An evaluation of elongation factor 1 alpha as a phylogenetic marker for eukaryotes.
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Elongation factor 1 alpha (EF-1 alpha) is a highly conserved ubiquitous protein involved in translation that has been suggested to have desirable properties for phylogenetic inference. To examine the utility of EF-1 alpha as a phylogenetic marker for eukaryotes, we studied three properties of EF-1 alpha trees: congruency with other phyogenetic markers, the impact of species sampling, and the degree of substitutional saturation occurring between taxa. Our analyses indicate that the EF-1 alpha tree is congruent with some other molecular phylogenies in identifying both the deepest branches and some recent relationships in the eukaryotic line of descent. However, the topology of the intermediate portion of the EF-1 alpha tree, occupied by most of the protist lineages, differs for different phylogenetic methods, and bootstrap values for branches are low. Most problematic in this region is the failure of all phylogenetic methods to resolve the monophyly of two higher-order protistan taxa, the Ciliophora and the Alveolata. JACKMONO analyses indicated that the impact of species sampling on bootstrap support for most internal nodes of the eukaryotic EF-1 alpha tree is extreme. Furthermore, a comparison of observed versus inferred numbers of substitutions indicates that multiple overlapping substitutions have occurred, especially on the branch separating the Eukaryota from the Archaebacteria, suggesting that the rooting of the eukaryotic tree on the diplomonad lineage should be treated with caution. Overall, these results suggest that the phylogenies obtained from EF-1 alpha are congruent with other molecular phylogenies in recovering the monophyly of groups such as the Metazoa, Fungi, Magnoliophyta, and Euglenozoa. However, the interrelationships between these and other protist lineages are not well resolved. This lack of resolution may result from the combined effects of poor taxonomic sampling, relatively few informative positions, large numbers of overlapping substitutions that obscure phylogenetic signal, and lineage-specific rate increases in the EF-1 alpha data set. It is also consistent with the nearly simultaneous diversification of major eukaryotic lineages implied by the "big-bang" hypothesis of eukaryote evolution. (+info)
Cloning, sequencing, and nucleolar targeting of the basal-body-binding nucleolar protein BN46/51.
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BN46/51 is an acidic protein found in the granular component of the nucleolus of the amebo-flagellate Naegleria gruberi. When Naegleria amebae differentiate into swimming flagellates, BN46/51 is found associated with the basal body complex at the base of the flagella. In order to determine the factors responsible for targeting BN46/51 to a specific subnucleolar region, cDNAs coding for both subunits were isolated and sequenced. Two clones, JG4.1 and JG12.1 representing the 46 kDa and 51 kDa subunits, respectively, were investigated in detail. JG12.1 encoded a polypeptide of 263 amino acids with a predicted size of 30.1 kDa that co-migrated with the 51 kDa subunit of BN46/51 when expressed in yeast. JG4.1 encoded a polypeptide of 249 amino acids with a predicted size of 28.8 kDa that co-migrated with the 46 kDa subunit of BN46/51. JG4.1 was identical to JG12.1 except for the addition of an aspartic acid between positions 94 and 95 of the JG12.1 sequence and the absence of 45 amino acids beginning at position 113. The predicted amino acid sequences were not closely related to any previously reported. However, the sequences did have 26-31% identity to a group of FKPBs (FK506 binding proteins) but lacked the peptidyl-prolyl cis-trans isomerase domain of the FKBPs. Both subunits contained two KKE and three KKX repeats found in other nucleolar proteins and in some microtubule binding proteins. Using 'Far Western' blots of nucleolar proteins, BN46/51 bound to polypeptides of 44 kDa and 74 kDa. The 44 kDa component was identified as the Naegleria homologue of fibrillarin. BN46/51 bound specifically to the nucleoli of fixed mammalian cells, cells which lack a BN46/51 related polypeptide. When the JG4.1 and JG12.1 cDNAs were expressed in yeast, each subunit was independently targeted to the yeast nucleolus. We conclude that BN46/51 represents a unique nucleolar protein that can form specific complexes with fibrillarin and other nucleolar proteins. We suggest that the association of BN46/51 with the MTOC of basal bodies may reflect its role in connecting the nucleolus with the MTOC activity for the mitotic spindle. This would provide a mechanism for nucleolar segregation during the closed mitosis of Naegleria amebae. (+info)
Expression of the Naegleria intron endonuclease is dependent on a functional group I self-cleaving ribozyme.
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NaSSU1 is a complex nuclear group I intron found in several species of Naegleria, consisting of a large self-splicing group I ribozyme (NaGIR2), which itself is interrupted by a small, group I-like ribozyme (NaGIR1) and an open reading frame (ORF) coding for a homing endonuclease. The GIR1 ribozyme cleaves in vitro transcripts of NaSSU1 at two internal processing sites about 400 nt downstream of the 5' end of the intron, proximal to the endonuclease ORF. Here we demonstrate that self-cleavage of the excised intron also occurs in vivo in Naegleria gruberi, generating an ORF-containing RNA that possesses a short leader with a sequence element likely to be involved in gene expression. To assess the functional significance of self-cleavage, we constructed a genetic system in Saccharomyces cerevisiae. First, a mutant yeast strain was selected with a mutation in all the rRNA genes, rendering the rDNA resistant to cleavage by the Naegleria endonuclease. Active endonuclease, which is otherwise lethal, could be expressed readily in these cells. Endonuclease activity also could be detected in extracts of yeast harboring plasmids in which the endonuclease ORF was embedded in its native context in the intron. Analysis of the RNA from these yeast cells showed that the excised intron RNA was processed as in N. gruberi. A mutant intron constructed to prevent self-cleavage of the RNA failed to express endonuclease activity. These results support the hypothesis that the NaGIR1-catalyzed self-cleavage of the intron RNA is a key event in expression of the endonuclease. (+info)
Functional characterization of isoschizomeric His-Cys box homing endonucleases from Naegleria.
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Several species within the amoeboflagellate genus Naegleria harbor an optional ORF containing group I introns in their nuclear small subunit ribosomal DNA. The different ORFs encode homing endonucleases with 65 to 95% identity at the amino-acid level. I-NjaI, I-NanI and I-NitI, from introns in Naegleria jamiesoni, N. andersoni and N. italica, respectively, were analyzed in more detail and found to be isoschizomeric endonucleases that recognize and cleave an approximal 19-bp partially symmetrical sequence, creating a pentanucleotide 3' overhang upon cleavage. The optimal conditions for cleavage activity with respect to temperature, pH, salt and divalent metal ions were investigated. The optimal cleavage temperature for all three endonucleases was found to be 37 degrees C and the activity was dependent on the concentration of NaCl with an optimum at 200 mM. Divalent metal ions, primarily Mg2+, are essential for Naegleria endonuclease activity. Whereas both Mn2+ and Ca2+ could substitute for Mg2+, but with a slower cleavage rate, Zn2+ was unable to support cleavage. Interestingly, the pH dependence of DNA cleavage was found to vary significantly between the I-NitI and I-NjaI/I-NanI endonucleases with optimal pH values at 6.5 and 9, respectively. Site-directed mutagenesis of conserved I-NjaI residues strongly supports the hypothesis that Naegleria homing endonucleases share a similar zinc-binding structure and active site with the His-Cys box homing endonuclease I-PpoI. (+info)
Identity of Naegleria strains isolated from organs of freshwater fishes.
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Eighteen Naegleria strains were isolated from organs of freshwater fishes belonging to 5 species. Morphometric study allowed the separation of the Naegleria strains from the non-vahlkampfiid amoeboflagellates, but was inadequate for species determination. Six strains, representatives of groups that had a slightly different cyst size, were selected and corresponding derived clones were subjected to sequence analysis and riboprinting restriction fragment length polymorphism (RFLP)-PCR analysis of the small subunit (SSU) rRNA genes. One strain isolated from the brain of a fish with systemic infection was characterised by an intronless 2 kb long SSU rRNA gene and was identified as N. australiensis. Another 5 strains had a 1.3 kb long group I intron in their SSU rRNA gene and, based on the SSU rRNA sequences and riboprints, RFLP-PCR patterns appeared in phylogenetic trees to be closely related to Naegleria clarki. (+info)
Characterization of the self-splicing products of two complex Naegleria LSU rDNA group I introns containing homing endonuclease genes.
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The two group I introns Nae.L1926 and Nmo.L2563, found at two different sites in nuclear LSU rRNA genes of Naegleria amoebo-flagellates, have been characterized in vitro. Their structural organization is related to that of the mobile Physarum intron Ppo.L1925 (PpLSU3) with ORFs extending the L1-loop of a typical group IC1 ribozyme. Nae.L1926, Nmo.L2563 and Ppo.L1925 RNAs all self-splice in vitro, generating ligated exons and full-length intron circles as well as internal processed excised intron RNAs. Formation of full-length intron circles is found to be a general feature in RNA processing of ORF-containing nuclear group I introns. Both Naegleria LSU rDNA introns contain a conserved polyadenylation signal at exactly the same position in the 3' end of the ORFs close to the internal processing sites, indicating an RNA polymerase II-like expression pathway of intron proteins in vivo. The intron proteins I-NaeI and I-NmoI encoded by Nae.L1926 and Nmo.L2563, respectively, correspond to His-Cys homing endonucleases of 148 and 175 amino acids. I-NaeI contains an additional sequence motif homologous to the unusual DNA binding motif of three antiparallel beta sheets found in the I-PpoI endonuclease, the product of the Ppo.L1925 intron ORF. (+info)
Use of multiplex PCR and PCR restriction enzyme analysis for detection and exploration of the variability in the free-living amoeba Naegleria in the environment.
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A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites. (+info)
Cultivation of pathogenic and opportunistic free-living amebas.
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Free-living amebas are widely distributed in soil and water, particularly members of the genera Acanthamoeba and NAEGLERIA: Since the early 1960s, they have been recognized as opportunistic human pathogens, capable of causing infections of the central nervous system (CNS) in both immunocompetent and immunocompromised hosts. Naegleria is the causal agent of a fulminant CNS condition, primary amebic meningoencephalitis; Acanthamoeba is responsible for a more chronic and insidious infection of the CNS termed granulomatous amebic encephalitis, as well as amebic keratitis. Balamuthia sp. has been recognized in the past decade as another ameba implicated in CNS infections. Cultivation of these organisms in vitro provides the basis for a better understanding of the biology of these amebas, as well as an important means of isolating and identifying them from clinical samples. Naegleria and Acanthamoeba can be cultured axenically in cell-free media or on tissue culture cells as feeder layers and in cultures with bacteria as a food source. Balamuthia, which has yet to be isolated from the environment, will not grow on bacteria. Instead, it requires tissue culture cells as feeder layers or an enriched cell-free medium. The recent identification of another ameba, Sappinia diploidea, suggests that other free-living forms may also be involved as causal agents of human infections. (+info)