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.

Objectives Investigate the feasibility of saliva sampling as a noninvasive and safer tool to detect SARS-CoV-2 and to compare its reproducibility and sensitivity with nasopharyngeal swab samples (NPS). The use of sample pools was also investigated. Methods 2107 paired samples were collected from asymptomatic health care and office workers in Mexico City. Sixty of these samples were also analyzed in two other independent laboratories for concordance analysis. Sample processing and analysis of virus genetic material were performed according to standard protocols described elsewhere. Pooling analysis was performed by analyzing the saliva pool and the individual pool components. Results The concordance between NPS and saliva results was 95.2% (Kappa: 0.727, p = 0.0001) and 97.9% without considering inconclusive results (Kappa: 0.852, p = 0.0001). Saliva had a lower number of inconclusive results than NPS (0.9% vs 1.9%). Furthermore, saliva shows a significantly higher concentration of both total RNA and viral copies than NPS. Comparison of our results with those of the other two laboratories shows 100% and 97% concordance. Saliva samples are stable without the use of any preservative, a positive SARS-CoV-2 sample can be detected 5, 10, and 15 days after collection when the sample is stored at 4 °C. Conclusions Our results indicate that saliva is as effective as NPS for the identification of SARS-CoV-2-infected asymptomatic patients, sample pooling facilitates the analysis of a larger number of samples with the benefit of cost reduction.

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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.

A simple model is put forward to explain the longknown 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.

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.

ABSTRACTSynthesis 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 inTrichomonas 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 (catflanked byT. vaginalisactin noncoding sequences). When cells were transfected with a plasmid bearing the original 3′ untranslated region (UTR) sequence containing the UAAA motif, the resultingcatmRNA 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 ofT. vaginalisproteins with the UAAA motif was shown by electrophoretic mobility shift assays. Based on our findings, we provide evidence that inT. vaginalisthe UAAA tetranucleotide has a role equivalent to that of the metazoan consensus AAUAAA polyadenylation signal.

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