Stromules: a characteristic cell-specific feature of plastid morphology

Stromules (stroma-filled tubules) are highly dynamic structures extending from the surface of all plastid types examined so far, including proplastids, chloroplasts, etioplasts, leucoplasts, amyloplasts, and chromoplasts. Stromules are usually 0.35-0.85 microm in diameter and of variable length, from short beak-like projections to linear or branched structures up to 220 mum long. They are enclosed by the inner and outer plastid envelope membranes and enable the transfer of molecules as large as Rubisco (approximately 560 kDa) between interconnected plastids. Stromules occur in all cell types, but stromule morphology and the proportion of plastids with stromules vary from tissue to tissue and at different stages of plant development. In general, stromules are more abundant in tissues containing non-green plastids, and in cells containing smaller plastids. The primary function of stromules is still unresolved, although the presence of stromules markedly increases the plastid surface area, potentially increasing transport to and from the cytosol. Other functions of stromules, such as transfer of macromolecules between plastids and starch granule formation in cereal endosperm, may be restricted to particular tissues and cell types.     

Effect of miracidial dose on adoptively transferred resistance to Schistosoma mansoni in the snail intermediate host, Biomphalaria glabrata

Adoptively transferred resistance to Schistosoma mansoni in the snail intermediate host Biomphalaria glabrata was measured as a function of miracidial challenge dose. Schistosome-susceptible snails implanted with the amebocyte-producing organ (APO) from resistant donors showed 29 and 39% prevalences of infection after challenge with 5 and 10 miracidia, respectively, but 68-83% prevalences when exposed to 25-200 miracidia. Prevalences in control (untampered) susceptible snails ranged from 97 to 100% at the different miracidial doses. Higher infection prevalences at elevated doses suggest that a range of transferred resistance occurs and possibly that low levels of APO-derived plasma factors or hemocytes in some recipients can be overwhelmed by larger numbers of parasites.     

Isolation of a unique membrane protein from Naegleria fowleri

Naegleria fowleri, an amoeboflagellate, is the causative agent of Primary Amoebic Meningoencephalitis, a fulminating disease of the central nervous system. In order to elucidate the mechanisms of pathogenicity of this amoeba, a cDNA expression library was prepared from N. fowleri RNA. A specific protein was found to be expressed from a cDNA clone designated Mp2CL5. Northern blot analysis showed that the Mp2CL5 mRNA was expressed in pathogenic N. fowleri but was not expressed in non-pathogenic Naegleria species nor in Acanthamoeba. Western blot analysis using anti-N. fowleri antiserum demonstrated that IPTG-induced Escherichia coli Mp2CL5 expressed a 23-kDa recombinant protein. The Mp2CL5 recombinant protein was histidine-tagged and purified to homogeneity from E. coli. A polyclonal rabbit antiserum was prepared against the purified Mp2CL5 recombinant protein. This antibody was used to further characterize the Mp2CL5 native protein expressed by N. fowleri. Western blot analysis in conjunction with immunofluorescence microscopy demonstrated the presence of a native protein of 17 kDa on the plasma membrane of N. fowleri trophozoites. The native N. fowleri protein was expressed in the logarithmic phase of trophozoite growth and the production of this protein increased through the stationary phase of growth. Studies are in progress to examine further its role as a virulence factor.     

MAGIC database and interfaces: an integrated package for gene discovery and expression

The rapidly increasing rate at which biological data is being produced requires a corresponding growth in relational databases and associated tools that can help laboratories contend with that data. With this need in mind, we describe here a Modular Approach to a Genomic, Integrated and Comprehensive (MAGIC) Database. This Oracle 9i database derives from an initial focus in our laboratory on gene discovery via production and analysis of expressed sequence tags (ESTs), and subsequently on gene expression as assessed by both EST clustering and microarrays. The MAGIC Gene Discovery portion of the database focuses on information derived from DNA sequences and on its biological relevance. In addition to MAGIC SEQ-LIMS, which is designed to support activities in the laboratory, it contains several additional subschemas. The latter include MAGIC Admin for database administration, MAGIC Sequence for sequence processing as well as sequence and clone attributes, MAGIC Cluster for the results of EST clustering, MAGIC Polymorphism in support of microsatellite and single-nucleotide-polymorphism discovery, and MAGIC Annotation for electronic annotation by BLAST and BLAT. The MAGIC Microarray portion is a MIAME-compliant database with two components at present. These are MAGIC Array-LIMS, which makes possible remote entry of all information into the database, and MAGIC Array Analysis, which provides data mining and visualization. Because all aspects of interaction with the MAGIC Database are via a web browser, it is ideally suited not only for individual research laboratories but also for core facilities that serve clients at any distance.     

Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin

Post-translational histone modifications regulate epigenetic switching between different chromatin states. Distinct histone modifications, such as acetylation, methylation and phosphorylation, define different functional chromatin domains, and often do so in a combinatorial fashion. The centromere is a unique chromosomal locus that mediates multiple segregation functions, including kinetochore formation, spindle-mediated movements, sister cohesion and a mitotic checkpoint. Centromeric (CEN) chromatin is embedded in heterochromatin and contains blocks of histone H3 nucleosomes interspersed with blocks of CENP-A nucleosomes, the histone H3 variant that provides a structural and functional foundation for the kinetochore. Here, we demonstrate that the spectrum of histone modifications present in human and Drosophila melanogaster CEN chromatin is distinct from that of both euchromatin and flanking heterochromatin. We speculate that this distinct modification pattern contributes to the unique domain organization and three-dimensional structure of centromeric regions, and/or to the epigenetic information that determines centromere identity.     

Evaluating hypotheses of deuterostome phylogeny and chordate evolution with new LSU and SSU ribosomal DNA data

We investigated evolutionary relationships among deuterostome subgroups by obtaining nearly complete large-subunit ribosomal RNA (LSU rRNA)-gene sequences for 14 deuterostomes and 3 protostomes and complete small-subunit (SSU) rRNA-gene sequences for five of these animals. With the addition of previously published sequences, we compared 28 taxa using three different data sets (LSU only, SSU only, and combined LSU + SSU) under minimum evolution (with LogDet distances), maximum likelihood, and maximum parsimony optimality criteria. Additionally, we analyzed the combined LSU + SSU sequences with spectral analysis of LogDet distances, a technique that measures the amount of support and conflict within the data for every possible grouping of taxa. Overall, we found that (1) the LSU genes produced a tree very similar to the SSU gene tree, (2) adding LSU to SSU sequences strengthened the bootstrap support for many groups above the SSU-only values (e.g., hemichordates plus echinoderms as Ambulacraria; lancelets as the sister group to vertebrates), (3) LSU sequences did not support SSU-based hypotheses of pterobranchs evolving from enteropneusts and thaliaceans evolving from ascidians, and (4) the combined LSU + SSU data are ambiguous about the monophyly of chordates. No tree-building algorithm united urochordates conclusively with other chordates, although spectral analysis did so, providing our only evidence for chordate monophyly. With spectral analysis, we also evaluated several major hypotheses of deuterostome phylogeny that were constructed from morphological, embryological, and paleontological evidence. Our rRNA-gene analysis refutes most of these hypotheses and thus advocates a rethinking of chordate and vertebrate origins.     

Juvenile hormone and octopamine in the regulation of division of labor in honey bee colonies

Forager honey bees have high circulating levels of juvenile hormone (JH) and high brain levels of octopamine, especially in the antennal lobes, and treatment with either of these compounds induces foraging. Experiments were performed to determine whether octopamine acts more proximally than JH to affect the initiation of foraging behavior. Bees treated with octopamine became foragers more rapidly than bees treated with the JH analog methoprene. Bees treated with methoprene showed an increase in antennal lobe levels of octopamine, especially after 12 days. Bees with no circulating JH (corpora allata glands removed) treated with octopamine became foragers in similar numbers to bees with intact corpora allata. These results suggest that JH affects the initiation of foraging at least in part by increasing brain levels of octopamine, but octopamine can act independently of JH. Effects of JH that are not related to octopamine also are possible, as bees treated with both octopamine and methoprene were more likely to become foragers than bees treated with only octopamine or methoprene.     

Comparative sequence analysis of the symbiosis island of Mesorhizobium loti strain R7A

The Mesorhizobium loti strain R7A symbiosis island is a 502-kb chromosomally integrated element which transfers to nonsymbiotic mesorhizobia in the environment, converting them to Lotus symbionts. It integrates into a phenylalanine tRNA gene in a process mediated by a P4-type integrase encoded at the left end of the element. We have determined the nucleotide sequence of the island and compared its deduced genetic complement with that reported for the 611-kb putative symbiosis island of M. loti strain MAFF303099. The two islands share 248 kb of DNA, with multiple deletions and insertions of up to 168 kb interrupting highly conserved colinear DNA regions in the two strains. The shared DNA regions contain all the genes likely to be required for Nod factor synthesis, nitrogen fixation, and island transfer. Transfer genes include a trb operon and a cluster of potential tra genes which are also present on the strain MAFF303099 plasmid pMLb. The island lacks plasmid replication genes, suggesting that it is a site-specific conjugative transposon. The R7A island encodes a type IV secretion system with strong similarity to the vir pilus from Agrobacterium tumefaciens that is deleted from MAFF303099, which in turn encodes a type III secretion system not found on the R7A island. The 414 genes on the R7A island also include putative regulatory genes, transport genes, and an array of metabolic genes. Most of the unique hypothetical genes on the R7A island are strain-specific and clustered, suggesting that they may represent other acquired genetic elements rather than symbiotically relevant DNA.