Nucleotide sequence of a DNA region comprising the gene for elongation factor 1α (EF-1α) from the ultrathermophilic archaeotePyrococcus woesei: Phylogenetic implications

Summary The gene encoding elongation factor 1 (EF-1, 1290 bp) of the ultrathermophilic, sulfur-reducing archaeotePyrococcus woesei was localized within aBglII fragment of chromosomal DNA. Sequence analysis showed that the EF-1 gene is the upstream unit of a three-gene cluster comprising the genes for ribosomal protein S10 (306 bp) and transfer RNA_ser (GGA). The three genes follow each other immediately in the order EF-1·S10·tRNA_ser after a putative promoter located 55 bp upstream of the EF-1 gene. Alignment of the derived EF-1 sequence with the corresponding sequences from Eukarya, Bacteria/organelles, and with available archaeal sequences (Sulfolobus, Thermococcus, Methanococcus, Halobacterium) showed thatPyrococcus EF-1 is highly homologous (89% identity) toThermococcus celer EF-1, both being strikingly more similar to eukaryotic EF-1 than to bacterial EF-Tu. Unrooted dendrograms computed from aligned sequences by distance matrix and DNA parsimony methods, including evolutionary parsimony, showed the Archaea to be a monophyletic-holophyletic cluster closer to Eukarya than to Bacteria. Both distance matrix and DNA parsimony-although not evolutionary parsimony-support the partition of the known archaeal lineages between the kingdoms Crenarchaeota and Euryarchaeota, and the affiliation of thePyrococcus-Thermococcus lineage to the Euryarchaeota, of which it is the most primitive offspring. A closer relation ofPyrococcus to Euryarchaeota than to Crenarchaeota was also inferred from sequence analysis of S10 ribosomal proteins.     

Geobiological analysis using whole genome-based tree building applied to the Bacteria, Archaea, and Eukarya

We constructed genomic trees based on the presence and absence of families of protein‐encoding genes observed in 55 prokaryotic and five eukaryotic genomes. There are features of the genomic trees that are not congruent with typical rRNA phylogenetic trees. In the bacteria, for example, Deinococcus radiodurans associates with the Gram‐positive bacteria, a result that is also seen in some other phylogenetic studies using whole genome data. In the Archaea, the methanogens plus Archaeoglobus form a united clade and the Euryarchaeota are divided with the two Thermoplasma genomes and Halobacterium sp. falling below the Crenarchaeota. While the former appears to be an accurate representation of methanogen‐relatedness, the misplacement of Halobacterium may be an artefact of parsimony. These results imply the last common ancestor of the Archaea was not a methanogen, leaving sulphur reduction as the most geochemically plausible metabolism for the base of the archaeal crown group. It also suggests that methanogens were not a component of the Earth's earliest biosphere and that their origin occurred sometime during the Archean. In the Eukarya, the parsimony analysis of five Eukaryotes using the Crenarchaeota as an outgroup seems to counter the Ecdysozoa hypothesis, placing Caenorhabditis elegans (Nematoda) below the common ancestor of Drosophila melanogaster (Arthropoda) and Homo sapiens (Chordata) even when efforts are made to counter the possible effects of a faster rate of sequence evolution for the C. elegans genome. Further analysis, however, suggests that the gene loss of ‘animal’ genes is highest in C. elegans and is obscuring the relationships of these organisms.     

Reconstructing ancestral patterns of colonization and dispersal in the Hawaiian understory tree genus Psychotria (Rubiaceae): a comparison of parsimony and likelihood approaches

Systematic and biogeographical relationships within the Hawaiian clade of the pantropical understory shrub genus Psychotria (Rubiaceae) were investigated using phylogenetic analysis of 18S-26S ribosomal DNA internal (ITS) and external (ETS) transcribed spacers. Phylogenetic analyses strongly suggest that the Hawaiian Psychotria are monophyletic and the result of a single introduction to the Hawaiian Islands. The results of phylogenetic analyses of ITS and ETS partitions alone give slightly different topologies among basal lineages of the Hawaiian clade; however, such differences are not well supported. Relationships in the section Straussia clade in particular are not well resolved because of few nucleotide changes on internal branches, suggesting extremely rapid radiation in the lineage. Parsimony and likelihood reconstructions of ancestral geographical distributions using the topologies inferred from both parsimony and likelihood analysis of combined data and using different combinations of models and branch lengths gave highly congruent results. However, for one internal node (corresponding to the majority of the "greenwelliae" clade), parsimony reconstructions were unable to distinguish between three possible island states, whereas likelihood reconstructions resulted in clear ordering of possible states, with the island of Oàhu slightly more probable than other islands under all but one model and branch length combination considered (the Jukes-Cantor-like model with branch lengths inferred under parsimony, under which conditions Maui Nui is more probable). A pattern of colonization from oldest to youngest islands was inferred from the phylogeny, using maximum parsimony and maximum likelihood. Additionally, a much higher incidence of intraisland versus interisland speciation was inferred.     

The origin of land plants: Phylogenetic relationships among charophytes,bryophytes, and vascular plants inferred from complete small-subunit ribosomal RNA gene sequences

Complete nuclear-encoded small-subunit 18S rRNA (=SSU rRNA) gene sequences were determined for the prasinophyte green alga Mantoniella squamata; the charophycean green algae Chara foetida, Coleochaete scutata, Klebsormidium flaccidum, and Mougeotia scalaris; the bryophytes Marchantia polymorpha, Fossombronia pusilla, and Funaria hygrometrica; and the lycopod Selaginella galleottii to get a better insight into the sequential evolution from green algae to land plants. The sequences were aligned with several previously published SSU rRNA sequences from chlorophytic and charophytic algae as well as from land plants to infer the evolutionary relationships for major evolutionary lineages within the Chlorobionta by distance matrix, maximum parsimony, and maximum likelihood analyses. Phylogenetic trees created by the different methods consistently placed the Charophyceae on the branch leading to the land plants. The Charophyceae were shown to be polyphyletic with the Charales (charalean algae) diverging earlier than the Coleochaetales, Klebsormidiales, Chlorokybales, and Zygnematales (charophycean algae) which branch from a point closer to the land plants in most analyses. Maximum parsimony and maximum likelihood analyses imply a successive evolution from charophycean algae, particularly Coleochaetales, to bryophytes, lycopods, and seed plants. In contrast, distance matrix methods group the bryophytes together with the charophycean algae, suggesting a separate evolution of these organisms compared with the club moss and the seed plants. Correspondence to.: V.A.R. Huss     

Somatic embryogenesis and plant regeneration from protoplasts of Cichorium intybus L. x Cichorium endivia L.

Somatic embryos and adult plants were regenerated from mesophyll protoplasts of a clone of chicory 474 (Cichorium intybus L. x Cichorium endivia L.). Embryos were obtained in three different ways:

Molecular evolution and secondary structural conservation in the B-cell lymphoma leukemia 2 (bcl-2) family of proto-oncogene products

The nature of the bcl-2 family of protooncogenes was analyzed by sequence alignment, secondary structure prediction, and phylogenetic techniques. Phylogenies were inferred from both the nucleic acid and amino acid sequences of the human, murine, rat, and chicken sequences for BCL-2 and BCL-X, human MCL1, murine A1, the nematode Caenorhabditis elegans and Caenorhabditis briggsiae ced-9 proteins, and the sequences BHRF1 from Epstein-Barr and LMW5-HL from African swine fever viruses. Both sequence alignment and secondary structure prediction techniques supported the conservation of both the overall secondary structure and the carboxy-terminal transmembrane domain in all members of the family. All the treeing methods employed (distance matrix, maximum likelihood, and parsimony) supported a tree in which the proapoptotic proteins BCL-2 and BCL-X represent the most recent additions to the group. All the trees also indicated that the viral proteins BHRF1 and LMW-HL arose from a common ancestor, an ancestor they shared in common with the pro-apoptotic control protein BAX, indicating that this function of BAX evolved only recently. The most ancient branches are represented by the nematode ced-9 protein and by the control genes MCL1 and A1, which in the treeing methods employed represent separate lineages within the most ancient grouping. These results demonstrate the evolution of a highly conserved family of developmental control genes from nematode to man—genes that encode proteins essential for normal development but which are highly conserved in terms of predicted structure and possible cellular localization. The evolutionary analysis also indicates that the family may be even larger than originally predicted and that other members are waiting to be discovered. Correspondence to: D. Lloyd Evans     

The influence of gene-environment interactions on GHR and IGF-1 expression and their association with growth in brook charr, Salvelinus fontinalis (Mitchill)

Background

Information transfer systems in Archaea, including many components of the DNA replication machinery, are similar to those found in eukaryotes. Functional assignments of archaeal DNA replication genes have been primarily based upon sequence homology and biochemical studies of replisome components, but few genetic studies have been conducted thus far. We have developed a tractable genetic system for knockout analysis of genes in the model halophilic archaeon, Halobacterium sp. NRC-1, and used it to determine which DNA replication genes are essential.

Results

Using a directed in-frame gene knockout method in Halobacterium sp. NRC-1, we examined nineteen genes predicted to be involved in DNA replication. Preliminary bioinformatic analysis of the large haloarchaeal Orc/Cdc6 family, related to eukaryotic Orc1 and Cdc6, showed five distinct clades of Orc/Cdc6 proteins conserved in all sequenced haloarchaea. Of ten orc/cdc6 genes in Halobacterium sp. NRC-1, only two were found to be essential, orc10, on the large chromosome, and orc2, on the minichromosome, pNRC200. Of the three replicative-type DNA polymerase genes, two were essential: the chromosomally encoded B family, polB1, and the chromosomally encoded euryarchaeal-specific D family, polD1/D2 (formerly called polA1/polA2 in the Halobacterium sp. NRC-1 genome sequence). The pNRC200-encoded B family polymerase, polB2, was non-essential. Accessory genes for DNA replication initiation and elongation factors, including the putative replicative helicase, mcm, the eukaryotic-type DNA primase, pri1/pri2, the DNA polymerase sliding clamp, pcn, and the flap endonuclease, rad2, were all essential. Targeted genes were classified as non-essential if knockouts were obtained and essential based on statistical analysis and/or by demonstrating the inability to isolate chromosomal knockouts except in the presence of a complementing plasmid copy of the gene.

Conclusion

The results showed that ten out of nineteen eukaryotic-type DNA replication genes are essential for Halobacterium sp. NRC-1, consistent with their requirement for DNA replication. The essential genes code for two of ten Orc/Cdc6 proteins, two out of three DNA polymerases, the MCM helicase, two DNA primase subunits, the DNA polymerase sliding clamp, and the flap endonuclease.     

Thermoacidophilic Sulfolobus species as source for extremozymes and as novel archaeal platform organisms

Sulfolobus species – thermoacidophilic Archaea – as sources for novel extremozymes and as novel platform organism for metabolic engineering and synthetic biology.

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Global Patterns of Protein Domain Gain and Loss in Superkingdoms

Domains are modules within proteins that can fold and function independently and are evolutionarily conserved. Here we compared the usage and distribution of protein domain families in the free-living proteomes of Archaea, Bacteria and Eukarya and reconstructed species phylogenies while tracing the history of domain emergence and loss in proteomes. We show that both gains and losses of domains occurred frequently during proteome evolution. The rate of domain discovery increased approximately linearly in evolutionary time. Remarkably, gains generally outnumbered losses and the gain-to-loss ratios were much higher in akaryotes compared to eukaryotes. Functional annotations of domain families revealed that both Archaea and Bacteria gained and lost metabolic capabilities during the course of evolution while Eukarya acquired a number of diverse molecular functions including those involved in extracellular processes, immunological mechanisms, and cell regulation. Results also highlighted significant contemporary sharing of informational enzymes between Archaea and Eukarya and metabolic enzymes between Bacteria and Eukarya. Finally, the analysis provided useful insights into the evolution of species. The archaeal superkingdom appeared first in evolution by gradual loss of ancestral domains, bacterial lineages were the first to gain superkingdom-specific domains, and eukaryotes (likely) originated when an expanding proto-eukaryotic stem lineage gained organelles through endosymbiosis of already diversified bacterial lineages. The evolutionary dynamics of domain families in proteomes and the increasing number of domain gains is predicted to redefine the persistence strategies of organisms in superkingdoms, influence the make up of molecular functions, and enhance organismal complexity by the generation of new domain architectures. This dynamics highlights ongoing secondary evolutionary adaptations in akaryotic microbes, especially Archaea.     

The 492-bp RM07 DNA Fragment from the Halophilic Archaea Confers Promoter Activity in All Three Domains of Life

A 492-bp DNA fragment, designated RM07, was isolated from the chromosomal DNA of the halophilic Archaea, Halobacterium halobium, and was shown to confer promoter activity in Escherichia coli. Sequence analysis revealed that RM07 contained three consensus sequences of the archaeal distal promoter element as well as the typical –35 and –10 box sequences of bacterial promoters. Promoter probe analysis confirmed that RM07 conferred promoter activity in all three domains of life: Archaea (Haloferax volcanii), Eukarya (Saccharomyces cerevisiae) and Bacteria (Escherichia coli). Deletion analysis and site-directed mutagenesis further identified the functional regions within RM07 required for promoter activity. This is the first report of a DNA fragment from Archaea that confers promoter activity in all three domains of life, suggesting that the promoter structure and activity may be viewed as a bridge narrowing the gaps among the different domains of life.     

Conserved and variable regions in the chromosomal and extrachromosomal DNA of halobacteria

Summary Total DNA from Halobacterium halobium and other halobacteria strains is separated into two fractions, FI and FII, which differ in their G+C content. FI DNA, which represents the major part of the genome is highly conserved in all purple-membrane-forming halobacteria. Fraction II (FII) consists in H. halobium of three DNA specimen: (a) the previously isolated plasmid pHH1, (b) a heterogeneous set of ccc-DNA molecules present in the cell in low copies, termed minor-circular DNA (MCD) and (c) a new type of more A-T rich DNA segments (chromosomal islands) which, as described here and by Pfeifer and Betlach (1985), are integrated in FI. Sequences homologous to pHH1 occur only in Halobacterium species closely related to H. halobium (like H. cutirubrum), whereas MCD sequences are present in all purple-membrane-forming halobacteria. The sequences of the newly identified chromosomal islands are only found like pHH1, in Halobacterium species, closely related to H. halobium. Total DNA from square halobacteria exhibits no extended homologies to FI or FII DNA from H. halobium. The only common DNA sequences found in all halobacteria are certain insertion elements (ISH), such as ISH26. Based on these data, halobacteria can be subdivided in at least three major groups.Dedicated to Prof. Dr. F. Lingens to his 60th anniversary     

Phylogeny of Selected Maxillopodan and Other Crustacean Taxa Based on 18S Ribosomal Nucleotide Sequences: A Preliminary Analysis

Relationships among the following taxa were examined based on 18S ribosomal RNA and DNA nucleotide sequences: Branchiopoda (Branchinecta packardi), Ostracoda (two podocopid species), Branchiura (Argulus nobilis), Pentastomida (Porocephalus crotali), Copepoda (Calanus pacificus, Macrocyclops albidus), Thoracica (Balanus eburneus, Calantica villosca), Acrothoracica (Trypetesa lampas), and Decapoda (Procambarus leonensis, Callinectes sapidus). Phylogenetic relationships were inferred by maximum parsimony and results evaluated by bootstrapping. With Branchinecta packardi as an outgroup, the following tree was inferred: the first branch leads to a node that joins a pentastome, a branchiuran and the two ostracodes; the second branch leads to a node that has the two copepods as a sister taxon to a clade that includes the acrothoracican and thoracicans; the last branch leads to a node joining the two decapods. The analyses suggest: (1) acrothoracicans diverged very early from the cirripede line and are not derived from a lepadomorph-like ancestor; (2) branchiurans are not related to either copepods or thecostracans but are closely allied with pentastomes; and (3) the Maxillopoda, broadly defined, is not a monophyletic taxon. These results differ from previous studies of these groups.     

Bayesian Estimation of Positively Selected Sites

In protein-coding DNA sequences, historical patterns of selection can be inferred from amino acid substitution patterns. High relative rates of nonsynonymous to synonymous changes (=d _N /d _S ) are a clear indicator of positive, or directional, selection, and several recently developed methods attempt to distinguish these sites from those under neutral or purifying selection. One method uses an empirical Bayesian framework that accounts for varying selective pressures across sites while conditioning on the parameters of the model of DNA evolution and on the phylogenetic history. We describe a method that identifies sites under diversifying selection using a fully Bayesian framework. Similar to earlier work, the method presented here allows the rate of nonsynonymous to synonymous changes to vary among sites. The significant difference in using a fully Bayesian approach lies in our ability to account for uncertainty in parameters including the tree topology, branch lengths, and the codon model of DNA substitution. We demonstrate the utility of the fully Bayesian approach by applying our method to a data set of the vertebrate -globin gene. Compared to a previous analysis of this data set, the hierarchical model found most of the same sites to be in the positive selection class, but with a few striking exceptions.     

A comparison of phylogenetic network methods using computer simulation

Background

We present a series of simulation studies that explore the relative performance of several phylogenetic network approaches (statistical parsimony, split decomposition, union of maximum parsimony trees, neighbor-net, simulated history recombination upper bound, median-joining, reduced median joining and minimum spanning network) compared to standard tree approaches, (neighbor-joining and maximum parsimony) in the presence and absence of recombination.

Principal Findings

In the absence of recombination, all methods recovered the correct topology and branch lengths nearly all of the time when the substitution rate was low, except for minimum spanning networks, which did considerably worse. At a higher substitution rate, maximum parsimony and union of maximum parsimony trees were the most accurate. With recombination, the ability to infer the correct topology was halved for all methods and no method could accurately estimate branch lengths.

Conclusions

Our results highlight the need for more accurate phylogenetic network methods and the importance of detecting and accounting for recombination in phylogenetic studies. Furthermore, we provide useful information for choosing a network algorithm and a framework in which to evaluate improvements to existing methods and novel algorithms developed in the future.     

Phylogenetic Position of Cryothecomonas Inferred from Nuclear-Encoded Small Subunit Ribosomal RNA

The systematic position of the genus Cryothecomonas has been determined from an analysis of the nuclear-encoded small subunit ribosomal RNA gene of Cryothecomonas longipes and two strains of Cryothecomonas aestivalis. Our phylogenetic trees inferred from maximum likelihood, distance and maximum parsimony methods robustly show that the genus Cryothecomonas clusters within the phylum Cercozoa, and is related to the sarcomonad flagellate Heteromita globosa. Morphological data supporting the taxonomic placement of Cryothecomonas near the sarcomonad flagellates has been compiled from the literature. The high number of nucleotide substitutions found between two morphologically indistinguishable strains of Cryothecomonas aestivalis suggests the possibility of cryptic species within Cryothecomonas aestivalis.     

Biosynthesis of δ-aminolevulinic acid from glutamate by Sulfolobus solfataricus

The extremely thermophilic, obligately aerobic bacterium Sulfolobus solfataricus forms the tetrapyrrole precursor, -aminolevulinic acid (ALA), from glutamate by the tRNA-dependent five-carbon pathway. This pathway has been previously shown to occur in plants, algae, and most prokaryotes with the exception of the -group of proteobacteria (purple bacteria). An alternative mode of ALA formation by condensation of glycine and succinyl-CoA occurs in animals, yeasts, fungi, and the -proteobacteria. Sulfolobus and several other thermophilic, sulfur-dependent bacteria, have been variously placed within a subgroup of archaea (archaebacteria) named crenarchaeotes, or have been proposed to comprise a distinct prokaryotic group designated eocytes. On the basis of ribosomal structure and certain other criteria, eocytes have been proposed as predecessors of the nuclear-cytoplasmic descent line of eukaryotes. Because aplastidic eukaryotes differ from most prokaryotes in their mode of ALA formation, and in view of the proposed affiliation of eocytes to eukaryotes, it was of interest to determine how eocytes form ALA. Sulfolobus extracts were able to incorporate label from [1-¹⁴C]glutamate, but not from [2-¹⁴C]glycine, into ALA. Glutamate incorporation was abolished by preincubation of the extract with RNase. Sulfolobus extracts contained glutamate-1-semialdehyde aminotransferase activity, which is indicative of the five-carbon pathway. Growth of Sulfolobus was inhibited by gabaculine, a mechanism-based inhibitor of glutamate-1-semialdehyde aminotransferase, an enzyme of the five-carbon ALA biosynthetic pathway. These results indicate that Sulfolobus uses the five-carbon pathway for ALA formation.Abbreviations AHA 4-amino-5-hexynoic acid - ALA -aminolevulinic acid, Gabaculine, 3-amino-2,3-dihydrobenzoic acid - GSA glutamate 1-semialdehyde     

Inferring angiosperm phylogeny from EST data with widespread gene duplication

Background

Most studies inferring species phylogenies use sequences from single copy genes or sets of orthologs culled from gene families. For taxa such as plants, with very high levels of gene duplication in their nuclear genomes, this has limited the exploitation of nuclear sequences for phylogenetic studies, such as those available in large EST libraries. One rarely used method of inference, gene tree parsimony, can infer species trees from gene families undergoing duplication and loss, but its performance has not been evaluated at a phylogenomic scale for EST data in plants.

Results

A gene tree parsimony analysis based on EST data was undertaken for six angiosperm model species and Pinus, an outgroup. Although a large fraction of the tentative consensus sequences obtained from the TIGR database of ESTs was assembled into homologous clusters too small to be phylogenetically informative, some 557 clusters contained promising levels of information. Based on maximum likelihood estimates of the gene trees obtained from these clusters, gene tree parsimony correctly inferred the accepted species tree with strong statistical support. A slight variant of this species tree was obtained when maximum parsimony was used to infer the individual gene trees instead.

Conclusion

Despite the complexity of the EST data and the relatively small fraction eventually used in inferring a species tree, the gene tree parsimony method performed well in the face of very high apparent rates of duplication.

     

A procedure for the enrichment and isolation of Halobacterium

A procedure for the specific enrichment and isolation of species of the genus Halobacterium was designed, based on the ability of Halobacterium cells to grow anaerobically by fermentation of l-arginine. None of the other genera of neutrophilic halophilic Archaea tested grew fermentatively on arginine. Using anaerobic enrichments in the presence of arginine, representatives of the genus Halobacterium were consistently isolated from saltern crystallizer ponds in Eilat (Israel) and San Francisco Bay (California), environments in which Halobacterium represents only a very small fraction of the halophilic archaeal community.     

Molecular systematics of the Eastern Fence Lizard (Sceloporus undulatus): a comparison of Parsimony, Likelihood, and Bayesian approaches

Phylogenetic analysis of large datasets using complex nucleotide substitution models under a maximum likelihood framework can be computationally infeasible, especially when attempting to infer confidence values by way of nonparametric bootstrapping. Recent developments in phylogenetics suggest the computational burden can be reduced by using Bayesian methods of phylogenetic inference. However, few empirical phylogenetic studies exist that explore the efficiency of Bayesian analysis of large datasets. To this end, we conducted an extensive phylogenetic analysis of the wide-ranging and geographically variable Eastern Fence Lizard (Sceloporus undulatus). Maximum parsimony, maximum likelihood, and Bayesian phylogenetic analyses were performed on a combined mitochondrial DNA dataset (12S and 16S rRNA, ND1 protein-coding gene, and associated tRNA; 3,688 bp total) for 56 populations of S. undulatus (78 total terminals including other S. undulatus group species and outgroups). Maximum parsimony analysis resulted in numerous equally parsimonious trees (82,646 from equally weighted parsimony and 335 from weighted parsimony). The majority rule consensus tree derived from the Bayesian analysis was topologically identical to the single best phylogeny inferred from the maximum likelihood analysis, but required approximately 80% less computational time. The mtDNA data provide strong support for the monophyly of the S. undulatus group and the paraphyly of "S. undulatus" with respect to S. belli, S. cautus, and S. woodi. Parallel evolution of ecomorphs within "S. undulatus" has masked the actual number of species within this group. This evidence, along with convincing patterns of phylogeographic differentiation suggests "S. undulatus" represents at least four lineages that should be recognized as evolutionary species.