RESUMO
Macronuclear chromosomes of ciliates, especially those of Spirotrichea, Armophorea and Phyllopharyngea, are extensively fragmented and their copy numbers vary significantly. A recent study suggested that parental RNA molecules regulate macronuclear copy number in offspring cells after conjugation. However, variations in patterns of macronuclear copy number during vegetative growth are not clear. Previous studies have reported macronuclear copy numbers of population averages, potentially masking individual variation. In the present investigation, we studied copy number variations among closely related species of Pseudokeronopsis and among individual cells during vegetative growth. We found that macronuclear copy numbers of Actin I, II in our Pseudokeronopsis populations are in the same range as in other spirotrichean species, but no close relationship is detected among morphologically related Pseudokeronopsis species. Copy numbers of three cells within each Pseudokeronopsis population range from 1.01 to 4.55 fold, suggesting that stochastic influences copy number during vegetative growth. Furthermore, the absence of a relationship between macronuclear copy numbers of Actin I and Actin II within Pseudokeronopsis is consistent with the fact that these genes are located on different gene-sized macronuclear chromosomes. Additionally, Actin II might have disappeared in P. carnea during evolution within the Actin gene family.
Assuntos
Actinas/metabolismo , Cilióforos/citologia , Cilióforos/fisiologia , Macronúcleo/químicaRESUMO
Only a limited number of studies exist on the life cycles of nonmodel ciliates such as Chilodonella uncinata (Cl: Phyllopharyngea). The handful of papers on this taxon indicate the presence of a heteromeric macronucleus, marked by separate DNA-rich and DNA-poor regions. Here, we study the life cycle of C. uncinata using confocal laser scanning microscopy with 4',6-diamidino-2-phenylindole staining, which allows us to differentiate nuclear dynamics of the micronucleus and the macronucleus during life-cycle stages. We photo-documented various stages and confirmed aspects of the development of the new macronucleus previously characterized by electron microscopy. We further reveal the heteromeric structure of the macronucleus with Z-stacks and three-dimensional (3D) reconstructions. We find no evidence for the presence of an endosome at the center of the macronucleus during vegetative growth. In addition to illustrating the life cycle of this ciliate, the approaches developed for this study will enable additional comparative analyses of nuclear dynamics using fluorescence microscopy.
Assuntos
Cilióforos/crescimento & desenvolvimento , Cilióforos/ultraestrutura , Estágios do Ciclo de Vida , Macronúcleo/química , Macronúcleo/ultraestrutura , Cilióforos/química , Imageamento Tridimensional , Microscopia ConfocalRESUMO
Tetrahymena thermophila is both a cell and an organism, which combines great intracellular complexity with a remarkable accessibility to investigation using many different approaches. In this review, we start with a description of the elaborate cortical organization of the Tetrahymena cell, and then proceed inward to consider the mitochondria and then the nuclei. For each of these cellular organelles and organelle-systems, first we familiarize the reader with its location in the cell and its structure and ultrastructure, and then we analyze the molecular mechanisms associated with organelle assembly, function, and subdivision. This analysis includes a molecular inventory of the organelle or organelle system, as well as a review of the consequences of modification, disruption or overexpression of important molecular components of each structure or system. Relevant comparisons to results obtained with other well-studied organisms, from Paramecium to Homo sapiens, are also included. Our goal is to provide investigators, in particular those who are new to this organism, both the background and the motivation to work with this model system and achieve further insight into its organization and dynamics.
Assuntos
Genes de Protozoários , Genoma Mitocondrial , Proteínas de Protozoários/química , Tetrahymena thermophila/ultraestrutura , Fator de Indução de Apoptose/química , Fator de Indução de Apoptose/genética , Membrana Celular/química , Forma do Núcleo Celular , Cílios/química , Fragmentação do DNA , Células Eucarióticas/química , Células Eucarióticas/ultraestrutura , Macronúcleo/química , Macronúcleo/genética , Micronúcleo Germinativo/química , Micronúcleo Germinativo/genética , Microscopia Eletrônica de Transmissão , Mitocôndrias/química , Mitocôndrias/genética , Transporte Proteico , Proteínas de Protozoários/genética , Tetrahymena thermophila/química , Tetrahymena thermophila/genéticaRESUMO
The ciliated protozoan Tetrahymena thermophila has been an important model system for biological research for many years. During that time, a variety of useful strains, including highly inbred stocks, a collection of diverse mutant strains, and wild cultivars from a variety of geographical locations have been identified. In addition, thanks to the efforts of many different laboratories, optimal conditions for growth, maintenance, and storage of Tetrahymena have been worked out. To facilitate the efficient use of Tetrahymena, especially by those new to the system, this chapter presents a brief description of many available Tetrahymena strains and lists possible resources for obtaining viable cultures of T. thermophila and other Tetrahymena species. Descriptions of commonly used media, methods for cell culture and maintenance, and protocols for short- and long-term storage are also presented.
Assuntos
Técnicas de Cultura de Células/métodos , Genoma de Protozoário , Laboratórios , Preservação Biológica/métodos , Tetrahymena/crescimento & desenvolvimento , Alelos , Bancos de Espécimes Biológicos , Sobrevivência Celular , Cromossomos/química , Cromossomos/genética , Conjugação Genética , Meios de Cultura/química , Macronúcleo/química , Macronúcleo/genética , Meiose , Micronúcleo Germinativo/química , Micronúcleo Germinativo/genética , Mutação , Especificidade da Espécie , Tetrahymena/química , Tetrahymena/genéticaRESUMO
The differentiation of germline and somatic genomes in Tetrahymena thermophila results in two independent systems of genetic transmission. One is the conserved, sexual Mendelian genetics system of the germline genome. The other is a random genetic assortment mechanism, which operates in the somatic genome during asexual propagation. This chapter describes both systems, their interplay, and how they are exploited to construct useful biological reagents and powerful tools, which can be used to answer a variety of experimental questions.
Assuntos
Cruzamentos Genéticos , DNA de Protozoário/genética , Genoma de Protozoário , Tetrahymena thermophila/genética , Técnicas de Cultura de Células , Divisão do Núcleo Celular , Mapeamento Cromossômico/métodos , Cromossomos/química , Cromossomos/genética , Conjugação Genética , Meios de Cultura/química , DNA de Protozoário/química , Heterozigoto , Homozigoto , Macronúcleo/química , Macronúcleo/genética , Micronúcleo Germinativo/química , Micronúcleo Germinativo/genética , Mutação , Fenótipo , Reprodução Assexuada , Tetrahymena thermophila/químicaRESUMO
Epitope tagging is a powerful approach used to enable investigations of a cellular component by elucidating its localization, interaction partners, and/or activity targets. Successful tag-based affinity purification yields a mixture of the molecule of interest, associated proteins and nucleic acids, and nonspecific background proteins and nucleic acids, many of which can depend on details of the protocol for enrichment. This chapter provides guidelines and considerations for designing an affinity purification experiment, beginning with construction of a strain expressing a tagged subunit. Common biochemical methods for detecting protein, RNA, and DNA in Tetrahymena thermophila are also discussed.
Assuntos
Marcadores de Afinidade/química , Mapeamento de Epitopos/métodos , Epitopos/química , Técnicas Imunológicas/métodos , Proteínas de Protozoários/isolamento & purificação , Tetrahymena thermophila/química , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Loci Gênicos , Macronúcleo/química , Macronúcleo/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Organismos Geneticamente Modificados/genética , Regiões Promotoras Genéticas , Proteínas de Protozoários/química , RNA de Protozoário/química , RNA de Protozoário/isolamento & purificação , Tetrahymena thermophila/genética , TransgenesRESUMO
Chilodonella uncinata, like all ciliates, contains two distinct nuclei in every cell: a germline micronucleus and a somatic macronucleus. During development of the macronucleus from a zygotic nucleus, the genome is processed in several ways, including elimination of internal sequences. In this study, we analyze micronuclear and macronuclear copies of beta-tubulin in C. uncinata and find at least four divergent paralogs of beta-tubulin in the macronucleus. We characterize the micronuclear version of one paralog and compare its internally eliminated sequences (IESs) with previously described IESs in this species. These comparisons reveal the presence of a conserved sequence motif within IESs. In addition, we compare the sequences of beta-tubulin from C. uncinata with other ciliates and to other alveolates in order to test the hypothesis that the mode of molecular evolution in ciliates obscures phylogenetic signal in protein-coding genes. We find that heterogeneous rates of substitution in beta-tubulin across ciliates result in unstable genealogies that are inconsistent with phylogenies based on small subunit rDNA genes and on ultrastructure. We discuss the implications of our findings for genome processing and protein evolution in ciliates.
Assuntos
Cilióforos/genética , Genoma de Protozoário , Macronúcleo/genética , Micronúcleo Germinativo/genética , Proteínas de Protozoários/genética , Tubulina (Proteína)/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cilióforos/classificação , Evolução Molecular , Macronúcleo/química , Micronúcleo Germinativo/química , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Especificidade da EspécieRESUMO
Rab proteins belong to the largest family of the Ras superfamily of small GTPase that play an important role in intracellular vesicular traffic. So far, almost 60 members of Rab family have been identified in mammalian cells. To further study the diversity and function of Rab protein in evolution, unicellular protozoa ciliates, Euplotes octocarinatus, were used in this study, Rab genes were screened by PCR method from macronuclear DNA of E. octocarinatus. Sixteen Rab genes were obtained. They share 87.6-99.5% identities. Highly conserved GTP-binding domains were found. There are some hot regions that diverse sharply in these genes as well.