Membrane Fusion: HAP2 Protein on a Short Leash

SummaryLocalized membrane fusion in the Tetrahymena conjugation junction generates pores that provide transient cytoplasmic continuity between the two partner cells. Without male gamete-specific fusion protein HAP2/GSC1, pores fail to form, fertilization is blocked, and pair stability is compromised

Microarray Analyses of Gene Expression during the Tetrahymena thermophila Life Cycle

The model eukaryote, Tetrahymena thermophila, is the first ciliated protozoan whose genome has been sequenced, enabling genome-wide analysis of gene expression.A genome-wide microarray platform containing the predicted coding sequences (putative genes) for T. thermophila is described, validated and used to study gene expression during the three major stages of the organism's life cycle: growth, starvation and conjugation.Of the approximately 27,000 predicted open reading frames, transcripts homologous to only approximately 5900 are not detectable in any of these life cycle stages, indicating that this single-celled organism does indeed contain a large number of functional genes. Transcripts from over 5000 predicted genes are expressed at levels >5x corrected background and 95 genes are expressed at >250x corrected background in all stages. Transcripts homologous to 91 predicted genes are specifically expressed and 155 more are highly up-regulated in growing cells, while 90 are specifically expressed and 616 are up-regulated during starvation. Strikingly, transcripts homologous to 1068 predicted genes are specifically expressed and 1753 are significantly up-regulated during conjugation. The patterns of gene expression during conjugation correlate well with the developmental stages of meiosis, nuclear differentiation and DNA elimination. The relationship between gene expression and chromosome fragmentation is analyzed. Genes encoding proteins known to interact or to function in complexes show similar expression patterns, indicating that co-ordinate expression with putative genes of known function can identify genes with related functions. New candidate genes associated with the RNAi-like process of DNA elimination and with meiosis are identified and the late stages of conjugation are shown to be characterized by specific expression of an unexpectedly large and diverse number of genes not involved in nuclear functions

Genome-Scale Analysis of Programmed DNA Elimination Sites in Tetrahymena thermophila

Genetically programmed DNA rearrangements can regulate mRNA expression at an individual locus or, for some organisms, on a genome-wide scale. Ciliates rely on a remarkable process of whole-genome remodeling by DNA elimination to differentiate an expressed macronucleus (MAC) from a copy of the germline micronucleus (MIC) in each cycle of sexual reproduction. Here we describe results from the first high-throughput sequencing effort to investigate ciliate genome restructuring, comparing Sanger long-read sequences from a Tetrahymena thermophila MIC genome library to the MAC genome assembly. With almost 25% coverage of the unique-sequence MAC genome by MIC genome sequence reads, we created a resource for positional analysis of MIC-specific DNA removal that pinpoints MAC genome sites of DNA elimination at nucleotide resolution. The widespread distribution of internal eliminated sequences (IES) in promoter regions and introns suggests that MAC genome restructuring is essential not only for what it removes (for example, active transposons) but also for what it creates (for example, splicing-competent introns). Consistent with the heterogeneous boundaries and epigenetically modulated efficiency of individual IES deletions studied to date, we find that IES sites are dramatically under-represented in the ∼25% of the MAC genome encoding exons. As an exception to this general rule, we discovered a previously unknown class of small (<500 bp) IES with precise elimination boundaries that can contribute the 3′ exon of an mRNA expressed during genome restructuring, providing a new mechanism for expanding mRNA complexity in a developmentally regulated manner

Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure

<p>Abstract</p> <p>Background</p> <p><it>Tetrahymena thermophila</it>, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a germline micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of <it>Tetrahymena</it>'s coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing.</p> <p>Results</p> <p>We addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified.</p> <p>Conclusion</p> <p>We report here significant progress in genome closure and reannotation of <it>Tetrahymena thermophila</it>. Our experience to date suggests that complete closure of the MAC genome is attainable. Using the new EST evidence, automated and manual curation has resulted in substantial improvements to the over 24,000 gene models, which will be valuable to researchers studying this model organism as well as for comparative genomics purposes.</p

Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome

The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymenas germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum.</p

Transcriptome Analysis of the Model Protozoan, Tetrahymena thermophila, Using Deep RNA Sequencing

Background: The ciliated protozoan Tetrahymena thermophila is a well-studied single-celled eukaryote model organism for cellular and molecular biology. However, the lack of extensive T. thermophila cDNA libraries or a large expressed sequence tag (EST) database limited the quality of the original genome annotation. Methodology/Principal Findings: This RNA-seq study describes the first deep sequencing analysis of the T. thermophila transcriptome during the three major stages of the life cycle: growth, starvation and conjugation. Uniquely mapped reads covered more than 96 % of the 24,725 predicted gene models in the somatic genome. More than 1,000 new transcribed regions were identified. The great dynamic range of RNA-seq allowed detection of a nearly six order-of-magnitude range of measurable gene expression orchestrated by this cell. RNA-seq also allowed the first prediction of transcript untranslated regions (UTRs) and an updated (larger) size estimate of the T. thermophila transcriptome: 57 Mb, or about 55 % of the somatic genome. Our study identified nearly 1,500 alternative splicing (AS) events distributed over 5.2 % of T. thermophila genes. This percentage represents a two order-of-magnitude increase over previous EST-based estimates in Tetrahymena. Evidence of stage-specific regulation of alternative splicing was also obtained. Finally, our study allowed us to completely confirm about 26.8 % of the genes originally predicted by the gene finder, to correct coding sequence boundaries an

Macronuclear Genome Sequence of the Ciliate Tetrahymena thermophila, a Model Eukaryote

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance

Tetrahymena Metallothioneins Fall into Two Discrete Subfamilies

BACKGROUND: Metallothioneins are ubiquitous small, cysteine-rich, multifunctional proteins which can bind heavy metals. METHODOLOGY/PRINCIPAL FINDINGS: We report the results of phylogenetic and gene expression analyses that include two new Tetrahymena thermophila metallothionein genes (MTT3 and MTT5). Sequence alignments of all known Tetrahymena metallothioneins have allowed us to rationalize the structure of these proteins. We now formally subdivide the known metallothioneins from the ciliate genus Tetrahymena into two well defined subfamilies, 7a and 7b, based on phylogenetic analysis, on the pattern of clustering of Cys residues, and on the pattern of inducibility by the heavy metals Cd and Cu. Sequence alignment also reveals a remarkably regular, conserved and hierarchical modular structure of all five subfamily 7a MTs, which include MTT3 and MTT5. The former has three modules, while the latter has only two. Induction levels of the three T. thermophila genes were determined using quantitative real time RT-PCR. Various stressors (including heavy metals) brought about dramatically different fold-inductions for each gene; MTT5 showed the highest fold-induction. Conserved DNA motifs with potential regulatory significance were identified, in an unbiased way, upstream of the start codons of subfamily 7a MTs. EST evidence for alternative splicing in the 3′ UTR of the MTT5 mRNA with potential regulatory activity is reported. CONCLUSION/SIGNIFICANCE: The small number and remarkably regular structure of Tetrahymena MTs, coupled with the experimental tractability of this model organism for studies of in vivo function, make it an attractive system for the experimental dissection of the roles, structure/function relationships, regulation of gene expression, and adaptive evolution of these proteins, as well as for the development of biotechnological applications for the environmental monitoring of toxic substances

Características clínico-epidemiológicas de pacientes hipertensos en un Consultorio Médico de Santa Clara

High blood pressure is a chronic non-transmittable disease, which is also a risk factor for the development of other clinical conditions. The incidence of arterial hypertension in the Cuban population is high.Aim: to characterize the evolution of arterial hypertension in a Family Doctor's Office.Methods: an observational, descriptive and cross-sectional study was carried out at the Family Doctor's Office 17-19 in the municipality of Santa Clara. The study covered the months of January to March 2020. Of the 256 hypertensive patients, a sample of 52 was selected by a simple random method.Results: Males predominated (53.84 %), together with the age group between 40 and 49 years (28.84 %). A total of 63.46 % of the patients were white-skinned. 51.61% presented risk factors. The risk factors with the highest incidence were smoking, followed by obesity and sedentary lifestyle.Conclusions: the most affected hypertensive patients are male. Most patients have a family history of high blood pressure. Smoking is a high incidence risk factor in the hypertensive population.Introducción: la hipertensión arterial es una enfermedad crónica no transmisible, que a la vez constituye un factor de riesgo para el desarrollo de otras enfermedades. La incidencia de la hipertensión arterial en la población de Cuba es alta.Objetivo: caracterizar el comportamiento de la hipertensión arterial en un Consultorio Médico de Familia.Métodos: se realizó un estudio observacional, descriptivo y transversal en el Consultorio Médico de Familia 17-19 del municipio Santa Clara. El período de estudio comprendió los meses de enero a marzo del 2020. La población fue de 256 hipertensos y se escogió una muestra de 52 hipertensos por muestreo aleatorio simple.Resultados: predominó el sexo masculino (53,84 %), y el grupo de edad entre 40 y 49 años (28,84 %). El 63,46 % de los pacientes fueron de color de la piel blanca. El 51,61 % presentaron factores de riesgo. Los factores de riesgo de mayor incidencia fueron el tabaquismo, seguido por la obesidad y el sedentarismo.Conclusiones: los pacientes hipertensos más afectados son los del sexo masculino. La mayor parte de los pacientes tienen antecedentes familiares de hipertensión arterial. El tabaquismo es un factor de riesgo de alta incidencia en la población hipertensa