ABSTRACT. Based on morphological characters, peritrich ciliates (Class Olygohymenophorea, Subclass Peritrichia) have been subdivided into the Orders Sessilida and Mobilida. Molecular phylogenetic studies on peritrichs have been restricted to members of the Order Sessilida. In order to shed more light into the evolutionary relationships within peritrichs, the complete small subunit rRNA (SSU rRNA) sequences of four mobilid species, Trichodina nobilis, Trichodina heterodentata, Trichodina reticulata, and Trichodinella myakkae were used to construct phylogenetic trees using maximum parsimony, neighbor joining, and Bayesian analyses. Whatever phylogenetic method used, the peritrichs did not constitute a monophyletic group: mobilid and sessilid species did not cluster together. Similarity in morphology but difference in molecular data led us to suggest that the oral structures of peritrichs are the result of evolutionary convergence. In addition, Trichodina reticulata, a Trichodina species with granules in the center of the adhesive disc, branched separately from its congeners, Trichodina nobilis and Trichodina heterodentata, trichodinids without such granules. This indicates that granules in the adhesive disc might be a phylogenetic character of high importance within the Family Trichodinidae.

Ciliates are very good models for studying post-translationally generated tubulin heterogeneity because they exhibit highly differentiated microtubular networks in combination with reduced genetic diversity. We have approached the analysis of tubulin heterogeneity in Paramecium through extensive isolation and characterization of monoclonal antibodies using various antigens and several immunization protocols. Eight monoclonal antibodies and 10 hybridoma supernatants were characterized by: i) immunoblotting on ciliate and pig brain tubulins as well as on peptide maps of Paramecium axonemal tubulin; ii) immunoblotting on ciliate tubulin fusion peptides generated in E coli, a procedure which allows in principle to discriminate antibodies that are directed against tubulin sequence (reactive on fusion peptides) from those directed against a post-translational epitope (non-reactive); and iii) immunofluorescence on Paramecium, 3T3 and PtK2 cells. Twelve antibodies labeled all microtubules in Paramecium cells and were found to be directed against tubulin primary sequences (nine of them being located in the alpha N-terminal domain, one in the beta C-terminal one, and two in alpha and beta central stretches). The remaining ones decorated only a specific subset of microtubules within the cell and were presumably directed against post-translational modifications. Among these, three antibodies are directed against an N-terminal acetylated epitope of alpha-tubulin whereas the epitopes of three other ones (TAP 952 degrees, AXO 58 and AXO 49 degrees) apparently correspond to still unidentified post-translational modifications, located in the C-terminal domain of both alpha- and beta-tubulins. The AXO 49 degrees specificity is similar to that of a previously described polyclonal serum raised against Paramecium axonemal tubulin [2]. The results are discussed in terms of identification and accessibility of the epitopes and immunogenicity of ciliate tubulin with reference to mammalian and ciliate tubulin sequences.

ABSTRACT. Peritrich ciliates have been traditionally subdivided into two orders, Sessilida and Mobilida within the subclass Peritrichia. However, all the existing small subunit (SSU) rRNA phylogenetic trees showed that the sessilids and mobilids did not branch together. To shed some light on this disagreement, we tested whether or not the classic Peritrichia is a monophyletic group by assessing the reliability of the SSU rRNA phylogeny in terms of congruency with a-tubulin phylogeny. For this purpose, we obtained 10 partial a-tubulin sequences from peritrichs and built phylogenetic trees based on a-tubulin nucleotide and amino acid data. A phylogenetic tree from the a-tubulin and SSU rRNA genes in combination was also constructed and compared with that from the SSU rRNA gene using a similar species sampling. Our results show that the mobilids and sessilids are consistently separated in all trees, which reinforces the idea that the peritrichs do not constitute a monophyletic group. However, in all a-tubulin gene trees, the urceolariids and trichodiniids do not group together, suggested mobilids may not be a monophyletic group.

Synthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit.

The use of PCR to amplify a specific virA gene fragment serves as a highly specific and sensitive method to detect virulent bacteria of the genus Shigella and enteroinvasiveEscherichia coli. Amplification of a 215-bp DNA band was obtained by using isolated genomic DNA of Shigella, individual cells of Shigella dysenteriae, and mayonnaise contaminated with S. dysenteriae. Moreover, a multiplex PCR with specific (virA) and bacterium-restricted (16S ribosomal DNA) primers generated an amplification product of approximately 755 bp for all bacteria tested and an additional 215-bp product for Shigella and enteroinvasive E. coli.

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The parasitic protozoan Entamoeba histolytica relies on a very dynamic cytoskeleton in order to invade and survive in host tissues. Identification of cytoskeletal elements is key to understanding these processes. Here we present the characterization of EhLimA, the first LIM protein of E. histolytica. EhLimA consists of a single LIM domain at its N terminus and exhibits the highest degree of homology with DdLimE from Dictyostelium discoideum. Immunofluorescence localization of EhLimA using anti-EhLimA antibodies revealed that EhLimA is highly concentrated at the plasma membrane of cells. Silencing or overexpression of the EhLimA gene did not have a significant effect on the growth or morphology of the parasite. EhLimA associates with the cytoskeleton as demonstrated by the enrichment of the protein in cytoskeleton fractions as well as in pull-down assays that revealed that cytoskeleton association involves interaction with actin. EhLimA binding to actin was shown to be dependent on the N-terminal LIM domain of EhLimA, as removal of even half of the LIM domain resulted in almost complete inhibition of the binding to actin. We also found that a portion of EhLimA floats to the lower-density regions of a sucrose gradient together with portions of the Gal-lectin light subunit and actin. Treatment of cells with the cholesterol-sequestering agent digitonin resulted in increased solubility of EhLimA. These results indicate that in addition to cytoskeletal association, EhLimA may also associate with lipid rafts in the parasite plasma membrane and suggest that EhLimA may be part of the molecular system connecting the actin cytoskeleton to membrane rafts.Many cellular functions are dependent on specific proteinprotein interactions. These protein-protein interactions are governed by a variety of protein binding domains one of which is the LIM domain. Since first described nearly 2 decades ago (21, 28, 57), LIM domain-containing proteins have been identified in a wide range of eukaryotes, including protozoa (16,30,45), plants (5,20,40), and yeast (Saccharomyces cerevisiae) (39), stressing the evolutionary importance of these proteins. In the parasitic protozoan Entamoeba histolytica, a few studies have revealed the presence of such proteins (17, 36), although to date, no LIM domain-containing proteins have been characterized in this organism. The name LIM is derived from the three developmentally regulated transcription factors, LIN-11 of Caenorhabditis elegans (21), Isl1 of rat (28), and MEC-3 of C. elegans (57) in which the LIM domain was initially identified. The LIM domain is a cysteine-rich motif consisting of two zinc finger-like modules and displaying the consensus sequence CX 2 CX 16-23 HX 2 CX 2 CX 2 CX 16-21 CX 2 (C/H/D/) (31). This domain is found in a variety of different proteins with diverse functions, including transcription factors and cytoskeleton-associated proteins, and may be found in association with other functional domains, such as homeodomains, protein kinase domains, or other protein binding domains (...