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Modification of the universally unmodified uridine-33 in a mitochondria-imported edited tRNA and the role of the anticodon arm structure on editing efficiency.

Editing of tRNA has a wide phylogenetic distribution among eukaryotes and in some cases serves to expand the decoding capacity of the target tRNA. We previously described C-to-U editing of the wobble position of the imported tRNA(Trp) in Leishmania mitochondria, which is essential for decoding UGA codons as tryptophan. Here we show the complete set of nucleotide modifications in the anticodon arm of the mitochondrial and cytosolic tRNA(Trp) as determined by electrospray ionization mass spectrometry. This analysis revealed extensive mitochondria-specific posttranscriptional modifications, including the first example of thiolation of U33, the "universally unmodified" uridine. In light of the known rigidity imparted on sugar conformation by thiolation, our discovery of a thiolated U33 suggests that conformational flexibility is not a universal feature of the anticodon structural signature. In addition, the in vivo analysis of tRNA(Trp) variants presented shows a single base-pair reversal in the anticodon stem of tRNA(Trp) is sufficient to abrogate editing in vivo, indicating that subtle changes in anticodon structure can have drastic effects on editing efficiency.

Anticodon↗

Twelve Group I introns in the same pre-rRNA transcript of the myxomycete Fuligo septica: RNA processing and evolution.

The ribosomal DNA region of the myxomycete Fuligo septica was investigated and found to contain 12 group I introns (four in the small subunit and eight in the large subunit ribosomal RNAs). We have performed molecular and phylogenetic analyses to provide insight into intron structure and function, intron-host biology, and intron origin and evolution. The introns vary in size from 398 to 943 nt, all lacking detectable open reading frames. Secondary structure models revealed considerable structural diversity, but all, except one (subclass IB), represent the common group IC1 intron subclass. In vitro splicing analysis revealed that 10 of the 12 introns were able to self-splice as naked RNA, but all 12 introns were able to splice out from the precursor rRNA in vivo as evaluated by reverse transcription PCR analysis on total F. septica RNA. Furthermore, RNA processing analyses in vitro and in vivo showed that 10 of 12 introns perform hydrolytic cleavage at the 3' splice site, as well as intron circularization. Full-length intron RNA circles were detected in vivo. The order of splicing was analyzed by a reverse transcription PCR approach on cellular RNA, but no strict order of intron excision could be detected. Phylogenetic analysis indicated that most Fuligo introns were distantly related to each other and were independently gained in ribosomal DNA during evolution.

Animals↗

Systematics of the Eucestoda: advances toward a new phylogenetic paradigm, and observations on the early diversification of tapeworms and vertebrates.

Evolutionary relationships of the Eucestoda have received intense but sporadic attention over the past century. Since 1996, the landscape has dramatically changed with respect to our knowledge of the phylogenetic relationships among the tapeworms. The 2nd International Workshop for Tapeworm Systematics (IWTS) held in Lincoln, Nebraska in October of that year provided the catalyst for development of novel hypotheses for inter- and intraordinal phylogeny. The working-group structure of the 2nd IWTS and results of phylogenetic studies are briefly introduced in the present manuscript. Higher-level phylogenies derived from parsimony analysis of independent data bases representing comparative morphology or molecular sequences were largely congruent and supported monophyly for the Eucestoda. The Caryophyllidea are basal; difossate forms such as the Pseudophyllidea are primitive; tetrafossates including the Tetraphyllidea, Proteocephalidea, Nippotaeniidea, Tetrabothriidea and Cyclophyllidea are derived; and hypotheses differed in the placement of the Trypanorhyncha and the Diphyllidea. These studies may provide a foundation for resolution of inter- and intra-ordinal relationships for the tapeworms. Additionally, the first comprehensive phylogenetic hypotheses for the Pseudophyllidea, Diphyllidea, Trypanorhyncha, the paraphyletic Tetraphyllidea + Lecanicephalidea, Proteocephalidea and Cyclophyllidea were developed during and subsequent to the 2nd IWTS. The stage is now set for continued and rapid advances in our understanding of the eucestodes. These studies have also served to re-emphasise the rich genealogical diversity of tapeworms and the temporally deep history for their origin. A co-evolutionary history and radiation of eucestodes may involve deep co-speciation with vertebrate host taxa, accompanied by some level of colonisation and extinction, extending into the Palaeozoic, minimally 350-420 million years ago.

Animals↗

Prevalence of virulence genes and clonality in Escherichia coli strains that cause bacteremia in cancer patients.

Phenotypic analysis of Escherichia coli strains causing bacteremia in cancer patients suggests that they possess specific virulence properties. To investigate this hypothesis, we compared the frequency of the virulence-related genes cnf1, cnf2, papC, hlyC, and iut in 155 E. coli strains isolated from hospitalized cancer patients with epidemiologically unrelated cases of bacteremia to their frequency in 70 E. coli strains isolated from the feces of healthy unrelated volunteers. Of the blood isolates, 24, 37, and 26% were positive for cnf1, papC, and hlyC, respectively, versus only 6, 17, and 6% of the fecal isolates (P < 0.05 in all instances). By contrast, 47% of both isolates carried the iut gene. The patients' clinical characteristics did not significantly influence these frequencies. The presence on various pathogenicity islands (PAIs) of a combination of the cnf1, papC, and hlyC genes on the chromosome was strongly suggested by Southern blotting of pulsed-field gel electrophoresis (PFGE) patterns with specific DNA probes. The phylogenetic relatedness among 60 strains carrying three, two, one, or no virulence genes and 6 ECOR strains included as references was determined by neighbor joining, the unweighted pair-group method with arithmetic mean, and Wagner analysis of the randomly amplified polymorphic DNA (RAPD) patterns generated by 11 primers. Identification of a major cluster including 96.4% of the strains carrying the cnf1, papC, and hlyC genes and ECOR subgroup B2 strains suggested that the virulent E. coli strains causing bacteremia in cancer patients are closely related to ECOR B2 strains. The presence in the E. coli population surveyed of a strong linkage disequilibrium, and especially of a highly significant correlation between PFGE and RAPD genetic distances, confirms that clonal propagation has a major impact on the E. coli population structure. Nevertheless, low bootstrap values in the phylogenetic tree suggested that frequent genetic exchange inhibits the individualization of discrete genetic lineages, which are stable on an evolutionary scale.

Animals↗

Use of circular permutation to assess six bulges and four loops of DNA-packaging pRNA of bacteriophage phi29.

A 120-base phage phi29 encoded RNA (pRNA) has a novel role in DNA packaging. This pRNA possesses five single-base bulges, one three-base bulge, one bifurcation bulge, one bulge loop, and two stem loops. Circularly permuted pRNAs (cpRNA) were constructed to examine the function of these bulges and loops as well as their adjacent sequences. Each of the five single-base bulges was nonessential. The bifurcation bulge could be deleted and replaced with a new opening to provide flexibility for maintaining an overall correct folding in three-way junction. All of these nonessential bulges or their adjacent bases could be used as new termini for cpRNAs. The three-base (C18C19A20) bulge was dispensable for procapsid binding, but was indispensable for DNA packaging. The secondary structure around this CCA bulge and the phylogenetically conserved bases within or around it were investigated. Bases A14C15U16 were confirmed, by compensatory modification, to pair with U103G102A101. A99 was needed only to allow the proper folding of CCA bulge in the appropriate sequence order and distance constraints. Beyond these, the seemingly phylogenetic conservation of other bases has little role in pRNA activity. Each of the three stem loops was essential for procapsid binding, DNA packaging, and phage assembly. Disruption of the middle of any one of the loops resulted in dramatic reductions in procapsid binding, subsequent DNA packaging, and phage assembly activities. However, disruption of the loops at sequences that were close to double-stranded regions of the RNA did not interfere with pRNA activity significantly. Our results suggest that double-stranded helical regions near these loops were most likely not involved in interactions with components of the DNA-packaging machinery. Instead, these regions appear to be merely present to serve as a scaffolding to display the single-stranded loops that are important for pRNA tertiary structure or for interaction with the procapsid or other packaging components.

Bacillus Phages↗

The internal transcribed spacer 2 database--a web server for (not only) low level phylogenetic analyses.

The internal transcribed spacer 2 (ITS2) is a phylogenetic marker which has been of broad use in generic and infrageneric level classifications, as its sequence evolves comparably fast. Only recently, it became clear, that the ITS2 might be useful even for higher level systematic analyses. As the secondary structure is highly conserved within all eukaryotes it serves as a valuable template for the construction of highly reliable sequence-structure alignments, which build a fundament for subsequent analyses. Thus, any phylogenetic study using ITS2 has to consider both sequence and structure. We have integrated a homology based RNA structure prediction algorithm into a web server, which allows the detection and secondary structure prediction for ITS2 in any given sequence. Furthermore, the resource contains more than 25,000 pre-calculated secondary structures for the currently known ITS2 sequences. These can be taxonomically searched and browsed. Thus, our resource could become a starting point for ITS2-based phylogenetic analyses and is therefore complementary to databases of other phylogenetic markers, which focus on higher level analyses. The current version of the ITS2 database can be accessed via http://its2.bioapps.biozentrum.uni-wuerzburg.de.

DNA, Ribosomal Spacer↗

Comparative avian phylogeography of Cameroon and equatorial Guinea mountains: implications for conservation.

We illustrate the use of Faith's 'Phylogenetic Diversity' measure to compare the phylogeographic structure of two bird species with patterns of avian endemism across six mountains in Cameroon and Equatorial Guinea. The Mountain Greenbul and Cameroon Blue-headed Sunbird showed phylogeographic patterns that together defined three biogeographic regions: Bioko, Mt. Cameroon, and the northern mountains of Cameroon. In contrast, the distributions of endemic species were largely a function of geographical distance, with close mountains sharing more endemic species than distant mountains. Moreover, for both species, populations on Mt. Cameroon were distinctive with respect to the ecologically relevant character bill size. Our results, while preliminary, illustrate the utility of a comparative approach for identifying geographical regions that harbour evolutionarily distinct populations and caution against using only the distributional patterns of endemics to prioritize regions for conservation. Results show that patterns of endemism may not be concordant with patterns of phylogenetic diversity nor morphological variation in a character important in fitness. While incorporation of additional species from unrelated taxa will be necessary to draw definitive conclusions about evolutionarily distinct regions, our preliminary results suggest a conservation approach for the Afromontane region of the Gulf of Guinea that would: (i) emphasize protection of both Bioko and Mt. Cameroon, thereby maximizing preservation of within-species phylogenetic and morphologic diversity; (ii) emphasize protection within the northern mountains to further conserve intraspecific phylogenetic diversity and maximize protection of endemic species.

Animals↗

Structure and function of telomerase RNA.

Maintenance of telomeres by the enzyme telomerase is essential for genomic stability and cell viability in ciliates, vertebrates and yeast. The minimal components of telomerase required for catalytic activity are the telomerase reverse transcriptase (TERT) protein and the template-containing telomerase RNA (TER). Recent studies have afforded significant advances in the biophysical characterization of telomerase RNAs from various species. The first TER structures have been reported, for regions of the catalytically essential pseudoknot and CR4/CR5 domains of human TER, and provide a structural basis for interpretation of mutational and biochemical data. The domains and interactions of the Tetrahymena thermophila telomerase holoenzyme RNA and protein components have been further characterized biochemically, and structures of the TER template boundary element and the N-terminal domain of T. thermophila TERT have been determined. Phylogenetic and biochemical analyses of yeast TERs have revealed core structural elements in common with ciliates and vertebrates, and the minimal domains required for function in vivo.

Animals↗

Common phylogeny of catalase-peroxidases and ascorbate peroxidases.

Catalase-peroxidases belong to Class I of the plant, fungal, bacterial peroxidase superfamily, together with yeast cytochrome c peroxidase and ascorbate peroxidases. Obviously these bifunctional enzymes arose via gene duplication of an ancestral hydroperoxidase. A 230-residues long homologous region exists in all eukaryotic members of Class I, which is present twice in both prokaryotic and archaeal catalase-peroxidases. The overall structure of eukaryotic Class I peroxidases may be retained in both halves of catalase-peroxidases, with major insertions in several loops, some of which may participate in inter-domain or inter-subunit interactions. Interspecies distances in unrooted phylogenetic trees, analysis of sequence similarities in distinct structural regions, as well as hydrophobic cluster analysis (HCA) suggest that one single tandem duplication had already occurred in the common ancestor prior to the segregation of the archaeal and eubacterial lines. The C-terminal halves of extant catalase-peroxidases clearly did not accumulate random changes, so prolonged periods of independent evolution of the duplicates can be ruled out. Fusion of both copies must have occurred still very early or even in the course of the duplication. We suggest that the sparse representatives of eukaryotic catalase-peroxidases go back to lateral gene transfer, and that, except for several fungi, only single copy hydroperoxidases occur in the eukaryotic lineage. The N-terminal halves of catalase-peroxidases, which reveal higher homology with the single-copy members of the superfamily, obviously are catalytically active, whereas the C-terminal halves of the bifunctional enzymes presumably control the access to the haem pocket and facilitate stable folding. The bifunctional nature of catalase-peroxidases can be ascribed to several unique sequence peculiarities conserved among all N-terminal halves, which most likely will affect the properties of both haem ligands.

Amino Acid Motifs↗

Molecular evolution of ependymin and the phylogenetic resolution of early divergences among euteleost fishes.

The rate and pattern of DNA evolution of ependymin, a single-copy gene coding for a highly expressed glycoprotein in the brain matrix of teleost fishes, is characterized and its phylogenetic utility for fish systematics is assessed. DNA sequences were determined from catfish, electric fish, and characiforms and compared with published ependymin sequences from cyprinids, salmon, pike, and herring. Among these groups, ependymin amino acid sequences were highly divergent (up to 60% sequence difference), but had surprisingly similar hydropathy profiles and invariant glycosylation sites, suggesting that functional properties of the proteins are conserved. Comparison of base composition at third codon positions and introns revealed AT-rich introns and GC-rich third codon positions, suggesting that the biased codon usage observed might not be due to mutational bias. Phylogenetic information content of third codon positions was surprisingly high and sufficient to recover the most basal nodes of the tree, in spite of the observation that pairwise distances (at third codon positions) were well above the presumed saturation level. This finding can be explained by the high proportion of phylogenetically informative nonsynonymous changes at third codon positions among these highly divergent proteins. Ependymin DNA sequences have established the first molecular evidence for the monophyly of a group containing salmonids and esociforms. In addition, ependymin suggests a sister group relationship of electric fish (Gymnotiformes) and Characiformes, constituting a significant departure from currently accepted classifications. However, relationships among characiform lineages were not completely resolved by ependymin sequences in spite of seemingly appropriate levels of variation among taxa and considerably low levels of homoplasy in the data (consistency index = 0.7). If the diversification of Characiformes took place in an "explosive" manner, over a relatively short period of time this pattern should also be observed using other phylogenetic markers. Poor conservation of ependymin's primary structure hinders the design of efficient primers for PCR that could be used in wide-ranging fish systematic studies. However, alternative methods like PCR amplification from cDNA used here should provide promising comparative sequence data for the resolution of phylogenetic relationships among other basal lineages of teleost fishes.

Amino Acid Sequence↗

Structural implications of novel diversity in eucaryal RNase P RNA.

Previous eucaryotic RNase P RNA secondary structural models have been based on limited diversity, representing only two of the approximately 30 phylogenetic kingdoms of the domain Eucarya. To elucidate a more generally applicable structure, we used biochemical, bioinformatic, and molecular approaches to obtain RNase P RNA sequences from diverse organisms including representatives of six additional kingdoms of eucaryotes. Novel sequences were from acanthamoeba (Acathamoeba castellanii, Balamuthia mandrillaris, Filamoeba nolandi), animals (Caenorhabditis elegans, Drosophila melanogaster), alveolates (Theileria annulata, Babesia bovis), conosids (Dictyostelium discoideum, Physarum polycephalum), trichomonads (Trichomonas vaginalis), microsporidia (Encephalitozoon cuniculi), and diplomonads (Giardia intestinalis). An improved alignment of eucaryal RNase P RNA sequences was assembled and used for statistical and comparative structural analysis. The analysis identifies a conserved core structure of eucaryal RNase P RNA that has been maintained throughout evolution and indicates that covariation in size occurs between some structural elements of the RNA. Eucaryal RNase P RNA contains regions of highly variable length and structure reminiscent of expansion segments found in rRNA. The eucaryal RNA has been remodeled through evolution as a simplified version of the structure found in bacterial and archaeal RNase P RNAs.

Acanthamoeba castellanii↗

New analysis of the phylogenetic change of collagen thermostability.

Recent data concerning the thermostability and the primary structure of type IV collagens, some invertebrate collagens, and for the stability of synthetic collagen-like polypeptides, show that our earlier analysis of the phylogenetic change of thermostability has some shortcomings. The results of the analysis were corrected and it has been shown that the dependence of denaturation temperature Td on 4-hydroxyproline content is hyperbolic and the total Gly-Pro-Hyp sequence content is a main, but not exclusive, factor influencing the change of collagen thermostability. It appears possible that the same mechanism underlies the thermostability of fibril-forming collagens of all animal life, ranging from Antarctic ice fish to at least one annelid (Alvinella pompejana) living at very high temperatures at the bottom of the ocean near thermal vents.

Animals↗

Evolutionary genetics and biogeographic structure of Rhizobium gallicum sensu lato, a widely distributed bacterial symbiont of diverse legumes.

We used phylogenetic and population genetics approaches to evaluate the importance of the evolutionary forces on shaping the genetic structure of Rhizobium gallicum and related species. We analysed 54 strains from several populations distributed in the Northern Hemisphere, using nucleotide sequences of three 'core' chromosomal genes (rrs, glnII and atpD) and two 'auxiliary' symbiotic genes (nifH and nodB) to elucidate the biogeographic history of the species and symbiotic ecotypes (biovarieties) within species. The analyses revealed that strains classified as Rhizobium mongolense and Rhizobium yanglingense belong to the chromosomal evolutionary lineage of R. gallicum and harbour symbiotic genes corresponding to a new biovar; we propose their reclassification as R. gallicum bv. orientale. The comparison of the chromosomal and symbiotic genes revealed evidence of lateral transfer of symbiotic information within and across species. Genetic differentiation analyses based on the chromosomal protein-coding genes revealed a biogeographic pattern with three main populations, whereas the 16S rDNA sequences did not resolve that biogeographic pattern. Both the phylogenetic and population genetic analyses showed evidence of recombination at the rrs locus. We discuss our results in the light of the contrasting views of bacterial species expressed by microbial taxonomist and evolutionary biologists.

Base Sequence↗

Some advantages and limitations of mitosis as a phylogenetic criterion.

The advantages and disadvantages of mitosis as a phylogenetic character are discussed. Mitosis is suggested to be a good character on the grounds that it is universally applicable and that it reflects features of the basic organism since it is unlikely to have arisen through multiple endosymbiotic events. Since mitosis is an absolutely essential feature of the cell cycle it is subject to a great deal of evolutionary pressure and one would not expect a priori that relatively inefficient mitotic apparatuses would persist. It is suggested that supposedly primitive mitotic apparatuses are not inefficient and that the evidence that they are functionally different from those of higher organisms is largely negative electron microscopic evidence. This type of evidence is not very satisfactory since it can result from poor preservation and consequently it should be continually retested. Another weakness of mitosis as a phylogenetic criterion (shared with other essentially morphological criteria) is that there is no easy way to relate structural change with genetic change. Thus, while mitosis is a useful phylogenetic character, its usefulness is limited.

Biological Evolution↗

Descent of a split RNA.

tmRNA is found in the usual one-piece form in most cyanobacteria, but a small clade has an unusual two-piece form, resulting from processing of a circularly permuted precursor RNA. Here, the secondary structure of the cyanobacterial one-piece tmRNA is established by phylogenetic sequence comparison; it deviates from most tmRNAs in having an extra (fifth) pseudoknot and a branched structure at the end of the tag reading frame. Patterns of sequence conservation between the two cyanobacterial tmRNA types suggest particular events in the evolution of the two-piece form. A simple gene permutation model of tandem duplication followed by loss of the outermost segments appears inadequate. An additional rearrangement is proposed, in which a structure impaired by deletion was regenerated at the expense of a neighboring structure.

Base Sequence↗

Structural evolution of the protein kinase-like superfamily.

The protein kinase family is large and important, but it is only one family in a larger superfamily of homologous kinases that phosphorylate a variety of substrates and play important roles in all three superkingdoms of life. We used a carefully constructed structural alignment of selected kinases as the basis for a study of the structural evolution of the protein kinase-like superfamily. The comparison of structures revealed a "universal core" domain consisting only of regions required for ATP binding and the phosphotransfer reaction. Remarkably, even within the universal core some kinase structures display notable changes, while still retaining essential activity. Hence, the protein kinase-like superfamily has undergone substantial structural and sequence revision over long evolutionary timescales. We constructed a phylogenetic tree for the superfamily using a novel approach that allowed for the combination of sequence and structure information into a unified quantitative analysis. When considered against the backdrop of species distribution and other metrics, our tree provides a compelling scenario for the development of the various kinase families from a shared common ancestor. We propose that most of the so-called "atypical kinases" are not intermittently derived from protein kinases, but rather diverged early in evolution to form a distinct phyletic group. Within the atypical kinases, the aminoglycoside and choline kinase families appear to share the closest relationship. These two families in turn appear to be the most closely related to the protein kinase family. In addition, our analysis suggests that the actin-fragmin kinase, an atypical protein kinase, is more closely related to the phosphoinositide-3 kinase family than to the protein kinase family. The two most divergent families, alpha-kinases and phosphatidylinositol phosphate kinases (PIPKs), appear to have distinct evolutionary histories. While the PIPKs probably have an evolutionary relationship with the rest of the kinase superfamily, the relationship appears to be very distant (and perhaps indirect). Conversely, the alpha-kinases appear to be an exception to the scenario of early divergence for the atypical kinases: they apparently arose relatively recently in eukaryotes. We present possible scenarios for the derivation of the alpha-kinases from an extant kinase fold.

Adenosine Triphosphate↗

Genes and peptides from the scorpion Centruroides sculpturatus Ewing, that recognize Na(+)-channels.

Sixteen different genes were cloned from the venomous glands of Centruroides sculpturatus Ewing using RNA extracted from scorpions collected in Tucson, Arizona. Based on the amino acid sequence similarities of the proteins coded by these genes, all together there are 22 different structural components in this venom, thought to be specific for Na(+)-channels. The genes reported contain signal peptides with 19 amino acid residues followed by mature peptides of 63-66 amino acid residues in length. One of them correspond to toxin I (CsEI), a known scorpion toxin specific for Na(+)-channels. Four different genes are almost identical to variant 1 (Csv1), presenting only one amino acid change from the original protein. For variant 2 (Csv2) four related genes were found, with only one amino acid change in their primary sequences. Another gene resembles to variant 3 (Csv3, the best known Centruroides sculpturatus toxin), with only three amino acid changes in their primary sequences. Additionally, two genes show variations only on the nucleotide sequence at level of the signal peptides, and several genes clearly show sequences that suggest post-transcriptional modifications, during the maturation process. A phylogenetic tree was generated with the primary structures available and three main divergent branches were found.

Animals↗

Cobra venom factor: structural homology with the third component of human complement.

The functional analogy between cobra venom factor (CVF), the complement-activating protein in cobra venom, and C3b, the activated form of the third complement component, prompted us to conduct a comparative analysis of structural properties of the two proteins derived from two phylogenetically distant species. We subjected CVF and human C3 and its physiologic cleavage products, C3b and C3c, to a variety of biochemical analyses. We report here structural similarities of these proteins, which include similarities in amino acid composition, far and near UV circular dichroism spectra, secondary structure, band patterns and pI values from isoelectric focusing, immunochemical cross-reactivity, ultrastructural morphology, and amino-terminal amino acid sequences. Analysis of these data reveals that, structurally, CVF resembles C3c more than C3b. We conclude that CVF is not the product of a convergent evolution, but is, in all likelihood, derived from a common C3 ancestor protein.

Amino Acid Sequence↗