exchange, Richard T. Marconi, D. Scott Samuels, Robert K. Landry, Claude F. Garon
Biological Sciences Faculty Publications
Analysis of the ospD gene has revealed that this gene is not universal among Lyme disease spirochete isolates. The gene was found to be carried by 90, 50, and 24% of the Borrelia garinii, B. afzelii, and B. burgdorferi isolates tested. Size variability in the ospD-encoding plasmid was also observed. Sequence analysis has demonstrated the presence of various numbers of a 17-bp repeated sequence in the upstream control (promoter) region of the gene. In addition, a region within the coding sequence where various insertions, deletions, and direct repeats occur was identified. ospD gene sequences from 31 different isolates were determined …
Identification Of Outer-Membrane Proteins Of Bartonella-Bacilliformis, Michael F. Minnick
Identification Of Outer-Membrane Proteins Of Bartonella-Bacilliformis, Michael F. Minnick
Biological Sciences Faculty Publications
Purification of the outer membrane of Bartonella bacilliformis by sucrose step gradient centrifugation and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) suggest that 14 proteins, ranging from 11.2 to 75.3 kDa, are located in the outer membrane of the pathogen. On the basis of M(r)s, eleven of these proteins have counterparts which are labeled by extrinsic radioiodination of intact bartonellae, and two of the proteins are visibly sensitive to extrinsic proteinase IC digestion in analysis by SDS-PAGE. While nearly all the extrinsically radioiodinated proteins could be immunoprecipitated with rabbit antibartonella hyperimmune serum, proteins of 31.5, 42, and 45 kDa …
Gyrb Mutations In Coumermycin A1-Resistant Borrelia Burgdorferi., D. Scott Samuels, Richard T. Marconi, Wai Mun Huang, Claude F. Garon
Gyrb Mutations In Coumermycin A1-Resistant Borrelia Burgdorferi., D. Scott Samuels, Richard T. Marconi, Wai Mun Huang, Claude F. Garon
Biological Sciences Faculty Publications
We have isolated and characterized mutants of Borrelia burgdorferi that are resistant to the antibiotic coumermycin A1, which targets the B subunit of DNA gyrase. Mutants had either 100- or 300-fold higher resistance to coumermycin A1 than wild-type B. burgdorferi. In each case, a single point mutation in the gyrB gene converted Arg-133 to Gly or Ile. Mutations in the homologous Arg residue of Escherichia coli DNA gyrase are also associated with resistance to coumarin antimicrobial agents.
Genetic And Phenotypic Diversity Of 2,4-Dichlorophenoxyacetic Acid (2,4-D)-Degrading Bacteria Isolated From 2,4-D-Treated Field Soils, J. O. Ka, William E. Holben, James M. Tiedje
Genetic And Phenotypic Diversity Of 2,4-Dichlorophenoxyacetic Acid (2,4-D)-Degrading Bacteria Isolated From 2,4-D-Treated Field Soils, J. O. Ka, William E. Holben, James M. Tiedje
Biological Sciences Faculty Publications
Forty-seven numerically dominant 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated at different times from 1989 through 1992 from eight agricultural plots (3.6 by 9.1 m) which were either not treated with 2,4-D or treated with 2,4-D at three different concentrations. Isolates were obtained from the most dilute positive most-probable-number tubes inoculated with soil samples from the different plots on seven sampling dates over the 3-year period. The isolates were compared by using fatty acid methyl ester (FAME) profiles, chromosomal patterns obtained by PCR amplification of repetitive extragenic palindromic (REP) sequences, and hybridization patterns obtained with probes for the tfd genes of …
Use Of Gene Probes To Aid In Recovery And Identification Of Functionally Dominant 2,4-Dichlorophenoxyacetic Acid-Degrading Populations In Soil, J. O. Ka, William E. Holben, James M. Tiedje
Use Of Gene Probes To Aid In Recovery And Identification Of Functionally Dominant 2,4-Dichlorophenoxyacetic Acid-Degrading Populations In Soil, J. O. Ka, William E. Holben, James M. Tiedje
Biological Sciences Faculty Publications
The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was applied to soils in microcosms, and degradation was monitored after each of five repeated additions. Total DNAs were isolated from soil bacterial communities after each 2,4-D treatment. The DNA samples were analyzed on slot blots and Southern blots by using a tfdA gene probe subcloned from plasmid pJP4 and a Spa probe derived from a different 2,4-D-degrading isolate, a Sphingomonas paucimobilis strain. 2,4-D applied to soil was quickly degraded by indigenous microbial populations. As determined by slot blot analyses of DNA from a Michigan soil, the increase in hybridization signal in response to 2,4-D …
Analysis Of Competition In Soil Among 2,4-Dichlorophenoxyacetic Acid-Degrading Bacteria, J. O. Ka, William E. Holben, James M. Tiedje
Analysis Of Competition In Soil Among 2,4-Dichlorophenoxyacetic Acid-Degrading Bacteria, J. O. Ka, William E. Holben, James M. Tiedje
Biological Sciences Faculty Publications
Competition among indigenous and inoculated 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was studied in a native Kansas prairie soil following 2,4-D additions. The soil was inoculated with four different 2,4-D-degrading strains at densities of 10(3) cells per g of soil; the organisms used were Pseudomonas cepacia DBO1(pJP4) and three Michigan soil isolates, strain 745, Sphingomonas paucimobilis 1443, and Pseudomonas pickettii 712. Following 2,4-D additions, total soil DNA was extracted and analyzed on Southern blots by using a tfdA gene probe which detected three of the strains and another probe that detected the fourth strain, S. paucimobilis 1443, which belongs to a different …
The Composition And Social-Organization Of Mixed-Species Flocks In A Tropical Deciduous Forest In Western Mexico, Richard L. Hutto
The Composition And Social-Organization Of Mixed-Species Flocks In A Tropical Deciduous Forest In Western Mexico, Richard L. Hutto
Biological Sciences Faculty Publications
I recorded the flocking propensity of birds within a tropical deciduous forest in western Mexico during the nonbreeding season, and determined the species composition of 57 mixed-species, canopy insectivore flocks. Each of 27 canopy insectivore species present on the study area was observed foraging in mixed-species flocks on at least half of the occasions that it was detected on bird surveys. The proportion of flocks within which a given species was detected could be predicted on the basis of its index of abundance, as determined from independently derived point count data. Therefore, flocks are not comprised of a special subset …
Two Crystal Forms Of The Extracellular Domain Of Type I Tumor Necrosis Factor Receptor, Lynn E. Rodseth, Barbara Brandhuber, Tracey Q. Devine, Michael J. Eck, Karin Hale, James H. Naismith, Stephen R. Sprang
Two Crystal Forms Of The Extracellular Domain Of Type I Tumor Necrosis Factor Receptor, Lynn E. Rodseth, Barbara Brandhuber, Tracey Q. Devine, Michael J. Eck, Karin Hale, James H. Naismith, Stephen R. Sprang
Biological Sciences Faculty Publications
The soluble extracellular domain of human type I tumor necrosis factor receptor (sTNFrI) is a 161 residue polypeptide found in serum and urine. This domain tightly binds tumor necrosis factors (TNF) α and β and, as part of the whole receptor, initiates the powerful biological effects of TNF. The extracellular domain, typical of other TNF receptor superfamily members, comprises four cysteine-rich motifs. We have obtained two crystal forms of the sTNFrI. One crystal form is grown at pH 3.7 with MgSO4 as the precipitant. These crystals are orthorhombic, space group P212121, with cell …
Crystallization And Preliminary Crystallographic Studies Of Giα1 And Mutants Of Giα1 In The Gtp And Gdp-Bound States, David E. Coleman, Ethan Lee, Mark B. Mixon, Maurine E. Linder, Albert M. Berghuis, Alfred G. Gilman, Stephen R. Sprang
Crystallization And Preliminary Crystallographic Studies Of Giα1 And Mutants Of Giα1 In The Gtp And Gdp-Bound States, David E. Coleman, Ethan Lee, Mark B. Mixon, Maurine E. Linder, Albert M. Berghuis, Alfred G. Gilman, Stephen R. Sprang
Biological Sciences Faculty Publications
Several different crystal forms of Giα1 have been grown and analyzed. Crystals of native protein containing bound GTPγS belong to space group P3121 or P3221 with cell dimensions a, b = 80·6 Åand c = 106·3 Å and diffract to a resolution of 1·9 Å using synchrotron radiation. Crystals of native protein containing bound GDP belong to space group I4 with cell dimensions a, b = 121·3 Å, and c = 67·7 Å and diffract to 3·0Å. Data sets from crystals grown using mutant proteins have also been obtained and characterized.