Protein phylogeny of translation elongation factor EF-1α suggests microsporidians are extremely ancient eukaryotes

Partial regions of the mRNA encoding a major part of translation elongation factor 1 (EF-1) from a mitochondrion-lacking protozoan,Glugea plecoglossi, that belongs to microsporidians, were amplified by polymerase chain reaction (PCR) and their primary structures were analyzed. The deduced amino acid sequence was highly divergent from typical EF-1's of eukaryotes, although it clearly showed a eukaryotic feature when aligned with homologs of the three primary kingdoms. Maximum likelihood (ML) analyses on the basis of six different stochastic models of amino acid substitutions and a maximum parsimony (MP) analysis consistently suggest that among eukaryotic species being analyzed,G. plecoglossi is likely to represent the earliest offshoot of eukaryotes. Microsporidians might be the extremely ancient eukaryotes which have diverged before an occurrence of mitochondrial symbiosis. Sequence availability: The nucleotide sequence data reported here appear in the GSDB, DDBJ, EMBL, and NCBI databases with the accession number D32139     

Details of Retropositional Genome Dynamics That Provide a Rationale for a Generic Division: The Distinct Branching of All the Pacific Salmon and Trout (Oncorhynchus) from the Atlantic Salmon and Trout (Salmo)

Salmonid species contain numerous short interspersed repetitive elements (SINEs), known collectively as the HpaI family, in their genomes. Amplification and successive integration of individual SINEs into the genomes have occurred during the evolution of salmonids. We reported previously a strategy for determining the phylogenetic relationships among the Pacific salmonids in which these SINEs were used as temporal landmarks of evolution. Here, we provide evidence for extensive genomic rearrangements that involved retropositions and deletions in a common ancestor of all the Pacific salmon and trout. Our results provide genetic support for the recent phylogenetic reassignment of steelhead and related species from the genus Salmo to the genus Oncorhynchus. Several other informative loci identified by insertions of HpaI SINEs have been isolated, and previously proposed branching orders of the Oncorhynchus species have been confirmed. The authenticity of our phylogenetic tree is supported both by the isolation of more than two informative loci per branching point and by the congruence of all our data, which suggest that the period between succesive speciations was sufficiently long for each SINE that had been amplified in the original species to become fixed in all individuals of that species.     

ATP-Activated Nonselective Cation Current in NG108-15 Cells

Abstract: ATP (1 mM) induced a biphasic increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), i.e., an initial transient increase decayed to a level of sustained increase, in NG108-15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), whereas the sustained increase was abolished by removal of external Ca²⁺. We examined the mechanism of the ATP-elicited sustained [Ca²⁺]_i increase using the fura-2 fluorescent method and the whole-cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 ± 0.8 mV (n = 10) in Tyrode external solution. The EC₅₀ of ATP was ～0.75 mM. The permeability ratio of various cations carrying this current was Na⁺ (defined as 1) > Li⁺ (0.92 ± 0.01; n = 5) > K⁺ (0.89 ± 0.03; n = 6) > Rb⁺ (0.55 ± 0.02; n = 6) > Cs⁺ (0.51 ± 0.01; n = 5) > Ca²⁺ (0.22 ± 0.03; n = 3) > N-methyl-d -glucamine (0.13 ± 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP-induced current was larger at lower concentrations of external Mg²⁺. ATP analogues that induced the current were 2-methylthio-ATP (2MeSATP), benzoylbenzoic-ATP, adenosine 5′-thiotriphosphate (ATPγS), and adenosine 5′-O-(2-thiodiphosphate), but not adenosine, ADP, α,β-methylene-ATP (AMPCPP), β,γ-methylene-ATP (AMPPCP), or UTP. Concomitant with the current data, 2MeSATP and ATPγS, but not AMPCPP or AMPPCP, increased the sustained [Ca²⁺]_i increase. We conclude that ATP activates a class of Ca²⁺-permeable nonselective cation channels via the P_2z receptor in NG108-15 cells.     

Selection of the best target site for ribozyme-mediated cleavage within a fusion gene for adenovirus E1A-associated 300 kDa protein (p300) and luciferase.

The cellular 300 kDa protein known as p300 is a target for the adenoviral E1A oncoprotein and it is thought to participate in prevention of the G0/G1 transition during the cell cycle, in activation of certain enhancers and in the stimulation of differentiation pathways. In order to determine the exact function of p300, as a first step we constructed a simple assay system for the selection of a potential target site of a hammerhead ribozyme in vivo. For the detection of ribozyme-mediated cleavage, we used a fusion gene (p300-luc) that consisted of the sequence encoding the N-terminal region of p300 and the gene for luciferase, as the reporter gene. We were also interested in the correlation of the GUX rule, for the triplet adjacent to the cleavage site, with ribozyme activity in vivo. Therefore, we selected five target sites that all included GUX The rank order of activities in vitro indeed followed the GUX rule; with respect to the kcat, a C residue as the third base (X) was the best, next came an A residue and a U residue was the worst (GUC > GUA > GUU). However, in vivo the tRNA(Val) promoter-driven ribozyme, targeted to a GUA located upstream of the initiation codon, had the highest inhibitory effect (96%) in HeLa S3 cells when the molar ratio of the DNA template for the target p300 RNA to that for the ribozyme was 1:4. Since the rank order of activities in vivo did not conform to the GUX rule, it is unlikely that the rate limiting step for cleavage of the p300-luc mRNA was the chemical step. This kind of ribozyme expression system should be extremely useful for elucidation of the function of p300 in vivo.     

Purification and characterization of microbial gellan lyase.

Gellan lyase was purified from the culture fluid of soil samples incubated in a medium containing gellan as a sole carbon source. The enzyme was a monomer with a molecular mass of 140 kDa and was most active at pH 7.5 and 45 degrees C. The enzyme was highly specific to gellan and lowered the viscosity of the polymer.     

Simultaneous determination of ascorbic acid and dehydroascorbic acid in fish tissues by high-performance liquid chromatography

An high-performance liquid chromatographic method with post-column derivatization has been developed for the simultaneous determination of ascorbic acid (AA) and dehydroascorbic acid (DHAA) in fish tissues. Extracted AA and DHAA were separated by a Shim-pack SCR-101H column within 20 min, reacted with sodium hydroxide containing sodium borohydride and monitored at 300 nm. The detection limits for both AA and DHAA were 0.1 μg/ml.     

Visualization of mitotic chromosomes in filamentous fungi by fluorescence staining and fluorescence in situ hybridization

Mitotic chromosomes of the plant pathogenic filamentous fungi Botrytis cinerea and Alternaria alternata were observed. Chromosomes prepared by the germ tube burst method were stained with the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI) to yield figures with good resolution. Using this method, component chromosomes were clearly distinguished and the chromosome number could be determined. Fluorescence in situ hybridization (FISH) was also successfully applied to the specimens, revealing one ribosomal RNA gene cluster, or nucleolus organizer region (NOR) in the genome of each fungus. A long attenuated chromatid thread expanding from a condensed metaphase chromosome, which had been called a thread-like structure in B. cinerea, was proved to be an NOR. This is the first report of the successful application of FISH to the chromosomes of filamentous fungi.     

Migration Behavior of Granule Cell Neurons in Cerebellar Cultures I. A PKH26 Labeling Study in Microexplant and Organotypic Cultures

Granule cells were dissociated from early postnatal mouse cerebella and labeled with a fluorescent dye probe PKH26. Small number of the labeled cells were mixed with cerebellar cortical microexplant cultures or transplanted into cerebellar cortical organotypic explants, and their time-dependent morphological changes during cultures were examined with fluorescence microscopy. Granule cell neurons first extended asymmetrical short bipolar processes in both cultures, and migrated actively in microexplant cultures. After elongation of symmetrically bipolar long and thin neurites, they sprouted short thick processes from cell bodies and migrated perpendicular to neurite bundles that were devoid of glia in microexplant cultures, or migrated vertically inward into the internal granular layer in the organotypic explant. During such migrations, they extended short thick processes in front and thin processes behind the cell body. The latter processes were connected to thin long neurites with T- or Y-shaped junctions in both cultures. Finally, they extended many short thick processes from cell bodies in both cultures. Such behaviors of granule cell neurons in microexplant cultures were, thus, similar to those in organotypic explant cultures despite of the absence of Bergmann glial cells. These migration patterns may be closely related to migration of granule cells in histogenesis of the cerebellar cortex.     

Migration Behavior of Granule Cell Neurons in Cerebellar Cultures. II. An Electron Microscopic Study

We examined the fine structure of migrating granule cell neurons in cerebellar microexplant cultures. Radially migrating bipolar cells extended microspikes or small filopodia from their soma and processes and frequently made contact with neighboring cells. These microspikes contained microfilaments but no microtubules. At the later phase of the migration, in which they had symmetrical bipolar long processes, filopodia extending from perikarial region of cells contained microtubules, suggesting that they are precursors of the future thick perpendicular processes. When cell bodies changed orientation from radial to perpendicular, microtubules that were nucleated from perinuclear centrioles frequently extended into both thick radial and perpendicular processes from the perikarial region. Bundles of 10nm intermediate filaments also appeared in these processes. During migration by the perpendicular contact guidance, many filopodia extending from both the thick leading processes and thin trailing processes made close contacts with the radial parallel neurite. These findings suggest that; 1) The direct contact of the filopodia from both the growth cones and their processes of the granule cells to the neurite bundle plays roles in both the parallel and perpendicular contact guidances. 2) The spacial and temporal changes of cytoskeletons and the association of microtubules with perinuclear centrioles are important for the formation of perpendicular processes and initiation of the perpendicular contact guidance.     

Identification of conserved domains in the Δ12 desaturases of cyanobacteria

Cyanobacterial genes for enzymes that desaturate fatty acids at the 12 position, designated desA, were isolated from Synechocystis PCC6714, Synechococcus PCC7002 and Anabaena variabilis by crosshybridization with a DNA probe derived from the desA gene of Synechocystis PCC6803. The genes of Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis encode proteins of 349, 347 and 350 amino acid residues, respectively. The transformation of Synechococcus PCC7942 with the desA genes from Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis was associated with the ability to introduce a second double bond at the 12 position of fatty acids. The amino acid sequence of the products of the desA genes revealed the presence of four conserved domains. Since one of the conserved domains was also found in the amino acid sequences of 3 desaturases of Brassica napus and mung bean, this domain may play an essential role in the introduction of a double bond into fatty acids bound to membrane lipids.Abbreviations X:Y(Z) fatty acid containing X carbon atoms with Y double bonds in the cis configuration at position Z counted from the carboxyl terminus