Phylogeography of Kandelia candel in East Asiatic mangroves based on nucleotide variation of chloroplast and mitochondrial DNAs

Vivipary with precocious seedlings in mangrove plants was thought to be a hindrance to long-range dispersal. To examine the extent of seedling dispersal across oceans, we investigated the phylogeny and genetic structure among East Asiatic populations of Kandelia candel based on organelle DNAs. In total, three, 28 and seven haplotypes of the chloroplast DNA (cpDNA) atpB-rbcL spacer, cpDNA trnL-trnF spacer, and mitochondrial DNA (mtDNA) internal transcribed spacer (ITS) were identified, respectively, from 202 individuals. Three data sets suggested consistent phylogenies recovering two differentiated lineages corresponding to geographical regions, i.e. northern South-China-Sea + East-China-Sea region and southern South-China-Sea region (Sarawak). Phylogenetically, the Sarawak population was closely related to the Ranong population of western Peninsula Malaysia instead of other South-China-Sea populations, indicating its possible origin from the Indian Ocean Rim. No geographical subdivision was detected within the northern geographical region. An analysis of molecular variance (AMOVA) revealed low levels of genetic differentiation between and within mainland and island populations (phiCT = 0.015, phiSC = 0.037), indicating conspicuous long-distance seedling dispersal across oceans. Significant linkage disequilibrium excluded the possibility of recurrent homoplasious mutations as the major force causing phylogenetic discrepancy between mtDNA and the trnL-trnF spacer within the northern region. Instead, relative ages of alleles contributed to non-random chlorotype-mitotype associations and tree inconsistency. Widespread distribution and random associations (chi2 = 0.822, P = 0.189) of eight hypothetical ancestral cytotypes indicated the panmixis of populations of the northern geographical region as a whole. In contrast, rare and recently evolved alleles were restricted to marginal populations, revealing some preferential directional migration.     

Crystal structures and enzymatic properties of three formyltransferases from archaea: environmental adaptation and evolutionary relationship

Formyltransferase catalyzes the reversible formation of formylmethanofuran from N(5)-formyltetrahydromethanopterin and methanofuran, a reaction involved in the C1 metabolism of methanogenic and sulfate-reducing archaea. The crystal structure of the homotetrameric enzyme from Methanopyrus kandleri (growth temperature optimum 98 degrees C) has recently been solved at 1.65 A resolution. We report here the crystal structures of the formyltransferase from Methanosarcina barkeri (growth temperature optimum 37 degrees C) and from Archaeoglobus fulgidus (growth temperature optimum 83 degrees C) at 1.9 A and 2.0 A resolution, respectively. Comparison of the structures of the three enzymes revealed very similar folds. The most striking difference found was the negative surface charge, which was -32 for the M. kandleri enzyme, only -8 for the M. barkeri enzyme, and -11 for the A. fulgidus enzyme. The hydrophobic surface fraction was 50% for the M. kandleri enzyme, 56% for the M. barkeri enzyme, and 57% for the A. fulgidus enzyme. These differences most likely reflect the adaptation of the enzyme to different cytoplasmic concentrations of potassium cyclic 2,3-diphosphoglycerate, which are very high in M. kandleri (>1 M) and relatively low in M. barkeri and A. fulgidus. Formyltransferase is in a monomer/dimer/tetramer equilibrium that is dependent on the salt concentration. Only the dimers and tetramers are active, and only the tetramers are thermostable. The enzyme from M. kandleri is a tetramer, which is active and thermostable only at high concentrations of potassium phosphate (>1 M) or potassium cyclic 2,3-diphosphoglycerate. Conversely, the enzyme from M. barkeri and A. fulgidus already showed these properties, activity and stability, at much lower concentrations of these strong salting-out salts.     

Theoretical Characterization of the Long-range Attraction between G-actin Molecules through the Excluded Volume Effect

One of the interactions between macromolecules is the attractive forcethrough the excluded volume effect. We studied the attraction betweenthe molecules of muscle protein, actin, in the two points by using theextended scaled particle theory (XSPT). I) we verified the basic assumptionused in the XSPT that topological elements which determine the analyticalexpression of the excluded volume are almost unchanged through the scalingdown of the solute molecule in the thought experiment. Results of thecomputational geometry method (-shape method) showed that thisassumption is valid even in the case of the actin molecule. II) wecalculated the attraction between actin monomer molecules, G-actin.Calculated differences of the values of the attraction potential of twomacromolecules between at contact and at one macromolecule apart by theXSPT is almost the same as those by the Asakura-Oosawa theory.     

A method for allelic replacement in Francisella tularensis

A vector for mutagenesis of Francisella tularensis was constructed based on the pUC19 plasmid. By inserting the sacB gene of Bacillus subtilis, oriT of plasmid RP4, and a chloramphenicol resistance gene of Shigella flexneri, a vector, pPV, was obtained that allowed specific mutagenesis. A protocol was developed that allowed introduction of the vector into the live vaccine strain, LVS, of F. tularensis by conjugation. As a proof of principle, we aimed to develop a specific mutant defective in expression of a 23-kDa protein (iglC) that we previously have shown to be prominently upregulated during intracellular growth of F. tularensis. A plasmid designated pPV-DeltaiglC was developed that contained only the regions flanking the encoding gene, iglC. By a double crossover event, the chromosomal iglC gene was deleted. However, the resulting strain, denoted DeltaiglC1, still had an intact iglC gene. Southern blot analysis verified that LVS harbors two copies for the iglC gene. The mutagenesis was therefore repeated and a mutant defective in both iglC alleles, designated DeltaiglC1+2, was obtained. The DeltaiglC1+2 strain, in contrast to DeltaiglC1, was shown to display impaired intracellular macrophage growth and to be attenuated for virulence in mice. The developed genetic system has the potential to provide a tool to elucidate virulence mechanisms of F. tularensis and the specific F. tularensis mutant illustrates the critical role of the 23-kDa protein, iglC, for the virulence of F. tularensis LVS.     

The caddisfly Ceraclea fulva and the freshwater sponge Ephydatia fluviatilis: a successful relationship

The association between the aquatic phases of the caddisfly Ceraclea fulva (Trichoptera, Leptoceridae) and the freshwater sponge Ephydatia fluviatilis (Porifera, Spongillidae) has been investigated by means of scanning and transmission electron microscopy (SEM, TEM). Ceraclea fulva habitually feeds on sponges and builds its case by using the siliceous spicules of the sponge, which are arranged side by side, inter-crossed, cemented with silk, and organised in layers. In the newly hatched larva, the case is strengthened exclusively by cemented siliceous spicules, while during growth, the insect adds sponge fragments to it. The fine organisation of the sponge tissues growing on the case proves that the sponge is functional. Inter-spaced, small protrusions, derived from the outermost compact silk layer, form a series of "bridges" enhancing case/sponge adhesion. Tube-case shape varies according to the aquatic developmental phase of the insect: in the mature larva and pupa, this shelter carries larger sponge fragments dorsally. The caddisfly acts as carrier of the sponge, thus facilitating its dispersal and the colonisation of new habitats. This justifies regarding this association as a successful mutualistic relationship, and not as a unilateral parasitic behaviour on the part of the insect.     

Hypervariable spacer regions are good sites for developing specific PCR-RFLP markers and PCR primers for screening actinorhizal symbionts

While the ribosomal RNA like highly conserved genes are good molecular chronometers for establishing phylogenetic relationships, they can also be useful in securing the amplification of adjoining hyper-variable regions. These regions can then be used for developing specific PCR primers or PCR-RFL profiles to be used as molecular markers. We report here the use of ITS region ofrrn operon ofFrankia for developing PCR-RFL profiles capable of discriminating between closely related frankiae. We have also made use of the ITS 1 region of the nuclearrrn operon ofAlnus nepalensis (D Don) for designing a PCR primer for specific amplification of nuclear DNA of this tree.     

Transmembrane domains in the functions of Fc receptors

In the present study, we use a novel method, PHDhtm, to predict the exact locations and extents of the transmembrane (TM) domains of multisubunit immunoglobulin Fc-receptors. Whereas most previous studies have used single residue hydrophobicity plots for characterizing of these domains, PHDhtm utilizes a system of neural networks and the evolutionary information contained in multiple alignments of related sequences to predict the above. Present PHDhtm application predicts TM domains of immunoglobulin Fc-receptors that in many cases differ significantly from those derived by using earlier methods. Comparisons of helical wheel projections of the presently derived TM domains from PHDhtm with those produced earlier reveal different hydrophobic moments as well as hydrophobic and hydrophilic surfaces. These differences probably alter the character of subunit association within the receptor complexes. This new algorithm can also be used for other membrane protein complexes and may advance both understanding the principles underlying such complexes formation and design of peptides that can interfere with such TM domain association so as to modulate specific cellular responses.     

An improved model of association for VH-VL immunoglobulin domains: asymmetries between VH and VL in the packing of some interface residues

The antibody-binding site is formed as a result of the association between VH and VL domains. Several studies have shown that this association plays an important role in the mechanism of antigen-antibody interaction (Stanfield et al. Structure 1: 83-93, 1993). Considering this, we propose that variations in the VH-VL association are part of the diversification strategy of the antibody repertoires. Previously, a model of association for VH-VL domains based on geometrical characteristics of the packing at the interface was developed by Chothia et al. (J. Mol. Biol. 186: 61-663, 1985). This model includes a common association form for antibodies and a three-layer structure for the interface. In the present work, a complementary model is introduced to account for the general geometrical restrictions of the VH-VL interface, and particular arrangements related to the chemical properties or the side-chain orientations of participating residues. Groups of residues assume common side-chain orientations, which are apparently related to particular functions of different interface zones. Analyses of amino acid usage and network are in agreement with the side-chain orientation patterns. Based on these observations, a three-zone model has evolved to illuminate geometrical and functional restrictions acting over the VH-VL interface. Additionally, this study has revealed the asymmetrical relationships between VH and VL residues important for the association of the two domains.     

Species association among predaceous and phytophagous apple mites (Acari: Eriophyidae, Phytoseiidae, Stigmaeidae, Tetranychidae)

Predator–predator, predator–prey, and prey–prey associations among nine species of mites were studied in a plot of 100 Red Delicious apple (Malus pumila Miller) trees from 1990 to 1997. In 1990, seven-year-old trees were inoculated with Panonychus ulmi (Koch), Tetranychus urticae Koch (Acari: Tetranychidae) or both, and sprayed with azinphosmethyl (alone or plus endosulfan), or nothing. The species Zetzellia mali (Ewing) (Acari: Stigmaeidae), Amblyseius andersoni Chant (Acari: Phytoseiidae), Eotetranychus sp., Bryobia rubrioculus (Scheuten) (Acari: Tetranychidae), and Aculus schlechtendali Nalepa (Acari: Eriophyidae) were already present or immigrated into plots, and Galendromus occidentalis (Nesbitt) and Typhlodromus pyri Scheuten (Acari: Phytoseiidae) were introduced. Yule's V association index was used to measure positive, neutral, or negative interspecific associations for each species pair, because of its robustness with spatially autocorrelated data. We found that pesticide and release treatments did not greatly affect the association results, but there were strong seasonal differences. Predator–predator associations were the strongest and most consistent, showing negative associations in the early and mid seasons, and neutral ones in late season. Negative associations of T. pyri with other predators were the strongest, which is consistent with evidence that this mite can detect other predators on a leaf. Predator–prey seasonal associations were mixed, with some positive and others negative, with most significant associations occurring in the mid season. One prey–prey interaction was positive, again in mid season, most likely because of similar habitat preferences.     

Carboxyl-terminal modification alters the subunit interactions and assembly pathways of normal and sickle hemoglobins

Parallel isofocusing studies established that carboxypeptidase A removal of the His-146 (HC3) and Tyr-145 (HC2) residues of heme subunits affected the assembly properties of both Des (A) and Des (S) with heme chains, albeit to differing degrees. Indeed, the rate of Des (A) oligomer dissociation (k ₁), as determined by visible spectroscopy, was 4.3-fold faster than that of its native (A) counterpart. Furthermore, Soret spectral studies have affirmed distinct rates of normal (HbA), sickle (HbS), and Des HbA hemoglobin assembly (k₂) from their and [Des (A)] heme-containing monomers. Matching kinetic analysis of Des (A) and Des (S) chain assembly (with an identical chain) revealed 4.6- and 7.8-fold faster combination rates than those seen for (A) and (S) chains, respectively. This 3-fold disparity in rates strongly supports the critical role of the -6 (A3) residue, and its amino-terminal region, in chain partner recognition and subsequent human hemoglobin assembly.