Fine oral filaments in Paramecium: a biochemical and immunological analysis.

In Paramecium, several kinds of the oral networks of fine filaments are defined at the ultrastructural level. Using the sodium chloride-treated oral apparatus of Paramecium as an antigen to produce monoclonal antibodies, we have begun to identify the proteins constituting these networks. Immunoblotting showed that all positive antibodies were directed against three bands (70-, 75-and 83-kD), which corresponded to quantitatively minor components of the antigen; there was no antibody specific for the quantitatively major components (58- and 62-kD). Immunolocalization with four of these antibodies directed against one or several of these three bands showed that these proteins are components of the fine filaments supporting the oral area; a decoration of the basal bodies and the outer lattice was also observed on the cortex. Immunofluorescence on interphase cells suggested that the three proteins colocalized on the left side of the oral apparatus, whereas only the 70-kD band was detected on the right side. During division, the antigens of the antibodies were detected at different stages after oral basal body assembly. The antibodies cross-reacted with the tetrins, which are oral filament-forming proteins in Tetrahymena, demonstrating that tetrin-related proteins are quantitatively minor components of the oral and the somatic cytoskeleton of Paramecium.

Early-branching or fast-evolving eukaryotes? An answer based on slowly evolving positions.

The current paradigm of eukaryotic evolution is based primarily on comparative analysis of ribosomal RNA sequences. It shows several early-emerging lineages, mostly amitochondriate, which might be living relics of a progressive assembly of the eukaryotic cell. However, the analysis of slow-evolving positions, carried out with the newly developed slow-fast method, reveals that these lineages are, in terms of nucleotide substitution, fast-evolving ones, misplaced at the base of the tree by a long branch attraction artefact. Since the fast-evolving groups are not always the same, depending on which macromolecule is used as a marker, this explains most of the observed incongruent phylogenies. The current paradigm of eukaryotic evolution thus has to be seriously re-examined as the eukaryotic phylogeny is presently best summarized by a multifurcation. This is consistent with the Big Bang hypothesis that all extant eukaryotic lineages are the result of multiple cladogeneses within a relatively brief period, although insufficiency of data is also a possible explanation for the lack of resolution. For further resolution, rare evolutionary events such as shared insertions and/or deletions or gene fusions might be helpful.

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.