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PubMed1998

New insights into the phylogeny of eukaryotes based on ciliate Hsp70 sequences.

The current framework of the eukaryotic phylogeny is based on the analysis of a comprehensive set of sequences of the small subunit ribosomal RNA. However, phylogenies based on protein-encoding genes are not completely congruent with this picture. Since congruence between different markers is the best tool to determine evolutionary history, we focused on Hsp70 (heat-shock protein of 70 kDa), a chaperone protein which is highly conserved and is a potentially reliable phylogenetic marker. We used a PCR-based approach to sequence Hsp70s in two distinct classes of Ciliates. Seven Hsp70s were identified from Paramecium tetraurelia (Oligohymenophora) and six Hsp70s from Euplotes aediculatus (Hypotricha), encompassing orthologous genes for all major Hsp70 classes of Eukaryotes, i.e., those localized in cytosol, in endoplasmic reticulum, and in mitochondria. Three independent phylogenies of eukaryotes, based on each set of orthologous genes, have been constructed using different tree reconstruction methods. A significant advantage of Hsp70s is the existence of outgroups close to Eukaryotes for these major classes, reducing the long-branch attraction artifact due to the outgroup. The monophyly of Ciliates is supported by good bootstrap proportions in the phylogenetic reconstructions, and this phylum is generally a sister-group of Sporozoa, forming the expected Alveolates clade. The Hsp70 seems to be a suitable phylogenetic marker since it recovers all the monophyletic groups, undoubtedly defined by morphological criteria. The Hsp70 trees are, however, notably different from the rRNA ones and do not show two aspects of the classical topology, i.e., the successive emergence of deeply branching groups and the vast assembly of the major eukaryotic groups, emerging at the tip of the tree, i.e., the "terminal crown". More precisely, the Hsp70 trees do not resolve the relationships between the major groups of Eukaryotes with confidence, in keeping with the hypothesis that all these groups emerged in a great radiation that occurred at the origin of all the extant Eukaryotes.

Amino Acid Sequence↗
PubMed2000

Iron hydrogenases and the evolution of anaerobic eukaryotes.

Hydrogenases, oxygen-sensitive enzymes that can make hydrogen gas, are key to the function of hydrogen-producing organelles (hydrogenosomes), which occur in anaerobic protozoa scattered throughout the eukaryotic tree. Hydrogenases also play a central role in the hydrogen and syntrophic hypotheses for eukaryogenesis. Here, we show that sequences related to iron-only hydrogenases ([Fe] hydrogenases) are more widely distributed among eukaryotes than reports of hydrogen production have suggested. Genes encoding small proteins which contain conserved structural features unique to [Fe] hydrogenases were identified on all well-surveyed aerobic eukaryote genomes. Longer sequences encoding [Fe] hydrogenases also occur in the anaerobic eukaryotes Entamoeba histolytica and Spironucleus barkhanus, both of which lack hydrogenosomes. We also identified a new [Fe] hydrogenase sequence from Trichomonas vaginalis, bringing the total of [Fe] hydrogenases reported for this organism to three, all of which may function within its hydrogenosomes. Phylogenetic analysis and hypothesis testing using likelihood ratio tests and parametric bootstrapping suggest that the [Fe] hydrogenases in anaerobic eukaryotes are not monophyletic. Iron-only hydrogenases from Entamoeba, Spironucleus, and Trichomonas are plausibly monophyletic, consistent with the hypothesis that a gene for [Fe] hydrogenase was already present on the genome of the common, perhaps also anaerobic, ancestor of these phylogenetically distinct eukaryotes. Trees where the [Fe] hydrogenase from the hydrogenosomal ciliate Nyctotherus was constrained to be monophyletic with the other eukaryote sequences were rejected using a likelihood ratio test of monophyly. In most analyses, the Nyctotherus sequence formed a sister group with a [Fe] hydrogenase on the genome of the eubacterium Desulfovibrio vulgaris. Thus, it is possible that Nyctotherus obtained its hydrogenosomal [Fe] hydrogenase from a different source from Trichomonas for its hydrogenosomes. We find no support for the hypothesis that components of the Nyctotherus [Fe] hydrogenase fusion protein derive from the mitochondrial respiratory chain.

Amino Acid Sequence↗
PubMed2006

Succession and regulation factors of small eukaryote community composition in a lacustrine ecosystem (Lake Pavin).

The structure and dynamics of small eukaryotes (cells with a diameter less than 5 microm) were studied over two consecutive years in an oligomesotrophic lake (Lake Pavin in France). Water samples were collected at 5 and 30 m below the surface; when the lake was stratified, these depths corresponded to the epilimnion and hypolimnion. Changes in small-eukaryote structure were analyzed using terminal restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of the 18S rRNA genes. Terminal restriction fragments from clones were used to reveal the dominant taxa in T-RFLP profiles of the environmental samples. Spumella-like cells (Chrysophyceae) did not dominate the small eukaryote community identified by molecular techniques in lacustrine ecosystems. Small eukaryotes appeared to be dominated by heterotrophic cells, particularly Cercozoa, which represented nearly half of the identified phylotypes, followed by the Fungi-LKM11 group (25%), choanoflagellates (10.3%) and Chrysophyceae (8.9%). Bicosoecida, Cryptophyta, and ciliates represented less than 9% of the community studied. No seasonal reproducibility in temporal evolution of the small-eukaryote community was observed from 1 year to the next. The T-RFLP patterns were related to bottom-up (resources) and top-down (grazing) variables using canonical correspondence analysis. The results showed a strong top-down regulation of small eukaryotes by zooplankton, more exactly, by cladocerans at 5 m and copepods at 30 m. Among bottom-up factors, temperature had a significant effect at both depths. The concentrations of nitrogenous nutrients and total phosphorus also had an effect on small-eukaryote dynamics at 5 m, whereas bacterial abundance and dissolved oxygen played a more important structuring role in the deeper zone.

Animals↗
PubMed2006

An actin homolog of the archaeon Thermoplasma acidophilum that retains the ancient characteristics of eukaryotic actin.

Actin, a central component of the eukaryotic cytoskeleton, plays a crucial role in determining cell shape in addition to several other functions. Recently, the structure of the archaeal actin homolog Ta0583, isolated from the archaeon Thermoplasma acidophilum, which lacks a cell wall, was reported by Roeben et al. (J. Mol. Biol. 358:145-156, 2006). Here we show that Ta0583 assembles into bundles of filaments similar to those formed by eukaryotic actin. Specifically, Ta0583 forms a helix with a filament width of 5.5 nm and an axial repeating unit of 5.5 nm, both of which are comparable to those of eukaryotic actin. Eukaryotic actin shows a greater resemblance to Ta0583 than to bacterial MreB and ParM in terms of polymerization characteristics, such as the requirement for Mg(2+), critical concentration, and repeating unit size. Furthermore, phylogenetic analysis also showed a closer relationship between Ta0583 and eukaryotic actin than between MreB or ParM and actin. However, the low specificity of Ta0583 for nucleotide triphosphates indicates that Ta0583 is more primitive than eukaryotic actin. Taken together, our results suggest that Ta0583 retains the ancient characteristics of eukaryotic actin.

Actins↗
PubMed2000

A hypothesis for DNA viruses as the origin of eukaryotic replication proteins.

The eukaryotic replicative DNA polymerases are similar to those of large DNA viruses of eukaryotic and bacterial T4 phages but not to those of eubacteria. We develop and examine the hypothesis that DNA virus replication proteins gave rise to those of eukaryotes during evolution. We chose the DNA polymerase from phycodnavirus (which infects microalgae) as the basis of this analysis, as it represents a virus of a primitive eukaryote. We show that it has significant similarity with replicative DNA polymerases of eukaryotes and certain of their large DNA viruses. Sequence alignment confirms this similarity and establishes the presence of highly conserved domains in the polymerase amino terminus. Subsequent reconstruction of a phylogenetic tree indicates that these algal viral DNA polymerases are near the root of the clade containing all eukaryotic DNA polymerase delta members but that this clade does not contain the polymerases of other DNA viruses. We consider arguments for the polarity of this relationship and present the hypothesis that the replication genes of DNA viruses gave rise to those of eukaryotes and not the reverse direction.

Amino Acid Sequence↗
PubMed1998

Role of DNA-membrane interactions in prokaryote-to-eukaryote transition: an hypothesis.

A model system of experiments to consider the problem of the origin of eukaryotic cells as well as the prokaryote-to-eukaryote transition was investigated, in terms of the role of nucleic acid-membrane interactions. It was thought worthwhile to consider the importance of DNA-membrane contacts for the organization of the prokaryotic nucleoid. The model for the fusion of four proto-eukaryotic cells was proposed to clarify the prokaryote-to-eukaryote transition as well as the formation of the nuclear pores of eukaryotes from the Bayer's junctions of proto-eukaryotes. The basic requirements following from the cell fusion model suggest such orientation of the cells involved. The obstacles for division of the ancestor cell were excluded by merging. Enormous advantages to the cell metabolism due to the fusion of four proto-eukaryotic cells and an intensive growth of the inner membranous structures resulted.

Cell Fusion↗
PubMedDate not supplied

[Aspects of bacteriophage-eukaryotic cell relationships].

The present work deals with bacteriophage--eukaryotic cell interrelationship, including:--Data on the possible presence of bacteriophages in calf serum and viral vaccines.--Methodological problems of research on phage--eukaryotic cell relations.--Data concerning the effects of phage infection on eukaryotic cells (demonstration of phage infection of the eukaryotic cell; integration of phage genetic material in the genome of the eukaryotic cell; coding of phage proteins in phage-infected eukaryotic cells; modified multiplication parameters and transformed potential of some phage-infected eukaryotic cells).--Indications for solving some of the problems.

Bacteriophages↗
PubMed2004

Construction and identification of eukaryotic expression vector of human full-length PLCgamma1 gene.

OBJECTIVE: To construct the eukaryotic expression vector of human full-length PLCgamma1 gene for further study of the role of PLCgamma1 in cancer invasion. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) technique was used to amplify human full-length PLCgamma1 gene from MG63 cells with a pair of specific primers containing the restriction sites for HindIII and NotI. After purification, the product of RT-PCR was digested with HindIII and NotI before insertion into the corresponding sites of eukaryotic expression vector pLNCX2, yielding the recombinant plasmid pLNCX2/PLCgamma1. PCR, restriction endonuclease analysis and DNA sequencing were performed to identify the recombinant eukaryotic expression vector pLNCX2/PLCgamma1. RT-PCR and Western blotting were used to detect the expression of the PLCgamma1 gene in LoVo cells after transient transfection via Lipofectamine TM 2000. RESULTS: A 3 878-bp full-length PLCgamma1 gene fragment was successfully amplified by RT-PCR and inserted into eukaryotic expression vector pLNCX2. After digestion by HindIII and NotI, the recombinant eukaryotic expression vector pLNCX2/PLCgamma1 yielded a 3 878-bp fragment (PLCgamma1 gene) and a 6 100 bp fragment (vector). HindIII-BglII digestion was also done to verify the correctness of the recombinant plasmid, resulting in the identification of the fragments as expected. Sequencing analysis further confirmed the results. In addition, RT-PCR and Western blotting verified that the PLCgamma1 could overexpress in LoVo cells after transfection with recombinant eukaryotic expression vector pLNCX2/PLCgamma1. CONCLUSION: The recombinant eukaryotic expression vector pLNCX2/PLCgamma1 has been constructed successfully.

Base Sequence↗
PubMed2005

Genetic diversity of microbial eukaryotes in anoxic sediment around fumaroles on a submarine caldera floor based on the small-subunit rDNA phylogeny.

Recent culture-independent molecular analyses have shown the diversity and ecological importance of microbial eukaryotes (protists) in various marine environments. In the present study we directly extracted DNA from anoxic sediment near active fumaroles on a submarine caldera floor at a depth of 200 m and constructed genetic libraries of PCR-amplified eukaryotic small-subunit (SSU) rDNA. By sequencing cloned SSU rDNA of the libraries and their phylogenetic analyses, it was shown that most sequences have affiliations with known major lineages of eukaryotes (Cercozoa, Alveolata, stramenopiles and Opisthokonta). In particular, some sequences were closely related to those of representatives of eukaryotic parasites, such as Phagomyxa and Cryothecomonas of Cercozoa, Pirsonia of stramenopiles and Ichthyosporea of Opisthokonta, although it is not clear whether the organisms occur in free-living or parasitic forms. In addition, other sequences did not seem to be related to any described eukaryotic lineages suggesting the existence of novel eukaryotes at a high-taxonomic level in the sediment. The community composition of microbial eukaryotes in the sediment we surveyed was different overall from those of other anoxic marine environments previously investigated.

Animals↗
PubMedDate not supplied

Eukaryotes have "two-component" signal transducers.

The eukaryotic proteins discussed above share a number of amino acid sequence features with prokaryotic members of the two-component signal transducer family. ETR1 and Sln1 each contain putative HPK and receivers, while Skn7 has similarity to receivers only. The other proteins--phytochromes, BCKDH kinase and the dr6 gene product--appear to be distant relatives of the two-component family because they have less-conserved motifs, although even biochemically defined prokaryotic family members can lack conserved motifs (Parkinson and Kofoid, 1992). The eukaryotic and prokaryotic proteins also show similar diversity with respect to the arrangements of the components; transmitters and receivers can lie within the same protein (for example, ETR1 and Sln1 in eukaryotes and BarA and LemA in prokaryotes) or receivers can lie on a separate protein (for example, Skn7 in yeast and CheY in bacteria (Stock et al., 1985)). The new eukaryotic members of the two-component family raise a number of questions, especially since their functions are not yet clearly defined. The identities of the signals transduced are unknown, except for the red light signal for phytochrome. Ethylene is presumably the signal for ETR1, but this has not been demonstrated directly. It also remains to be seen whether eukaryotic two-component modules are functionally homologous to the prokaryotic two-component systems; eukaryotic modules could be utilized in uncharacterized ways, possibly in combination with different signalling components. Among the eukaryotic proteins discussed here, BCKDH kinase has the best-understood function, yet its substrate is not an orthodox response regulator, and moreover, the substrate is phosphorylated at serine residues.

Arabidopsis↗
PubMed2005

A phylogenomic inventory of meiotic genes; evidence for sex in Giardia and an early eukaryotic origin of meiosis.

Sexual reproduction in eukaryotes is accomplished by meiosis, a complex and specialized process of cell division that results in haploid cells (e.g., gametes). The stereotypical reductive division in meiosis is a major evolutionary innovation in eukaryotic cells, and delineating its history is key to understanding the evolution of sex. Meiosis arose early in eukaryotic evolution, but when and how meiosis arose and whether all eukaryotes have meiosis remain open questions. The known phylogenetic distribution of meiosis comprises plants, animals, fungi, and numerous protists. Diplomonads including Giardia intestinalis (syn. G. lamblia) are not known to have a sexual cycle; these protists may be an early-diverging lineage and could represent a premeiotic stage in eukaryotic evolution. We surveyed the ongoing G. intestinalis genome project data and have identified, verified, and analyzed a core set of putative meiotic genes-including five meiosis-specific genes-that are widely present among sexual eukaryotes. The presence of these genes indicates that: (1) Giardia is capable of meiosis and, thus, sexual reproduction, (2) the evolution of meiosis occurred early in eukaryotic evolution, and (3) the conserved meiotic machinery comprises a large set of genes that encode a variety of component proteins, including those involved in meiotic recombination.

Animals↗
PubMed2005

Monophyly of primary photosynthetic eukaryotes: green plants, red algae, and glaucophytes.

Between 1 and 1.5 billion years ago, eukaryotic organisms acquired the ability to convert light into chemical energy through endosymbiosis with a Cyanobacterium (e.g.,). This event gave rise to "primary" plastids, which are present in green plants, red algae, and glaucophytes ("Plantae" sensu Cavalier-Smith). The widely accepted view that primary plastids arose only once implies two predictions: (1) all plastids form a monophyletic group, as do (2) primary photosynthetic eukaryotes. Nonetheless, unequivocal support for both predictions is lacking (e.g.,). In this report, we present two phylogenomic analyses, with 50 genes from 16 plastid and 15 cyanobacterial genomes and with 143 nuclear genes from 34 eukaryotic species, respectively. The nuclear dataset includes new sequences from glaucophytes, the less-studied group of primary photosynthetic eukaryotes. We find significant support for both predictions. Taken together, our analyses provide the first strong support for a single endosymbiotic event that gave rise to primary photosynthetic eukaryotes, the Plantae. Because our dataset does not cover the entire eukaryotic diversity (but only four of six major groups in), further testing of the monophyly of Plantae should include representatives from eukaryotic lineages for which currently insufficient sequence information is available.

Bayes Theorem↗
PubMed1994

Processing of eukaryotic ribosomal RNA.

In summary, it can be argued that the understanding of eukaryotic rRNA processing is no less important than the understanding of mRNA maturation, since the capacity of a cell to carry out protein synthesis is controlled, in part, by the abundance of ribosomes. Processing of pre-rRNA is highly regulated, involving many cellular components acting either alone or as part of a complex. Some of these components are directly involved in the modification and cleavage of the precursor rRNA, while others direct the packaging of the rRNA into ribosome subunits. As is the case for pre-mRNA processing, snoRNPs are clearly involved in eukaryotic rRNA processing, and have been proposed to assemble with other proteins into at least one complex called a "processosome" (17), which carries out the ordered processing of the pre-rRNA and its assembly into ribosomes. The formation of a processing complex clearly makes possible the regulation required to coordinate the abundance of ribosomes with the physiological and developmental changes of a cell. It may be that eukaryotic rRNA processing is even more complex than pre-mRNA maturation, since pre-rRNA undergoes extensive nucleotide modification and is assembled into a complex structure called the ribosome. Undoubtedly, features of the eukaryotic rRNA-processing pathway have been conserved evolutionarily, and the genetic approach available in yeast research (6) should provide considerable knowledge that will be useful for other investigators working with higher eukaryotic systems. Interestingly, it was originally hoped that the extensive work and understanding of bacterial ribosome formation would provide a useful paradigm for the process in eukaryotes. However, although general features of ribosome structure and function are highly conserved between bacterial and eukaryotic systems, the basic strategy in ribosome biogenesis seems to be, for the most part, distinctly different. Thus, the detailed molecular mechanisms for rRNA processing in each kingdom will have to be independently deciphered in order to elucidate the features and regulation of this important process for cell survival.

Animals↗
PubMed1993

DNA topoisomerase V is a relative of eukaryotic topoisomerase I from a hyperthermophilic prokaryote.

The DNA topoisomerases are ubiquitous enzymes that fulfil vital roles in the replication, transcription and recombination of DNA by carrying out DNA-strand passage reactions. Here we characterize a prokaryotic counterpart to the eukaryotic topoisomerase I in the hyperthermophilic methanogen Methanopyrus kandleri. The new enzyme, called topoisomerase V, has the following properties in common with eukaryotic topoisomerase I, which distinguish it from all other known prokaryotic topoisomerases: (1) its activity is Mg(2+)-independent; (2) it relaxes both negatively and positively supercoiled DNA; (3) it makes a covalent complex with the 3' end of the broken DNA strand; and (4) it is recognized by antibody raised against human topoisomerase I. Eukaryotic-like enzymes have been discovered in some hyperthermophilic prokaryotes, namely the eocytes and the extremely thermophilic archaebacteria, and hyperthermophilic homologues of eukaryotic DNA polymerase-alpha, transcription factor IIB and DNA ligase have all been reported. Thus our findings support the idea that some essential parts of the eukaryotic transcription-translation and replication machineries were in place before the emergence of eukaryotes, and that the closest living relatives of eukaryotes may be hyperthermophiles.

Base Sequence↗
PubMed1999

The Tangled Web: Gene Genealogies and the Origin of Eukaryotes.

Accessing data from the genomes of organisms (individual genes) and analyzing these data using sophisticated alignment and phylogenetic methods led to the expectation that we would be able to paint a clear picture of the evolution of eukaryotes. Previous analyses based on morphology and ultrastructure failed to pinpoint both the sister taxon to eukaryotes and the branching order of eukaryotic lineages. However, the expectation that molecular data would provide resolution has not been met since a growing number of gene genealogies present conflicting hypotheses for the origin and diversification of eukaryotes. Instead of reconstructing a simple bifurcating tree of life, these gene genealogies have generated a complex picture of eukaryotic genomes whereby ancient lateral transfers (of individual genes or perhaps even entire genomes) has tangled the evolutionary history of eukaryotes. Resolution of these conflicting genealogies comes in recognizing that eukaryotes are chimeric, containing genetic information from multiple ancestral lineages.

Journal Article↗
PubMed2003

Recognition of translation initiation sites of eukaryotic genes based on an EM algorithm.

With the rapid increase of DNA databases of human and other eukaryotic model organisms, a large great number of genes need to be distinguished from the DNA databases. Exact recognition of translation initiation sites (TISs) of eukaryotic genes is very important to understand the translation initiation process, predict the detailed structure of eukaryotic genes, and annotate uncharacterized sequences. The problem has not been solved satisfactorily, especially for recognizing TISs of the eukaryotic genes with shorter first exons. It is an important task for extracting new features and finding new powerful algorithms for recognizing TISs of eukaryotic genes. In this paper, the important characteristics of shorter flanking fragments around TISs are extracted and an expectation-maximization (EM) algorithm based on incomplete data is used to recognize TISs of eukaryotic genes. The accuracy is up to 87.8% over a six-fold cross-validation test. The result shows that the identification variables are effectively extracted and the EM algorithm is a powerful tool to predict the TISs of eukaryotic genes. The algorithm also can be applied to other classification or clustering tasks in bioinformatics.

Algorithms↗
PubMed1992

Metal-regulated transcription in eukaryotes.

This review has summarized many of the major aspects of metal-regulated gene transcription in eukaryotic organisms as they are currently understood at the mechanistic level. Clearly, metals represent a class of important transcriptional effector molecules which regulate gene expression in different ways and both by activation or repression of gene transcription. To date, studies of metal-regulated transcription in fungi have resulted in the most detailed description of the structure, function and mechanisms of action of eukaryotic metal-responsive transcription factors. Recently, significant progress has been made in higher eukaryotic systems through the biochemical detection and purification of MRE binding proteins which may represent MRTFs. Additionally, perhaps fungi will be exploited for their genetics and ease of manipulation to clone and functionally analyze cDNAs for MRTFs from higher eukaryotes. The isolation of cDNAs for higher eukaryotic MRTFs will provide important tools for answering a number of interesting questions in metal-regulated gene transcription. How do higher eukaryotes activate MT gene transcription in response to a broad range of environmental metals? What are the tissue distributions of MRTFs and how does their activity correlate with the exposure of different tissues to varying concentrations of metals? What are the identities of other genes regulated by MRTFs and why are such genes metal-responsive? A comprehensive understanding of the detailed mechanisms for metal-regulated transcription will ultimately require an understanding of how eukaryotic cells sense, transport, distribute and remove metals from their environment. These questions provide an interesting and exciting area of investigation for geneticists, physiologists, molecular biologists, biophysicists and biochemists now and in the future.

Animals↗
PubMed2000

Evolution of the eukaryotic translation termination system: origins of release factors.

Accurate translation termination is essential for cell viability. In eukaryotes, this process is strictly maintained by two proteins, eukaryotic release factor 1 (eRF1), which recognizes all stop codons and hydrolyzes peptidyl-tRNA, and eukaryotic release factor 3 (eRF3), which is an elongation factor 1alpha (EF-1alpha) homolog stimulating eRF1 activity. To retrace the evolution of this core system, we cloned and sequenced the eRF3 genes from Trichomonas vaginalis (Parabasalia) and Giardia lamblia (Diplomonada), which are generally thought to be "early-diverging eukaryotes," as well as those from two ciliates (Oxytricha trifallax and Euplotes aediculatus). We also determined the sequence of the eRF1 gene for G. lamblia. Surprisingly, the G. lamblia eRF3 appears to have only one domain, corresponding to EF-1alpha, while other eRF3s (including the T. vaginalis protein) have an additional N-terminal domain, of 66-411 amino acids. Considering this novel eRF3 structure and our extensive phylogenetic analyses, we suggest that (1) the current translation termination system in eukaryotes evolved from the archaea-like version, (2) eRF3 was introduced into the system prior to the divergence of extant eukaryotes, including G. lamblia, and (3) G. lamblia might be the first eukaryotic branch among the organisms considered.

Amino Acid Sequence↗