RESUMEN
The contractile vacuole complex (CVC) is a dynamic and morphologically complex membrane organelle, comprising a large vesicle (bladder) linked with a tubular reticulum (spongiome). CVCs provide key osmoregulatory roles across diverse eukaryotic lineages, but probing the mechanisms underlying their structure and function is hampered by the limited tools available for in vivo analysis. In the experimentally tractable ciliate Tetrahymena thermophila, we describe four proteins that, as endogenously tagged constructs, localize specifically to distinct CVC zones. The DOPEY homolog Dop1p and the CORVET subunit Vps8Dp localize both to the bladder and spongiome but with different local distributions that are sensitive to osmotic perturbation, whereas the lipid scramblase Scr7p colocalizes with Vps8Dp. The H+-ATPase subunit Vma4 is spongiome specific. The live imaging permitted by these probes revealed dynamics at multiple scales including rapid exchange of CVC-localized and soluble protein pools versus lateral diffusion in the spongiome, spongiome extension and branching, and CVC formation during mitosis. Although the association with DOP1 and VPS8D implicate the CVC in endosomal trafficking, both the bladder and spongiome might be isolated from bulk endocytic input.
Asunto(s)
Tetrahymena thermophila , Vacuolas , Vacuolas/metabolismo , Endosomas , Proteínas/metabolismo , MitosisRESUMEN
Ciliated protozoans perform extreme forms of programmed somatic DNA rearrangement during development. The model ciliate Tetrahymena thermophila removes 34% of its germline micronuclear genome from somatic macronuclei by excising thousands of internal eliminated sequences (IESs), a process that shares features with transposon excision. Indeed, piggyBac transposon-derived genes are necessary for genome-wide IES excision in both Tetrahymena (TPB2 [Tetrahymena piggyBac-like 2] and LIA5) and Paramecium tetraurelia (PiggyMac). T. thermophila has at least three other piggyBac-derived genes: TPB1, TPB6, and TPB7 Here, we show that TPB1 and TPB6 excise a small, distinct set of 12 unusual IESs that disrupt exons. TPB1-deficient cells complete mating, but their progeny exhibit slow growth, giant vacuoles, and osmotic shock sensitivity due to retention of an IES in the vacuolar gene DOP1 (Dopey domain-containing protein). Unlike most IESs, TPB1-dependent IESs have piggyBac-like terminal inverted motifs that are necessary for excision. Transposon-like excision mediated by TPB1 and TPB6 provides direct evidence for a transposon origin of not only IES excision machinery but also IESs themselves. Our study highlights a division of labor among ciliate piggyBac-derived genes, which carry out mutually exclusive categories of excision events mediated by either transposon-like features or RNA-directed heterochromatin.
Asunto(s)
Elementos Transponibles de ADN/genética , Reordenamiento Génico/genética , Genes Protozoarios/genética , Genoma de Protozoos/genética , Proteínas Protozoarias/metabolismo , Tetrahymena thermophila/genética , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Estadios del Ciclo de Vida , Proteínas Protozoarias/genética , Tetrahymena thermophila/crecimiento & desarrollo , Vacuolas/genéticaRESUMEN
Recognition of self and nonself is important for outcrossing organisms, and different mating types establish the barrier against self-mating. In the unicellular ciliate T. thermophila, mating type determination requires complex DNA rearrangements at a single mat locus during conjugation to produce a type-specific gene pair (MTA and MTB) for 1 of 7 possible mating types. Surprisingly, we found that decreased expression of the DNA breakage-repair protein Ku80 at late stages of conjugation generated persistent selfing phenotype in the progeny. DNA analysis revealed multiple mating-type gene pairs as well as a variety of mis-paired, unusually arranged mating-type genes in these selfers that resemble some proposed rearrangement intermediates. They are found also in normal cells during conjugation and are lost after 10 fissions but are retained in Ku mutants. Silencing of TKU80 or TKU70-2 immediately after conjugation also generated selfing phenotype, revealing a hidden DNA rearrangement process beyond conjugation. Mating reactions between the mutant and normal cells suggest a 2-component system for self-nonself-recognition through MTA and MTB genes.
Asunto(s)
ADN Protozoario/genética , Reordenamiento Génico , Autoantígeno Ku/genética , Proteínas Protozoarias/genética , Tetrahymena thermophila/genética , Conjugación Genética , Cruzamientos Genéticos , ADN Protozoario/metabolismo , Expresión Génica , Silenciador del Gen , Autoantígeno Ku/metabolismo , Fenotipo , Proteínas Protozoarias/metabolismo , Reproducción , Tetrahymena thermophila/metabolismoRESUMEN
Eukaryotic cells pack their genomic DNA into euchromatin and heterochromatin. Boundaries between these domains have been shown to be set by boundary elements. In Tetrahymena, heterochromatin domains are targeted for deletion from the somatic nuclei through a sophisticated programmed DNA rearrangement mechanism, resulting in the elimination of 34% of the germline genome in â¼10,000 dispersed segments. Here we showed that most of these deletions occur consistently with very limited variations in their boundaries among inbred lines. We identified several potential flanking regulatory sequences, each associated with a subset of deletions, using a genome-wide motif finding approach. These flanking sequences are inverted repeats with the copies located at nearly identical distances from the opposite ends of the deleted regions, suggesting potential roles in boundary determination. By removing and testing two such inverted repeats in vivo, we found that the ability for boundary maintenance of the associated deletion were lost. Furthermore, we analyzed the deletion boundaries in mutants of a known boundary-determining protein, Lia3p and found that the subset of deletions that are affected by LIA3 knockout contained common features of flanking regulatory sequences. This study suggests a common mechanism for setting deletion boundaries by flanking inverted repeats in Tetrahymena thermophila.
Asunto(s)
ADN Protozoario/genética , Eliminación de Gen , Heterocromatina/química , Proteínas Protozoarias/genética , Tetrahymena thermophila/genética , Secuencias de Aminoácidos , Núcleo Celular/metabolismo , ADN Protozoario/metabolismo , Eucromatina/química , Regulación de la Expresión Génica , Reordenamiento Génico , Genoma de Protozoos , Dominios ProteicosRESUMEN
Ciliated protozoa extensively remodel their somatic genomes during nuclear development, fragmenting their chromosomes and removing large numbers of internal eliminated sequences (IESs). The sequences eliminated are unique and repetitive DNAs, including transposons. Recent studies have identified transposase proteins that appear to have been domesticated and are used by these cells to eliminate DNA not wanted in the somatic macronucleus. This DNA elimination process is guided by meiotically produced small RNAs, generated in the germline nucleus, that recognize homologous sequences leading to their removal. These scan RNAs are found in complexes with PIWI proteins. Before they search the developing genome for IESs to eliminate, they scan the parental somatic nucleus and are removed from the pool if they match homologous sequences in that previously reorganized genome. In Tetrahymena, the scan RNAs target heterochromatin modifications to mark IESs for elimination. This DNA elimination pathway in ciliates shares extensive similarity with piRNA-mediated silencing of metazoans and highlights the remarkable ability of homologous RNAs to shape developing genomes.
Asunto(s)
Cilióforos/genética , ADN Protozoario/genética , Genoma de Protozoos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromosomas/genética , Cromosomas/metabolismo , Cilióforos/metabolismo , Elementos Transponibles de ADN , ADN Protozoario/metabolismo , Epigénesis Genética , Regulación de la Expresión Génica , Reordenamiento Génico , Heterocromatina/genética , Heterocromatina/metabolismo , ARN Protozoario/genética , ARN Protozoario/metabolismo , Transposasas/metabolismoRESUMEN
The maintenance of chromosome integrity is crucial for genetic stability. However, programmed chromosome fragmentations are known to occur in many organisms, and in the ciliate Tetrahymena the five germline chromosomes are fragmented into hundreds of minichromosomes during somatic nuclear differentiation. Here, we showed that there are different fates of these minichromosomes after chromosome breakage. Among the 326 somatic minichromosomes identified using genomic data, 50 are selectively eliminated from the mature somatic genome. Interestingly, many and probably most of these minichromosomes are eliminated during the growth period between 6 and 20 doublings right after conjugation. Genes with potential conjugation-specific functions are found in these minichromosomes. This study revealed a new mode of programmed DNA elimination in ciliates similar to those observed in parasitic nematodes, which could play a role in developmental gene regulation.
Asunto(s)
Rotura Cromosómica , Cromosomas/genética , Telómero/genética , Tetrahymena thermophila/genética , Animales , Núcleo Celular/genética , Inestabilidad Cromosómica/genética , Bases de Datos Genéticas , Expresión Génica/genética , Biblioteca Genómica , Células Germinativas/crecimiento & desarrollo , Tetrahymena thermophila/crecimiento & desarrolloRESUMEN
Bi-directional non-coding transcripts and their â¼29-nt small RNA products are known to guide DNA deletion in Tetrahymena, leading to the removal of one-third of the genome from developing somatic nuclei. Using an antibody specific for long double-stranded RNAs (dsRNAs), we determined the dynamic subcellular distributions of these RNAs. Conjugation-specific dsRNAs were found and show sequential appearances in parental germline, parental somatic nuclei and finally in new somatic nuclei of progeny. The dsRNAs in germline nuclei and new somatic nuclei are likely transcribed from the sequences destined for deletion; however, the dsRNAs in parental somatic nuclei are unexpected, and PCR analyses suggested that they were transcribed in this nucleus. Deficiency in the RNA interference (RNAi) pathway led to abnormal aggregations of dsRNA in both the parental and new somatic nuclei, whereas accumulation of dsRNAs in the germline nuclei was only seen in the Dicer-like gene mutant. In addition, RNAi mutants displayed an early loss of dsRNAs from developing somatic nuclei. Thus, long dsRNAs are made in multiple nuclear compartments and some are linked to small RNA production whereas others might participate in their regulations.
Asunto(s)
Núcleo Celular/fisiología , ARN Bicatenario/metabolismo , ARN Protozoario/metabolismo , Reordenamiento Génico , Genoma de Protozoos , Heterocromatina/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Transporte de ARN , ARN Bicatenario/genética , ARN Protozoario/genética , TetrahymenaRESUMEN
Breakage-fusion-bridge cycles contribute to chromosome instability and generate large DNA palindromes that facilitate gene amplification in human cancers. The prevalence of large DNA palindromes in cancer is not known. Here, by using a new microarray-based approach called genome-wide analysis of palindrome formation, we show that palindromes occur frequently and are widespread in human cancers. Individual tumors seem to have a nonrandom distribution of palindromes in their genomes, and a subset of palindromic loci is associated with gene amplification. This indicates that the location of palindromes in the cancer genome can serve as a structural platform that supports subsequent gene amplification. Genome-wide analysis of palindrome formation is a new approach to identify structural chromosome aberrations associated with cancer.
Asunto(s)
ADN de Neoplasias/genética , Amplificación de Genes , Neoplasias/genética , Línea Celular Tumoral , Humanos , Neoplasias/patología , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Autophagy is an evolutionarily conserved mechanism for the degradation of cellular components, but its role in enucleation during differentiation has not been established. Tetrahymena thermophila is a unicellular eukaryote with two functionally distinct nuclei, the somatic (macro-) and the germ line (micro-) nuclei. These nuclei are produced during sexual reproduction (conjugation), which involves differentiation and selective degradation of several specific nuclei. To examine the role of autophagy in nuclear degradation, we studied the function of two ATG8 genes in Tetrahymena. Through fluorescent protein tagging, we found that both proteins are targeted to degrading nuclei at specific stages, with some enrichment on the nuclear periphery, suggesting the formation of autophagosomes surrounding these nuclei. In addition, ATG8 knockout mutant cells showed a pronounced delay in nuclear degradation without apparently preventing the completion of other developmental events. This evidence provided direct support for a critical role for autophagy in programmed nuclear degradation. The results also showed differential roles for two ATG8 genes, with ATG8-65 playing a more significant role in starvation than ATG8-2, although both are important in nuclear degradation.
Asunto(s)
Autofagia/genética , Macronúcleo/metabolismo , Micronúcleo Germinal/metabolismo , Proteínas Protozoarias/fisiología , Tetrahymena thermophila/fisiología , Secuencia de Aminoácidos , Secuencia Conservada , ADN Protozoario/metabolismo , Viabilidad Microbiana , Datos de Secuencia Molecular , Transporte de Proteínas , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Reproducción , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismoRESUMEN
Histone H3K27me3 modification is an important regulator for development and gene expression. In Tetrahymena thermophila, the complex chromatin dynamics of H3K27me3 marks during nuclear development suggested that an H3K27me3 demethylase might exist. Here, we report an H3K27me3 demethylase homolog, JMJ1, in Tetrahymena. During conjugation, JMJ1 expression is upregulated and the protein is localized first in the parental macronucleus and then in the new macronucleus. In conjugating cells, knockdown of JMJ1 expression resulted in a severe reduction in the production of progeny, suggesting that JMJ1 is essential for Tetrahymena conjugation. Furthermore, knockdown of JMJ1 resulted in increased H3K27 trimethylation in the new macronucleus and reduced transcription of genes related to DNA elimination, while the DNA elimination process was also partially blocked. Knockdown of the H3K27 methyltransferase EZL2 but not that of EZL1 partially restored progeny production in JMJ1-knockdown cells and reduced abnormal H3K27me3 accumulation in the new macronucleus. Taken together, these results demonstrate a critical role for JMJ1 in regulating H3K27me3 during conjugation and the importance of JMJ1 in regulating gene expression in the new macronucleus but not in regulating the formation of heterochromatin associated with programmed DNA deletion.
Asunto(s)
Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proteínas Protozoarias/metabolismo , Tetrahymena thermophila/metabolismo , Secuencia de Aminoácidos , Western Blotting , Cloruro de Cadmio/farmacología , Inmunoprecipitación de Cromatina , Rotura Cromosómica , Biología Computacional , Conjugación Genética , ADN Protozoario/genética , ADN Protozoario/metabolismo , Técnicas de Silenciamiento del Gen , Heterocromatina/genética , Heterocromatina/metabolismo , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Macronúcleo/enzimología , Macronúcleo/genética , Macronúcleo/metabolismo , Metilación , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Filogenia , Proteínas Protozoarias/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Tetrahymena thermophila/enzimología , Tetrahymena thermophila/genética , Transcripción Genética , Activación TranscripcionalRESUMEN
Centromere-drive is a process where centromeres compete for transmission through asymmetric "female" meiosis for inclusion into the oocyte. In symmetric "male" meiosis, all meiotic products form viable germ cells. Therefore, the primary incentive for centromere-drive, a potential transmission bias, is believed to be missing from male meiosis. In this article, we consider whether male meiosis also bears the primary cost of centromere-drive. Because different taxa carry out different combinations of meiotic programs (symmetric + asymmetric, symmetric only, asymmetric only), it is possible to consider the evolutionary consequences of centromere-drive in the context of these differing systems. Groups with both types of meiosis have large, rapidly evolving centromeric regions, and their centromeric histones (CenH3s) have been shown to evolve under positive selection, suggesting roles as suppressors of centromere-drive. In contrast, taxa with only symmetric male meiosis have shown no evidence of positive selection in their centromeric histones. In this article, we present the first evolutionary analysis of centromeric histones in ciliated protozoans, a group that only undergoes asymmetric "female" meiosis. We find no evidence of positive selection acting on CNA1, the CenH3 of Tetrahymena species. Cytological observations of a panel of Tetrahymena species are consistent with dynamic karyotype evolution in this lineage. Our findings suggest that defects in male meiosis, and not mitosis or female meiosis, are the primary selective force behind centromere-drive suppression. Our study raises the possibility that taxa like ciliates, with only female meiosis, may therefore undergo unsuppressed centromere drive.
Asunto(s)
Centrómero/genética , Histonas/metabolismo , Meiosis/genética , Tetrahymena/genética , Tetrahymena/metabolismo , Secuencia de Aminoácidos , Animales , Evolución Molecular , Femenino , Regulación de la Expresión Génica , Genes Protozoarios , Masculino , Datos de Secuencia Molecular , Filogenia , Transporte de Proteínas , Alineación de Secuencia , Tetrahymena/clasificaciónRESUMEN
Amplification of large chromosomal regions (gene amplification) is a common somatic alteration in human cancer cells and often is associated with advanced disease. A critical event initiating gene amplification is a DNA double-strand break (DSB), which is immediately followed by the formation of a large DNA palindrome. Large DNA palindromes are frequent and nonrandomly distributed in the genomes of cancer cells and facilitate a further increase in copy number. Although the importance of the formation of large DNA palindromes as a very early event in gene amplification is widely recognized, it is not known how a DSB is resolved to form a large DNA palindrome and whether any local DNA structure determines the location of large DNA palindromes. We show here that intrastrand annealing following a DNA double-strand break leads to the formation of large DNA palindromes and that DNA inverted repeats in the genome determine the efficiency of this event. Furthermore, in human Colo320DM cancer cells, a DNA inverted repeat in the genome marks the border between amplified and nonamplified DNA. Therefore, an early step of gene amplification is a regulated process that is facilitated by DNA inverted repeats in the genome.
Asunto(s)
ADN/genética , ADN/metabolismo , Amplificación de Genes/genética , Genoma Humano/genética , Neoplasias/genética , Animales , Secuencia de Bases , Células CHO , Cromosomas Humanos Par 1/genética , Cricetinae , Cricetulus , ADN/química , Daño del ADN/genética , Humanos , Metotrexato/farmacología , Datos de Secuencia MolecularRESUMEN
Unlike in other eukaryotes, in which it causes gene silencing, RNA interference (RNAi) has been linked to programmed DNA deletion in the ciliate Tetrahymena thermophila. Here we have developed an efficient method to inducibly express double-stranded RNA hairpins and demonstrated that they cause gene silencing through targeted mRNA degradation in all phases of the life cycle, including growth, starvation, and mating. This technique offers a new tool for gene silencing in this model organism. Induction of RNA hairpins causes dramatic upregulation of Dicer and Argonaute family genes, revealing a system capable of rapidly responding to double-stranded RNA. These hairpins are processed into 23- to 24-nucleotide (nt) small RNAs, which are distinctly different from the 28- to 30-nt small RNAs known to be associated with DNA deletion. Thus, two different small RNA pathways appear to be responsible for gene silencing and DNA deletion. Surprisingly, expression of the RNA hairpin also causes targeted DNA deletion during conjugation, although at low efficiencies, which suggests a possible crossover of these two molecular paths.
Asunto(s)
Silenciador del Gen , MicroARNs/metabolismo , ARN/metabolismo , Tetrahymena thermophila/genética , Animales , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/fisiologíaRESUMEN
The macronucleus of the binucleate ciliate Tetrahymena thermophila contains fragmented and amplified chromosomes that do not have centromeres, eliminating the possibility of mitotic nuclear division. Instead, the macronucleus divides by amitosis with random segregation of these chromosomes without detectable chromatin condensation. This amitotic division provides a special opportunity for studying the roles of mitotic proteins in segregating acentric chromatin. The Smc4 protein is a core component of the condensin complex that plays a role in chromatin condensation and has also been associated with nucleolar segregation, DNA repair, and maintenance of the chromatin scaffold. Mutants of Tetrahymena SMC4 have remarkable characteristics during amitosis. They do not form microtubules inside the macronucleus as normal cells do, and there is little or no bulk DNA segregation during cell division. Nevertheless, segregation of nucleoli to daughter cells still occurs, indicating the independence of this process and bulk DNA segregation in ciliate amitosis.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Segregación Cromosómica , Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Protozoarias/metabolismo , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo , Adenosina Trifosfatasas/genética , Animales , Secuencia de Bases , Nucléolo Celular/metabolismo , Nucléolo Celular/ultraestructura , ADN Protozoario/genética , Proteínas de Unión al ADN/genética , Genes Protozoarios , Hibridación Fluorescente in Situ , Microscopía Electrónica , Microscopía Fluorescente , Microtúbulos/metabolismo , Complejos Multiproteicos/genética , Mutación , Filogenia , Proteínas Protozoarias/genética , Tetrahymena thermophila/crecimiento & desarrollo , Tetrahymena thermophila/ultraestructuraAsunto(s)
Epistasis Genética , Reordenamiento Génico/genética , Genoma de Protozoos/genética , Macronúcleo/genética , Oxytricha/genética , ARN Protozoario/genética , Animales , ADN Protozoario/genética , ADN Protozoario/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Oxytricha/citología , Oxytricha/crecimiento & desarrollo , ARN Protozoario/metabolismo , Moldes GenéticosRESUMEN
Ciliated protozoans present several features of chromosome segregation that are unique among eukaryotes, including their maintenance of two nuclei: a germline micronucleus, which undergoes conventional mitosis and meiosis, and a somatic macronucleus that divides by an amitotic process. To study ciliate chromosome segregation, we have identified the centromeric histone gene in the Tetrahymena thermophila genome (CNA1). CNA1p specifically localizes to peripheral centromeres in the micronucleus but is absent in the macronucleus during vegetative growth. During meiotic prophase of the micronucleus, when chromosomes are stretched to twice the length of the cell, CNA1p is found localized in punctate spots throughout the length of the chromosomes. As conjugation proceeds, CNA1p appears initially diffuse, but quickly reverts to discrete dots in those nuclei destined to become micronuclei, whereas it remains diffuse and is gradually lost in developing macronuclei. In progeny of germline CNA1 knockouts, we see no defects in macronuclear division or viability of the progeny cells immediately following the knockout. However, within a few divisions, progeny show abnormal mitotic segregation of their micronucleus, with most cells eventually losing their micronucleus entirely. This study reveals a strong dependence of the germline micronucleus on centromeric histones for proper chromosome segregation.
Asunto(s)
Segregación Cromosómica , Histonas/metabolismo , Micronúcleo Germinal/genética , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo , Animales , Eliminación de Gen , Expresión Génica , Histonas/deficiencia , Histonas/genética , Micronúcleo Germinal/metabolismo , Mitosis , Filogenia , Tetrahymena thermophila/citología , Factores de TiempoRESUMEN
Ciliates are unicellular eukaryotes known for their cellular complexity and wide range of natural habitats. How they adapt to their niches and what roles they play in ecology remain largely unknown. The genus Tetrahymena is among the best-studied groups of ciliates and one particular species, Tetrahymena thermophila, is a well-known laboratory model organism in cell and molecular biology, making it an excellent candidate for study in protist ecology. Here, based on cytochrome c oxidase subunit I (COX1) gene barcoding, we identify a total of 19 different putative Tetrahymena species and two closely related Glaucoma lineages isolated from distinct natural habitats, of which 13 are new species. These latter include 11 Tetrahymena species found in the bladder traps of Utricularia plants, the most species-rich and widely distributed aquatic carnivorous plant, thus revealing a previously unknown but significant symbiosis of Tetrahymena species living among the microbial community of Utricularia bladder traps. Additional species were collected using an artificial trap method we have developed. We show that diverse Tetrahymena species may live even within the same habitat and that their populations are highly dynamic, suggesting that the diversity and biomass of species worldwide is far greater than currently appreciated.
Asunto(s)
Ciclooxigenasa 1/genética , Lamiales/parasitología , Análisis de Secuencia de ADN/métodos , Tetrahymena/clasificación , Organismos Acuáticos/parasitología , Carnivoría , Código de Barras del ADN Taxonómico , Filogenia , Filogeografía , Proteínas Protozoarias/genética , Simbiosis , Taiwán , Tetrahymena/genética , Tetrahymena/aislamiento & purificaciónAsunto(s)
Cromosomas/metabolismo , Cilióforos/genética , ADN Protozoario/metabolismo , Amplificación de Genes/fisiología , Dosificación de Gen/fisiología , Genes Protozoarios/fisiología , Macronúcleo/metabolismo , Micronúcleo Germinal/metabolismo , ARN Protozoario/metabolismo , ARN Nuclear Pequeño/metabolismo , Animales , Cromosomas/genética , Cilióforos/metabolismo , Variaciones en el Número de Copia de ADN/fisiología , ADN Protozoario/genética , Epigénesis Genética/fisiología , Genoma de Protozoos/fisiología , Macronúcleo/genética , Micronúcleo Germinal/genética , Modelos Genéticos , ARN Protozoario/genética , ARN Nuclear Pequeño/genéticaRESUMEN
Although the presence of endosymbiotic rickettsial bacteria, specifically Candidatus Megaira, has been reported in diverse habitats and a wide range of eukaryotic hosts, it remains unclear how broadly Ca. Megaira are distributed in a single host species. In this study we seek to address whether Ca. Megaira are present in most, if not all isolates, of the parasitic ciliate Ichthyophthirius multifiliis. Conserved regions of bacterial 16S rRNA genes were either PCR amplified, or assembled from deep sequencing data, from 18 isolates/populations of I. multifiliis sampled worldwide (Brazil, Taiwan, and USA). We found that rickettsial rRNA sequences belonging to three out of four Ca. Megaira subclades could be consistently detected in all I. multifiliis samples. I. multifiliis collected from local fish farms tend to be inhabited by the same subclade of Ca. Megaira, whereas those derived from pet fish are often inhabited by more than one subclade of Ca. Megaira. Distributions of Ca. Megaira in I. multifiliis thus better reflect the travel history, but not the phylogeny, of I. multifiliis. In summary, our results suggest that I. multifiliis may be dependent on this endosymbiotic relationship, and the association between Ca. Megaira and I. multifiliis is more diverse than previously thought.