X-ray structure of recombinant horse L-chain apoferritin at 2.0 Å resolution: implications for stability and function

The X-ray structure of recombinant horse L-chain (rL) apoferritin, solved at 2.0?Å resolution with a final R factor of 17.9%, gives evidence that the residue at position 93 in the sequence is a proline and not a leucine, as found in earlier sequencing studies. The structure is isomorphous with other apoferritin structures, and we thus draw particular attention to those structural features which can be related to the stability and function of the protein. Analysis of hydrogen bonding and salt bridge interactions shows that dimers and tetramers are the most stable molecular entities within the protein shell: a result confirming earlier biophysical experiments. The stability of horse rL apoferritin to both dissociation into subunits at acidic pH values and to complete unfolding in guanidine chloride solutions is compared with that of other apoferritins. This emphasizes the role played by the salt bridge in the stability of this protein family. The horse rL apoferritin is significantly more resistant to denaturation than horse spleen ferritin, which in turn is more resistant than any human rH apoferritins, even those for which a salt bridge is restored. Finally, this structure determination not only establishes that a preformed pocket exists in L-chain apoferritin, at a site known to be able to bind porphyrin, but also underlines the particular function of a cluster of glutamic acids (E53, E56, E57 and E60) located at the entrance of this porphyrin-binding pocket.     

A motile but non-swarming mutant of Proteus mirabilis lacks FlgN, a facilitator of flagella filament assembly

A Tn phoA mutant of Proteus mirabilis was isolated, which had lost the ability to swarm, yet was still motile. The transposon had inserted into flgN , a flagella gene encoding a 147-amino-acid protein of undefined function. Proteus flgN is arranged in an operon with the class III anti-σ²⁸ gene, flgM , flanked by the class II genes, flgA , flgBCD and flhBA , and a novel putative virulence-related gene. The flgN mutation caused a substantial reduction in cell surface-associated flagellin, particularly during differentiation to the normally hyperflagellated swarm cell. This was not due to an effect on flagella gene expression or a typical defect in the flagella export apparatus as there was no class III gene downregulation by FlgM feedback, or intracellular flagellin accumulation. Loss of FlgN nevertheless caused a severe reduction in the incorporation of pulse-labelled flagellin into the membrane/flagellum fraction of differentiating cells. Substantial amounts of both non-oligomeric flagellin and flagellin degradation products appeared in the extracellular medium, although the few mature filaments made by the mutant were no more sensitive to proteolysis than those of the wild type. FlgN appeared soluble and active in the cytosol. The data suggest that the function of FlgN is to facilitate the initiation of flagella filament assembly, a role that may be especially critical in attaining the much higher concentration of surface flagellin required for swarming. Proteus FlgN has leucine zipper-like motifs arranged on potential amphipathic helices, a feature conserved in cytosolic chaperones for the exported substrates of flagella-related type III virulence systems. While gel filtration of FlgN from the soluble cell fraction did not establish an interaction with flagellin, it indicated that FlgN may associate with an unknown component and/or form an oligomer.     

Retroviral Oligonucleotide Distributions Correlate with Biased Nucleotide Compositions of Retrovirus Sequences,Suggesting a Duplicative Stepwise Molecular Evolution

A computer-assisted analysis was made of 24 complete nucleotide sequences selected from the vertebrate retroviruses to represent the ten viral groups. The conclusions of this analysis extend and strengthen the previously made hypothesis on the Moloney murine leukemia virus: The evolution of the nucleotide sequence appears to have occurred mainly through at least three overlapping levels of duplication: (1) The distributions of overrepresented (3–6)-mers are consistent with the universal rule of a trend toward TG/CT excess and with the persistence of a certain degree of symmetry between the two strands of DNA. This suggests one or several original tandemly repeated sequences and some inverted duplications. (2) The existence of two general core consensuses at the level of these (3–6)-mers supports the hypothesis of a common evolutionary origin of vertebrate retroviruses. Consensuses more specific to certain sequences are compatible with phylogenetic trees established independently. The consensuses could correspond to intermediary evolutionary stages. (3) Most of the (3–6)-mers with a significantly higher than average frequency appear to be internally repeated (with monomeric or oligomeric internal iterations) and seem to be at least partly the cause of the bias observed by other researchers at the level of retroviral nucleotide composition. They suggest a third evolutionary stage by slippage-like stepwise local duplications. Received: 3 January 1996 / Accepted: 27 March 1996     

Excess of deletions of maternal origin in the DiGeorge/Velo-cardio-facial syndromes. A study of 22 new patients and review of the literature

We have determined the parental origin of the deleted chromosome 22 in 29 cases of DiGeorge syndrome (DGS) using a CA-repeat mapping within the commonly deleted region, and in one other case by using a chromosome 22 short arm heteromorphism. The CA-repeat was informative in 21 out of 29 families studied and the deleted chromosome was of maternal origin in 16 cases (72%). When these data are pooled with recent results from the literature, 24 de novo DGS, velo-cardio-facial syndrome (VCFS) and isolated conotruncal cardiac disease deletions are found to be of maternal origin and 8 of paternal origin, yielding a ² of 8 with a probability level lower than 0.01. These data, and review of the literature on familial DGS/VCFS and isolated conotruncal cardiopathies suggest that there is a strong tendency for the 22q11.2 deletions to be of maternal origin.     

Neutralization of radiation damping by selective feedback on a 400 MHz NMR spectrometer

Summary Radiation damping is a phenomenon well known among NMR spectroscopists of proteins as a source of undesirable features, especially in high-field and high-Q probe NMR. In this paper, we present an electronic neutralization network which dramatically reduces radiation damping. It detects the radiation field profile and feeds back into the probe an rf field with identical amplitude and opposite phase. Experimental results of a practical implementation carried out on a 400 MHz Bruker spectrometer are shown.     

Histological and ultrastructural studies of Beauveria bassiana infection in Leptinotarsa decemlineta larvae during ecdysis

Studies of Leptinotarsa decemlineata larvae infected by Beauveria bassiana during ecdysis have enabled us to define the modes of fungal penetration employed to enter the ecdysial cuticle. We have observed the mechanically active passage of the penetrant hyphae and have followed the growth of the filaments and blastospore formation in the molting fluid. The attack of the new integument and its consequent alteration and the entry into the body cavity have also been studied. The infection develops rapidly in some of the larvae which die in premolt, while others are able to molt. Conditions rendered abnormal due to the presence of the fungus cause integumentary injuries which serve as an important factor in pathogenesis since they enhance the entry of fungal elements and bacteria thereby inducing septicemia. Contaminated larvae are able to molt, showing no signs of injury or disease, and survive for a long time, until the fungus finally invades the organism and causes death. This postponement of mortality shows that molting and hemocytic reactions are, to a certain extent, an effective defense mechanism. These last observations can be useful in the understanding of pathological processes associated with a hidden phase of fungal infection.     

Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis.

Summary A collection of about 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coli SK1592 by using the poly(dA)·poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Detection of cloned recombinant DNA molecules was based on the insertional inactivation of the Tc gene occurring at the unique BamHI cleavage site present in the vector plasmid.Thirty individual clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. CCC-recombinant plasmids extracted from E. coli were pooled in lots of 100 and used to transform auxotrophic mutants of B. subtilis 168. Complementation of these auxotrophic mutations was observed for several markers such as thr, leuA, hisA, glyB and purB. In several cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Loss of genetic markers from the hybrid plasmids did not occur when a rec ^- recipient strain of B. subtilis was used.Abbreviations Ap^R resistance to ampicillin - Tc^R resistance to tetracycline - Cm^R resistance to chloramphenicol - CCC covalently closed circular duplex - Mdal magadalton     

Lagomorph-constant region IgG allotypes: Shared e determinants inOchotona sp

The search for C allotypes as defined by the markers common to domestic rabbits (Oryctolagus) was extended to additional lagomorphs of the familiesLeporidae andOchotonidae. None of theOchotona sera obtained from Afghanistan and Iran (Ochotona rufescens) exhibited the d11, d12, or e14 allotypes. The e15 marker was detected only by radioimmune binding assays. Comparative inhibition of binding assays revealed similarities with other lagomorphs with respect to the e15i determinant. As in some variants of hare e15 markers, the e15j determinant is absent inOchotona. The results provided a revised phylogenetic scheme for the evolution of the C gene on the basis of the e-series allotypes.     

Amber mutations in Escherichia coli essential genes: isolation of mutants affected in the ribosomes.

Summary A method to obtain amber mutations in ribosomal protein genes is described. It relies on the P1-mediated localized mutagenesis (Hong and Ames, 1971) and on the fact that the recipient strain contains (a) an efficient but genetically unstable suppressor, (b) a particular thermoinducible prophage which kills suppressor hosts at 42° C. Exposure of these bacteria to the high temperature yields frequent suppressor-free derivatives while none will be found if the strain carries an amber mutation in an essential gene. Eleven mutants have been isolated by this method, of which at least six appear to carry amber mutations. All of them map close to, and to the right of spcA, in a region which codes mostly for ribosomal proteins. Three mutants were studied biochemically; all three show defective ribosomal assembly in vivo upon loss of suppression.