Reconstructing evolutionary trees from DNA and protein sequences: paralinear distances.
(33/36)
The reconstruction of phylogenetic trees from DNA and protein sequences is confounded by unequal rate effects. These effects can group rapidly evolving taxa with other rapidly evolving taxa, whether or not they are genealogically related. All algorithms are sensitive to these effects whenever the assumptions on which they are based are not met. The algorithm presented here, called paralinear distances, is valid for a much broader class of substitution processes than previous algorithms and is accordingly less affected by unequal rate effects. It may be used with all nucleic acid, protein, or other sequences, provided that their evolution may be modeled as a succession of Markov processes. The properties of the method have been proven both analytically and by computer simulations. Like all other methods, paralinear distances can fail when sequences are misaligned or when site-to-site sequence variation of rates is extensive. To examine the usefulness of paralinear distances, the "origin of the eukaryotes" has been investigated by the analysis of elongation factor Tu sequences with a variety of sequence alignments. It has been found that the order in which sequences are pairwise aligned strongly determines the topology which is reconstructed by paralinear distances (as it does for all other reconstruction methods tested). When the parts of the alignment that are unaffected by alignment order are analyzed, paralinear distances strongly select the eocyte topology. This provides evidence that the eocyte prokaryotes are the closest prokaryotic relatives of the eukaryotes. (+info)
Evidence for farnesol-mediated isoprenoid synthesis regulation in a halophilic archaeon, Haloferax volcanii.
(34/36)
Farnesol strongly inhibited growth of a halophilic archaeon, Haloferax volcanii, with an IC50 value of only 2 microM (0.4 microgram/ml) in rich medium and 50 nM (0.01 microgram/ml) in minimal medium without lysis. Other isoprenoid alcohols such as isopentenol, dimethylallyl alcohol, geraniol, and geranylgeraniol at 500 microM did not affect its growth. Mevalonate, which is the precursor of all isoprenoid membrane lipids in archaea, led to recovery of the growth inhibition of H. volcanii, but acetate had no such effect. Farnesol inhibited incorporation of acetate, but not mevalonate, into the lipid fraction. These results suggest that farnesol inhibited the biosynthetic pathway from acetate (acetyl-CoA) to mevalonate. Farnesol is known to be derived from the important intermediate of isoprenoids, farnesyl diphosphate (FPP), and found in neutral lipid fraction from this archaeon. Moreover, the cell-free extracts from H. volcanii could phosphorylate farnesol with ATP to generate farnesyl monophosphate and FPP. We conclude that farnesol-mediated isoprenoid synthesis regulation system by controlling farnesol concentration is present in H. volcanii. (+info)
Seryl-tRNA synthetase from the extreme halophile Haloarcula marismortui--isolation, characterization and sequencing of the gene and its expression in Escherichia coli.
(35/36)
The seryl-tRNA synthetase from the extreme halophilic archaebacterium Haloarcula marismortui, belonging to the group Euryarchaeota, has been purified and its hyperhalophilic behavior demonstrated by activity and stability tests in KCl, NaCl and MgCl2 solutions. Although the natural external environment of this archaebacterium is rich in sodium ions and poor in potassium ions, the converse being the case in the bacterial cytosol. there is no large significant difference in activity and stability in vitro of the enzyme between solutions of NaCl and KCl. Low, but not high, concentrations of MgCl2 stabilize the enzyme. The enzyme aminoacylates tRNA from Escherichia coli even under the high salt conditions of the assay. A fluorescence study indicated that low salt denaturation of the hyperhalophilic enzyme is a biphasic process. The hyperhalophilic enzyme demonstrated immunological reactivity with antisera against the catalytic domain of the homologous E. coli enzyme. The gene coding for the H. marismortui enzyme has been isolated and sequenced. The derived amino acid sequence is the first of a hyperhalophilic aminoacyl-tRNA synthetase. The wild-type gene and a mutant gene with a deletion of the halophile-specific insertion were expressed in E. coli using the T7 RNA polymerase and the Thiofusion expression systems. None of the expressed proteins were enzymically active. A structural model has been produced by comparison with other seryl-tRNA synthetases which illustrates the high negative-charge density of the surface of the hyperhalophilic enzyme. (+info)
Characterization and subunit structure of the ATP synthase of the halophilic archaeon Haloferax volcanii and organization of the ATP synthase genes.
(36/36)
The archaeal ATPase of the halophile Haloferax volcanii synthesizes ATP at the expense of a proton gradient, as shown by sensitivity to the uncoupler carboxyl cyanide p-trifluoromethoxyphenylhydrazone, to the ionophore nigericin, and to the proton channel-modifying reagent N,N'-dicyclohexylcarbodiimide. The conditions for an optimally active ATP synthase have been determined. We were able to purify the enzyme complex and to identify the larger subunits with antisera raised against synthetic peptides. To identify additional subunits of this enzyme complex, we cloned and sequenced a gene cluster encoding five hydrophilic subunits of the A1 part of the proton-translocating archaeal ATP synthase. Initiation, termination, and ribosome-binding sequences as well as the result of a single transcript suggest that the ATPase genes are organized in an operon. The calculated molecular masses of the deduced gene products are 22. 0 kDa (subunit D), 38.7 kDa (subunit C), 11.6 kDa (subunit E), 52.0 kDa (subunit B), and 64.5 kDa (subunit A). The described operon contains genes in the order D, C, E, B, and A; it contains no gene for the hydrophobic, so-called proteolipid (subunit c, the proton-conducting subunit of the A0 part). This subunit has been isolated and purified; its molecular mass as deduced by SDS-polyacrylamide gel electrophoresis is 9.7 kDa. (+info)