Analysis of a ubiquitous promoter element in a primitive eukaryote: early evolution of the initiator element.
Typical metazoan core promoter elements, such as TATA boxes and Inr motifs, have yet to be identified in early-evolving eukaryotes, underscoring the extensive divergence of these organisms. Towards the identification of core promoters in protists, we have studied transcription of protein-encoding genes in one of the earliest-diverging lineages of Eukaryota, that represented by the parasitic protist Trichomonas vaginalis. A highly conserved element, comprised of a motif similar to a metazoan initiator (Inr) element, surrounds the start site of transcription in all examined T. vaginalis genes. In contrast, a metazoan-like TATA element appears to be absent in trichomonad promoters. We demonstrate that the conserved motif found in T. vaginalis protein-encoding genes is an Inr promoter element. This trichomonad Inr is essential for transcription, responsible for accurate start site selection, and interchangeable between genes, demonstrating its role as a core promoter element. The sequence requirements of the trichomonad Inr are similar to metazoan Inrs and can be replaced by a mammalian Inr. These studies show that the Inr is a ubiquitous, core promoter element for protein-encoding genes in an early-evolving eukaryote. Functional and structural similarities between this protist Inr and the metazoan Inr strongly indicate that the Inr promoter element evolved early in eukaryotic evolution. (+info)
An evaluation of elongation factor 1 alpha as a phylogenetic marker for eukaryotes.
Elongation factor 1 alpha (EF-1 alpha) is a highly conserved ubiquitous protein involved in translation that has been suggested to have desirable properties for phylogenetic inference. To examine the utility of EF-1 alpha as a phylogenetic marker for eukaryotes, we studied three properties of EF-1 alpha trees: congruency with other phyogenetic markers, the impact of species sampling, and the degree of substitutional saturation occurring between taxa. Our analyses indicate that the EF-1 alpha tree is congruent with some other molecular phylogenies in identifying both the deepest branches and some recent relationships in the eukaryotic line of descent. However, the topology of the intermediate portion of the EF-1 alpha tree, occupied by most of the protist lineages, differs for different phylogenetic methods, and bootstrap values for branches are low. Most problematic in this region is the failure of all phylogenetic methods to resolve the monophyly of two higher-order protistan taxa, the Ciliophora and the Alveolata. JACKMONO analyses indicated that the impact of species sampling on bootstrap support for most internal nodes of the eukaryotic EF-1 alpha tree is extreme. Furthermore, a comparison of observed versus inferred numbers of substitutions indicates that multiple overlapping substitutions have occurred, especially on the branch separating the Eukaryota from the Archaebacteria, suggesting that the rooting of the eukaryotic tree on the diplomonad lineage should be treated with caution. Overall, these results suggest that the phylogenies obtained from EF-1 alpha are congruent with other molecular phylogenies in recovering the monophyly of groups such as the Metazoa, Fungi, Magnoliophyta, and Euglenozoa. However, the interrelationships between these and other protist lineages are not well resolved. This lack of resolution may result from the combined effects of poor taxonomic sampling, relatively few informative positions, large numbers of overlapping substitutions that obscure phylogenetic signal, and lineage-specific rate increases in the EF-1 alpha data set. It is also consistent with the nearly simultaneous diversification of major eukaryotic lineages implied by the "big-bang" hypothesis of eukaryote evolution. (+info)
Activity of disulfiram (bis(diethylthiocarbamoyl)disulphide) and ditiocarb (diethyldithiocarbamate) against metronidazole-sensitive and -resistant Trichomonas vaginalis and Tritrichomonas foetus.
Clinical resistance of Trichomonas vaginalis to metronidazole is best correlated with MIC values measured under aerobic conditions. Under these conditions both disulfiram (bis(diethylthiocarbamoyl)disulphide), and its first mammalian metabolite, ditiocarb (diethyldithiocarbamate), showed high levels of activity against metronidazole-sensitive (disulfiram MIC, 0.1-0.7 microM; ditiocarb MIC, 0.3-9 microM) and -resistant (MICs 0.2-1.3 microM and 1.2-9 microM respectively) isolates. Tritrichomonas foetus was also sensitive-the MICs for seven metronidazole-sensitive isolates were 0.1-1.0 microM for disulfiram and 1.0-6.9 microM for ditiocarb; those for two highly metronidazole-resistant strains were 0.3-1.3 microM and 0.6-6 microM respectively. Under anerobic conditions most strains became highly resistant to both compounds. Surprisingly, disulfiram was consistently more active than ditiocarb. (+info)
Influence of growth conditions on RNA levels in relation to activity of core metabolic enzymes in the parasitic protists Trypanosoma brucei and Trichomonas vaginalis.
Levels of mRNAs encoding metabolic enzymes and their cellular activities were measured on continuous culture samples of the parasitic protists Trypanosoma brucei and Trichomonas vaginalis. The organisms were grown in chemostats at varying growth rates under glucose limitation or in the presence of excess glucose (EG), resulting in extensive adaptation of the cellular activities of glycolytic enzymes. rRNA and mRNA for beta-tubulin were monitored as controls. In Trypanosoma brucei levels of all RNAs showed a biphasic dependence on growth rate (= dilution rate D), with a sharp increase at higher D values. Cellular RNA levels of Trichomonas vaginalis rate-limited by glucose decreased slightly with increasing D. In EG-grown cells the opposite trend was observed. Equal levels for both carbon regimes were observed at intermediate D values. In both species the ratio between rRNA and mRNA encoding beta-tubulin was constant, independent of the carbon regime. mRNA encoding metabolic enzymes showed varying degrees of correlation with rRNA and beta-tubulin mRNA. In contrast, there was little to no correlation between mRNA levels and the activities of the enzymes they encode, even though only one of these is allosterically regulated. The data indicate that RNA levels in Trypanosoma brucei and Trichomonas vaginalis are determined by growth rate and in the latter species by the availability of the carbon and energy source. Rates of synthesis of metabolic enzymes are most likely regulated at the post-transcriptional level. (+info)
Convergent evolution of Trichomonas vaginalis lactate dehydrogenase from malate dehydrogenase.
Lactate dehydrogenase (LDH) is present in the amitochondriate parasitic protist Trichomonas vaginalis and some but not all other trichomonad species. The derived amino acid sequence of T. vaginalis LDH (TvLDH) was found to be more closely related to the cytosolic malate dehydrogenase (MDH) of the same species than to any other LDH. A key difference between the two T. vaginalis sequences was that Arg91 of MDH, known to be important in coordinating the C-4 carboxyl of oxalacetate/malate, was replaced by Leu91 in LDH. The change Leu91Arg by site-directed mutagenesis converted TvLDH into an MDH. The reverse single amino acid change Arg91Leu in TvMDH, however, gave a product with no measurable LDH activity. Phylogenetic reconstructions indicate that TvLDH arose from an MDH relatively recently. (+info)
Delayed versus immediate bedside inoculation of culture media for diagnosis of vaginal trichomonosis.
A comparison of delayed versus immediate inoculation of culture medium for the diagnosis of trichomonosis was conducted. The sensitivities of the two methods were 100 and 97.4%, respectively. Delayed inoculation of culture medium for women without evidence of trichomonosis on direct microscopic examination is a valid diagnostic procedure. (+info)
Iron modulates phenotypic variation and phosphorylation of P270 in double-stranded RNA virus-infected Trichomonas vaginalis.
Trichomonas vaginalis infected with a double-stranded RNA virus undergoes phenotypic variation on the basis of surface versus cytoplasmic expression of the immunogenic protein P270. Examination of batch cultures by flow cytofluorometry with monoclonal antibody (MAb) to P270 yields both fluorescent and nonfluorescent trichomonads. Greater numbers and intensity of fluorescent organisms with surface P270 reactive with MAb were evident in parasites grown in medium depleted of iron. Placement of iron-limited organisms in medium supplemented with iron gave increased numbers of nonfluorescent trichomonads. Purified subpopulations of trichomonads with and without surface P270 obtained by fluorescence-activated cell sorting reverted to nonfluorescent and fluorescent phenotypes when placed in high- and low-iron media, respectively. No similar regulation by iron of P270 was evident among virus-negative T. vaginalis isolates or virus-negative progeny trichomonads derived from virus-infected isolates. Equal amounts of P270 were detectable by MAb on immunoblots of total proteins from identical numbers of parasites grown in low- and high-iron media. Finally, P270 was found to be highly phosphorylated in high-iron parasites. Iron, therefore, plays a role in modulating surface localization of P270 in virus-harboring parasites. (+info)
Identification of Trichomonas vaginalis alpha-actinin as the most common immunogen recognized by sera of women exposed to the parasite.
A study on presence of antibodies to Trichomonis vaginalis in serum was done on a group of 500 pregnant, asymptomatic Angolan women. A serologic screening, done by ELISA, revealed that 41% of the women had IgG and IgM against the parasite. Analysis of sera by immunoblotting revealed that 94.4% of sera with anti-T. vaginalis IgG class antibodies were reactive against a common immunogenic protein of 115 kDa. The common immunogen was identified as the protozoan alpha-actinin. All sera recognizing the 115-kDa antigen were reactive against both native and recombinant T. vaginalis alpha-actinin and nonreactive against human alpha-actinin. The findings presented in this work offer a new tool for epidemiologic studies and open new perspectives for vaccination. (+info)