Desferrioxamine-dependent iron transport in Erwinia amylovora CFBP1430: cloning of the gene encoding the ferrioxamine receptor FoxR

Iron deprivation of Erwinia amylovora CFBP1430, a species causing fire blight on Pomoïdeae, was shown to induce the production of siderophores of the desferrioxamine (dfo) family and two outer membrane polypeptides with apparent molecular weight of about 70 and 80 kDa, respectively. Cyclic dfo E was characterized as the major metabolite. Phage MudII_pR13 insertional mutagenesis and screening on CAS-agar medium yielded three dfo non-producing and one overproducing clones. These clones failed to grow in the presence of the Fe(III) chelator EDDHA and were determined further as dfo and ferrioxamine transport negative mutants, respectively. The transport mutant which appeared to lack the 70 kDa polypeptide in the outer membrane allowed the purification of dfo E. Growth under iron limitation of dfo negative mutants was stimulated with ferrioxamine E and B but not with other ferrisiderophores tested. The host DNA sequence flanking the left terminal part of the MudII_pR13 prophage responsible for the transport mutation was cloned and used to probe a parental gene library by DNA-DNA hybridization. Two recombinant cosmids restoring the transport mutation to normal were identified. Both cosmids also conferred the ability to utilize ferrioxamine B and E as iron sources on a FhuE¹ mutant of Escherichia coli. This correlated with the production of an additional polypeptide of 70 kDa in the outer membrane of E. coli transconjugants, thus confirming that this protein serves the ferrioxamine receptor function (FoxR) in E. amylovora.R. Kachadourian and A. Dellagi have contributed equally to this work.     

Fluorescence in situ hybridization with cosmid clones for the detection of human cytomegalovirus DNA in peripheral blood leukocytes

 Radioactive in situ hybridization techniques or enzymatic detection procedures of hapten-modified human cytomegalovirus (HCMV) probes have been widely used for studying the infection of peripheral blood leukocytes with HCMV. This report describes significant improvements in terms of signal resolution which can be obtained by applying a highly sensitive fluorescence in situ hybridization (FISH) technique in conjunction with a large subgenomic HCMV DNA probe. Three cosmid clones spanning 119.1 kb of the HCMV genome (230 kb) were used to construct the digoxigenin-11-dUTP-labeled probe which was found to be superior to a total HCMV probe representing the entire genome. Crucial hybridization parameters were analyzed systematically in order to ensure optimal resolution power and sensitivity. The protocol was successfully applied to HCMV-infected fibroblasts and peripheral blood leukocytes of 12 transplant patients and unambiguously facilitated the precise intracellular localization of HCMV genomes in infected cells. Because of its excellent resolution properties, accompanied by the virtual absence by any background staining, we recommend the use of this protocol as a sensitive approach for further virological analyses of the interactions between HCMV and peripheral blood leukocytes at the single-cell level. Accepted: 16 February 1996     

Glial cells phagocytose neuronal debris during the metamorphosis of the central nervous system in Drosophila melanogaster

 Using electron microscopy we demonstrate that degenerating neurons and cellular debris resulting from neuronal reorganization are phagocytosed by glial cells in the brain and nerve cord of the fruitfly Drosophila melanogaster during the first few hours following pupariation. At this stage several classes of glial cells appear to be engaged in intense phagocytosis. In the cell body rind, neuronal cell bodies are engulfed and phagocytosed by the same glial cells that enwrap healthy neurons in this region. In the neuropil, cellular debris in tracts and synaptic centres resulting from metamorphic re-differentiation of larval neurons is phagocytosed by neuropil-associated glial cells. Phagocytic glial cells are hypertrophied, produce large amounts of lysosome-like bodies and contain a large number of mitochondria, condensed chromatin bodies, membranes and other remains from neuronal degeneration in phagosomes. Received: 23 January 1996 / Accepted in revised form: 21 May 1996     

Immunogold Localisation of the Intermediate Chain within the Protein Complex of Cytoplasmic Dynein

It was our goal to determine the location of the intermediate chain within the complex of cytoplasmic dynein by immunoelectron microscopy. To do so we generated two monoclonal antibodies (m74-1 and m74-2) specific for the intermediate chain. Both antibodies recognised the intermediate chain by sodium dodecyl sulphate–polyacrylamide gel electrophoresis immunoblot and ELISA assays of native and denatured proteins. When sucrose density gradient-purified cytoplasmic dynein from bovine brain was incubated with the gold-conjugated monoclonal antibodies, m74-1 and m74-2, and examined by negative staining, the gold label was found opposite the globular heads at the base of the V-shaped stalk of the motor complex. The labelling of the intermediate chain is the first mapping of a component within cytoplasmic dynein. The identification of the intermediate chain at the base of the complex supports a possible docking function of the intermediate chain.     

Arginine side chain assignments in uniformly 15N-labeled proteins using the novel 2D HE(NE)HGHH experiment

A novel 2D NMR experiment, 2D HE(NE)HGHH, is presented for the assignment ofarginine side chain ¹H and ¹⁵N resonances inuniformly ¹⁵N-labeled proteins. Correlations between¹H, ¹Hand ¹H are established on the basis of³J(¹⁵N,¹H) heteronuclear scalarcoupling constants, and sequence-specific assignments are obtained by overlapof these fragments with ¹H chemical shiftsobtained by assignment procedures starting from the polypeptide backbone.Since guanidino protons exchange quite rapidly with the bulk water, the 2DHE(NE)HGHH pulse scheme has been optimized to avoid saturation and dephasingof the water magnetization during the course of the experiment. As anillustration, arginine side chain assignments are presented for two uniformly¹⁵N-labeled proteins of 7 and 23 kDa molecular weight.     

Retrofitting YACs for direct DNA transfer into plant cells

The utility of plant YAC libraries prepared in conventional YAC vectors would be dramatically increased if these YACs could be used directly for plant transformation. A pair of vectors that allow clones from YAC libraries to be modified (retrofitted) for plant transformation by direct DNA transfer methods, such as particle bombardment or electroporation, has been developed. Modification of the YAC is achieved in two sequential yeast transformation steps by taking advantage of the homologous recombination system in yeast. Using this approach, two plant-selectable marker genes and DNA sequence elements required for copy number amplification in yeast can be introduced into YACs present in yeast strain AB1380. The utility of these vectors is demonstrated by retrofitting YACs that contain inserts ranging in size from 80 to 700 kb. The 6- to 12-fold increase in copy number of these modified YACs facilitates the isolation of YAC DNA for direct DNA transformation methods. Retrofitted YACs were used for particle bombardment to examine the efficiency with which their large DNA inserts are transferred into plant cells. The availability of these retrofitting vectors should facilitate the transfer of YAC DNA inserts into plant cells and thus help bridge the gap between existing mapping techniques and plant transformation procedures.     

A pulmonary influenza virus infection in SCID mice can be cured by treatment with hemagglutinin-specific antibodies that display very low virus-neutralizing activity in vitro.

We have previously shown that a pulmonary influenza virus infection in SCID mice can be cured by treatment with monoclonal antibodies (MAbs) specific for the viral transmembrane protein hemagglutinin (HA) but not for matrix 2. Since both types of MAbs react with infected cells but only the former neutralizes the virus, it appeared that passive MAbs cured by neutralization of progeny virus rather than reaction with infected host cells. To prove this, we selected a set of four HA-specific MAbs, all of the immunoglobulin G2a isotype, which reacted well with native HA expressed on infected cells yet differed greatly (>10,000-fold) in virus neutralization (VN) activity in vitro, apparently because of differences in antibody avidity and accessibility of the respective determinants on the HA of mature virions. Since the VN activities of these MAbs in vitro were differentially enhanced by serum components, we determined their prophylactic activities in vivo and used them as measures of their actual VN activities in vivo. The comparison of therapeutic and prophylactic activities indicated that these MAbs cured the infection to a greater extent by VN activity (which was greatly enhanced in vivo) and to a lesser extent by reaction with infected host cells. Neither complement- nor NK cell-dependent mechanisms were involved in the MAb-mediated virus clearance.     

Larval Planktotrophy—A Primitive Trait in the Bilateria?

The concept of Gösta Jägersten of a primary biphasic metazoan life-cycle, consisting of a planktotrophic larva and a benthic adult, forms the basis for several theories on metazoan phylogeny. In this paper the assumed planktotrophic life-style of the larva is critically analyzed and reconsidered. It is shown, in particular for the Mollusca, that a biphasic life-cycle with a lecithotrophic larva is probably the plesiomorphic condition. Character distribution and structural data suggest a parallel evolution of the downstream collecting system used in planktotrophic larvae or filter-feeding adults of gastropods, bivalves and other spiralian or aschelminth taxa. In the basic metazoans (Parazoa, Placozoa, coelenterates) direct or lecithotrophic development dominates by far. For the acoelomate (Platyhelminthes, Gnathostomulida) and pseudocoelomate taxa direct development is probably the plesiomorphic condition. The structural similarities of the upstream collecting system in tentaculate and deuterostome phyla may also be explained by parallel events of heterochrony out of an ancestor with adult filter-feeding. The main conclusion of this survey is that larval planktotrophy is likely to be secondary and not a plesiomorphic condition among the Bilateria. Accordingly, theories which are based on the assumed plesiomorphy of larval planktotrophy of the Bilateria, need careful reevaluation.     

Dinucleotide repeat polymorphism at the D4S2458 locus close to the PKD2 locus on human chromosome 4q

A new polymorphic CA repeat sequence was identified within the candidate region fot the autosomal dominant polycystic kidney disease type 2 (PKD2) locus. It should be a useful marker in the localization of this gene.