Imelda López-Villaseñor

Ribosomal RNA genes in eukaryotic microorganisms: witnesses of phylogeny?

The study of genomic organization and regulatory elements of rRNA genes in metazoan paradigmatic organisms has led to the most accepted model of rRNA gene organization in eukaryotes. Nevertheless, the rRNA genes of microbial eukaryotes have also been studied in considerable detail and their atypical structures have been considered as exceptions. However, it is likely that these organisms have preserved variations in the organization of a versatile gene that may be seen as living records of evolution. Here, we review the organization of the main rRNA transcription unit (rDNA) and the 5S rRNA genes (5S rDNA). These genes are reiterated in the genome of microbial eukaryotes and may be coded alone, in tandem repeats, linked to each other or linked to other genes. They may be found in the chromosome or extrachromosomally in linear or circular units. rDNA coding regions may contain introns, sequence insertions, protein-coding genes or additional spacers. The 5S rDNA can be found in tandem repeats or genetically linked to genes transcribed by RNA polymerases I, II or III. Available information from about a hundred microbial eukaryotes was used to review the unexpected diversity in the genomic organization of rRNA genes.

A simple model to explain three-base periodicity in coding DNA

A simple model is put forward to explain the long‐known three‐base periodicity in coding DNA. We propose the concept of same‐phase triplet clustering, i.e. a condition wherein a triplet appears several times in one phase without interruption by the two other possible phases. For instance, in the sequence (i): NTT_GNN_NTT_GNN_NTT_GNN_NNN_NTT_GNN (where N is any nucleotide but combinations producing TTG are excluded) there would be clustering of same‐phase TTG because this triplet appears uninterruptedly in phase 2. In contrast, in the sequence (ii): TTG_NTT_GNN_NNT_TGN_NNN_NTT_GNN there is no same‐phase clustering because neighboring TTGs are all in different phases. Observe also that in sequence (i) TTG triplets are separated by 3, 3 and 6 nucleotides (3n distances), while in sequence (ii) they are separated by 1, 4 and 5 nucleotides (non‐3n distances). In this work, we demonstrate that in coding DNA the 3n distances generated by (i)‐type sequences proportionally outnumber the non‐3n distances generated by (ii)‐type sequences, this condition would be the basis of three‐base periodicity. Randomized sequences had (i)‐ and (ii)‐type sequences too but clustering was statistically different. To prove our model we generated (i)‐type sequences in a randomized sequence by inducing clustering of same‐phase triplets. In agreement with the model this sequence displayed three‐base periodicity. Furthermore, two‐ and four‐base periodicities could also be induced by artificially inducing clustering of duplets and tetraplets.

Stationary phase in Trypanosoma cruzi epimastigotes as a preadaptive stage for metacyclogenesis

Trypanosoma cruzi is a species of parasitic protozoa that causes American trypanosomiasis or Chagas disease. These parasites go through a complex life cycle in Triatominae insects and vertebrate hosts. Epimastigotes are replicative forms that colonize the digestive tract of the vector and can be cultured in axenic media. The growth curve of epimastigotes allows assessment of differences in cells undergoing growth rate transitions from an exponential growth to a stationary phase. Since the classical descriptions of T. cruzi, it has been noted that the growth curve of epimastigotes in culture can give rise, in the stationary phase, to nonreplicating forms of metacyclic trypomastigotes. Metacyclogenesis therefore regards to the development process by which epimastigote transform into infective metacyclic trypomastigotes. In nature, these metacyclic forms allow the spread of Chagas disease when transmitted from an infected vector to a vertebrate host. This work reviews cellular phenomena that occur during the growth rate transitions of epimastigotes in culture, which may be related to very early physiological conditions for metacyclogenesis. Many of these events have not been thoroughly investigated. Their analysis can stimulate new hypotheses and future research in an important area not fully exploited.

The Trypanosoma cruzi nucleolus: a morphometrical analysis of cultured epimastigotes in the exponential and stationary phases

Our group is interested in rRNA and ribosome biogenesis in the parasitic protozoan Trypanosoma cruzi. Epimastigotes represent an extracellular replicative stage of T. cruzi and can be cultured in axenic media. The growth curve of epimastigotes allows assessment of potential differences in the nucleoli of cells undergoing growth-rate transitions. To establish cellular parameters for studying ribosome biogenesis in T. cruzi, a morphometric analysis of the nucleoli of cultured cells in the exponential and stationary phases was conducted. Electron micrograph-based measurements of nuclear sections from independent cells demonstrated that the nucleolar area is over twofold higher in exponentially growing cells, as compared with epimastigotes in the stationary phase. The granular component of the nucleoli of actively growing cells was the main structural element. Cycloheximide moderately reduced the apparent size of the nucleoli without an apparent disruption of their architecture. Our results provide a firm basis for the establishment of an experimental model to study the organization of the nucleolus during the growth and development of T. cruzi.

Comparative analyses among the Trichomonas vaginalis, Trichomonas tenax, and Tritrichomonas foetus 5S ribosomal RNA genes.

The 5S ribosomal RNA (5S rRNA) is an essential component of ribosomes. Throughout evolution, variation is found among 5S rRNA genes regarding their chromosomal localization, copy number, and intergenic regions. In this report, we describe and compare the gene sequences, motifs, genomic copy number, and chromosomal localization of the Trichomonas vaginalis, Trichomonas tenax, and Tritrichomonas foetus 5S rRNA genes. T. vaginalis and T. foetus have a single type of 5S rRNA-coding region, whereas two types were found in T. tenax. The sequence identities among the three organisms are between 94 and 97%. The intergenic regions are more divergent in sequence and size with characteristic species-specific motifs. The T. foetus 5S rRNA gene has larger and more complex intergenic regions, which contain either an ubiquitin gene or repeated sequences. The 5S rRNA genes were located in Trichomonads chromosomes by fluorescent in situ hybridization.

Electron Microscopy Analysis of the Nucleolus of Trypanosoma cruzi

The nucleolus is the main site for synthesis and processing of ribosomal RNA in eukaryotes. In mammals, plants, and yeast the nucleolus has been extensively characterized by electron microscopy, but in the majority of the unicellular eukaryotes no such studies have been performed. Here we used ultrastructural cytochemical and immunocytochemical techniques as well as three-dimensional reconstruction to analyze the nucleolus of Trypanosoma cruzi, which is an early divergent eukaryote of medical importance. In T. cruzi epimastigotes the nucleolus is a spherical intranuclear ribonucleoprotein organelle localized in a relatively central position within the nucleus. Dense fibrillar and granular components but not fibrillar centers were observed. In addition, nuclear bodies resembling Cajal bodies were observed associated to the nucleolus in the surrounding nucleoplasm. Our results provide additional morphological data to better understand the synthesis and processing of the ribosomal RNA in kinetoplastids.

Two Trichomonas vaginalis Loci Encoding for Distinct Cysteine Proteinases Show a Genomic Linkage with Putative Inositol Hexakisphosphate Kinase (IP6K2) or an ABC Transporter Gene

Trichomonas vaginalis is the causative agent of trichomonosis, oneof the most common sexually transmitted diseases in humans. Thisprotist has multiple proteinases mainly of the cysteine type (CPs)[15,16], and some of them participate in parasite virulence [1–3,13].Nevertheless, only four CP genes with homology to cathepsins L havebeen reported in T. vaginalis: tvcp1, tvcp2, tvcp3 and tvcp4 [12].The T. vaginalis genome is considered AþT rich [26] as themajority of protozoan genomes. However, in a GþC content analysisof coding regions of several trichomonads genes, it was observed thatthe GþC content of these genomic sequences could be used as a tool toidentify potential coding regions, since the intergenic regions are AþTricher than the coding regions [5].Examination of the 59UTR of all available protein-encoding genesfrom this protozoan studied to date has revealed a highly conservedTCA

Functional Analysis of Sequence Motifs Involved in the Polyadenylation of Trichomonas vaginalis mRNAs

ABSTRACT Synthesis of functional mRNA in eukaryotes involves processing of precursor transcripts, including the addition of a poly(A) tail at the 3′ end. A multiprotein complex recognizes a polyadenylation signal, generally the hexanucleotide AAUAAA in metazoans, to direct processing of the pre-mRNA. Based on sequence analysis of several cDNAs, we have previously suggested that the UAAA tetranucleotide (which may include the UAA translation stop codon) could be the polyadenylation signal in Trichomonas vaginalis, a parasitic protozoon that causes human trichomoniasis. This proposal is analyzed here with the aid of a transient-expression system of a reporter gene (cat flanked by T. vaginalis actin noncoding sequences). When cells were transfected with a plasmid bearing the original 3′ untranslated region (UTR) sequence containing the UAAA motif, the resulting cat mRNA was polyadenylated similarly to the endogenous actin mRNA. Base changes in the UAAA sequence produced alterations to the polyadenylation site of the reporter mRNAs, while nucleotide substitutions at either side of UAAA did not. Furthermore, relocation of the UAAA motif redirected the processing and polyadenylation of the reporter mRNA. In addition, a pre-mRNA cleavage site for polyadenylation was defined. Interaction of T. vaginalis proteins with the UAAA motif was shown by electrophoretic mobility shift assays. Based on our findings, we provide evidence that in T. vaginalis the UAAA tetranucleotide has a role equivalent to that of the metazoan consensus AAUAAA polyadenylation signal.

Identification and characterization of a surface-associated, subtilisin-like serine protease in Trichomonas vaginalis.

SUMMARY Trichomonas vaginalis is a protozoan parasite causing trichomonosis, a sexually transmitted infection in humans. This parasite has numerous proteases, most of which are cysteine proteases that appear to be involved in adherence and cytotoxicity of host cells. In this report we identify and characterize a putative subtilisin-like serine protease (SUB1). The sub1 gene encodes a 101-kDa protein. In silico analyses predict signal and pro-peptides at the N-terminus, and a transmembrane helix at the carboxy-terminal region. The sub1 gene was found as single copy by Southern analysis, albeit additional serine protease related genes are annotated in the T. vaginalis genome. The expression of sub1 could only be detected by RT-PCR and Ribonuclease Protection Assays, suggesting a low abundant mRNA. The sub1 gene transcription start site was correctly assigned by RPA. The transcript abundance was found to be modulated by the availability of iron in the growth medium. Antibodies raised to a specific SUB1 peptide recognized a single protein band (approximately 82 kDa) in Western blots, possibly representing the mature form of the protein. Immunofluorescence showed SUB1 on the trichomonad surface, and in dispersed vesicles throughout the cytoplasm. A bioinformatic analysis of genes annotated as serine proteases in the T. vaginalis genome is also presented. To our knowledge this is the first putative serine protease experimentally described for T. vaginalis.

The yeasts phosphorelay systems: a comparative view

Cells contain signal transduction pathways that mediate communication between the extracellular environment and the cell interior. These pathways control transcriptional programs and posttranscriptional processes that modify cell metabolism in order to maintain homeostasis. One type of these signal transduction systems are the so-called Two Component Systems (TCS), which conduct the transfer of phosphate groups between specific and conserved histidine and aspartate residues present in at least two proteins; the first protein is a sensor kinase which autophosphorylates a histidine residue in response to a stimulus, this phosphate is then transferred to an aspartic residue located in a response regulator protein. There are classical and hybrid TCS, whose difference consists in the number of proteins and functional domains involved in the phosphorelay. The TCS are widespread in bacteria where the sensor and its response regulator are mostly specific for a given stimulus. In eukaryotic organisms such as fungi, slime molds, and plants, TCS are present as hybrid multistep phosphorelays, with a variety of arrangements (Stock et al. in Annu Rev Biochem 69:183–215, 2000; Wuichet et al. in Curr Opin Microbiol 292:1039–1050, 2010). In these multistep phosphorelay systems, several phosphotransfer events take place between different histidine and aspartate residues localized in specific domains present in more than two proteins (Thomason and Kay, in J Cell Sci 113:3141–3150, 2000; Robinson et al. in Nat Struct Biol 7:626–633, 2000). This review presents a brief and succinct description of the Two-component systems of model yeasts, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Cryptococcus neoformans and Kluyveromyces lactis. We have focused on the comparison of domain organization and functions of each component present in these phosphorelay systems.