5,107 research outputs found

    The bromodomain-containing protein Ibd1 links multiple chromatin related protein complexes to highly expressed genes in Tetrahymena thermophila

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    Background: The chromatin remodelers of the SWI/SNF family are critical transcriptional regulators. Recognition of lysine acetylation through a bromodomain (BRD) component is key to SWI/SNF function; in most eukaryotes, this function is attributed to SNF2/Brg1. Results: Using affinity purification coupled to mass spectrometry (AP-MS) we identified members of a SWI/SNF complex (SWI/SNFTt) in Tetrahymena thermophila. SWI/SNFTt is composed of 11 proteins, Snf5Tt, Swi1Tt, Swi3Tt, Snf12Tt, Brg1Tt, two proteins with potential chromatin interacting domains and four proteins without orthologs to SWI/SNF proteins in yeast or mammals. SWI/SNFTt subunits localize exclusively to the transcriptionally active macronucleus (MAC) during growth and development, consistent with a role in transcription. While Tetrahymena Brg1 does not contain a BRD, our AP-MS results identified a BRD-containing SWI/SNFTt component, Ibd1 that associates with SWI/SNFTt during growth but not development. AP-MS analysis of epitope-tagged Ibd1 revealed it to be a subunit of several additional protein complexes, including putative SWRTt, and SAGATt complexes as well as a putative H3K4-specific histone methyl transferase complex. Recombinant Ibd1 recognizes acetyl-lysine marks on histones correlated with active transcription. Consistent with our AP-MS and histone array data suggesting a role in regulation of gene expression, ChIP-Seq analysis of Ibd1 indicated that it primarily binds near promoters and within gene bodies of highly expressed genes during growth. Conclusions: Our results suggest that through recognizing specific histones marks, Ibd1 targets active chromatin regions of highly expressed genes in Tetrahymena where it subsequently might coordinate the recruitment of several chromatin remodeling complexes to regulate the transcriptional landscape of vegetatively growing Tetrahymena cells.Comment: Published on BMC Epigenetics & Chromati

    Progeny of Germ Line Knockouts of \u3cem\u3eASI2\u3c/em\u3e, a Gene Encoding a Putative Signal Transduction Receptor in \u3cem\u3eTetrahymena Thermophila\u3c/em\u3e, Fail to Make the Transition from Sexual Reproduction to Vegetative Growth

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    The ciliated protozoan Tetrahymena has two nuclei: a germ line micronucleus and a somatic macronucleus. The transcriptionally active macronucleus has about 50 copies of each chromosome. At sexual reproduction (conjugation), the parental macronucleus is degraded and new macronucleus develops from a mitotic product of the zygotic micronucleus. Development of the macronucleus involves massive genome remodeling, including deletion of about 6000 specific internal eliminated sequences (IES) and multiple rounds of DNA replication. A gene encoding a putative signal transduction receptor, ASI2, (anlagen stage induced 2) is up-regulated during development of the new macronuclei (anlagen). Macronuclear ASI2 is nonessential for vegetative growth. Homozygous ASI2 germ line knockout cells with wild type parental macronuclei proceed through mating but arrest at late macronuclear anlagen development and die before the first post-conjugation fission. IES elimination occurs in these cells. Two rounds of postzygotic DNA replication occur normally in progeny of ASI2 germ line knockouts, but endoreduplication of the macronuclear genome is arrested. The germ line ASI2 null phenotype is rescued in a mating of a knockout strain with wild type cells

    Diverse Sequences within Tlr Elements Target Programmed DNA Elimination in \u3cem\u3eTetrahymena Thermophila\u3c/em\u3e

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    Tlr elements are a novel family of ~30 putative mobile genetic elements that are conïŹned to the germ line micronuclear genome in Tetrahymena thermophila. Thousands of diverse germ line-limited sequences, including the Tlr elements, are speciïŹcally eliminated from the differentiating somatic macronucleus. Macronucleusretained sequences ïŹ‚anking deleted regions are known to contain cis-acting signals that delineate elimination boundaries. It is unclear whether sequences within deleted DNA also play a regulatory role in the elimination process. In the current study, an in vivo DNA rearrangement assay was used to identify internal sequences required in cis for the elimination of Tlr elements. Multiple, nonoverlapping regions from the ~23-kb Tlr elements were independently sufïŹcient to stimulate developmentally regulated DNA elimination when placed within the context of ïŹ‚anking sequences from the most thoroughly characterized family member, Tlr1. Replacement of element DNA with macronuclear or foreign DNA abolished elimination activity. Thus, diverse sequences dispersed throughout Tlr DNA contain cis-acting signals that target these elements for programmed elimination. Surprisingly, Tlr DNA was also efïŹciently deleted when Tlr1 ïŹ‚anking sequences were replaced with DNA from a region of the genome that is not normally associated with rearrangement, suggesting that speciïŹc ïŹ‚anking sequences are not required for the elimination of Tlr element DNA

    Introducing the concept of biocatalysis in the classroom: The conversion of cholesterol to provitamin D 3

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    Biocatalysis is a fundamental concept in biotechnology. The topic integrates knowledge of several disciplines; therefore, it was included in the course “design and optimization of biological systems” which is offered in the biochemistry curricula. We selected the ciliate tetrahymena as an example of a eukaryotic system with potential for the biotransformation of sterol metabolites of industrial interest; in particular, we focused on the conversion of cholesterol to provitamin D3. The students work with wild type and recombinant strains and learn how sterol pathways could be modified to obtain diverse sterol moieties. During the course the students identify and measure the concentration of sterols. They also search for related genes by bioinformatic analysis. Additionally, the students compare biotransformation rates, growing the ciliate in plate and in a bioreactor. Finally, they use fluorescence microscopy to localize an enzyme involved in biotransformation. The last day each team makes an oral presentation, explaining the results obtained and responds to a series of key questions posed by the teachers, which determine the final mark. In our experience, this course enables undergraduate students to become acquainted with the principles of biocatalysis as well as with standard and modern techniques, through a simple and robust laboratory exercise, using a biological system for the conversion of valuable pharmaceutical moieties.Fil: De Luca, BelĂ©n M.. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂ­mica; ArgentinaFil: Nudel, Berta Clara. Consejo Nacional de Investigaciones CientĂ­ficas y TĂ©cnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂ­mica; ArgentinaFil: Gonzalez, Rodrigo Horacio. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂ­mica; ArgentinaFil: Nusblat, Alejandro David. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂ­mica; Argentin

    Nucleus-specific linker histones Hho1 and Mlh1 form distinct protein interactions during growth, starvation and development in Tetrahymena thermophila

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    Chromatin organization influences most aspects of gene expression regulation. The linker histone H1, along with the core histones, is a key component of eukaryotic chromatin. Despite its critical roles in chromatin structure and function and gene regulation, studies regarding the H1 protein-protein interaction networks, particularly outside of Opisthokonts, are limited. The nuclear dimorphic ciliate protozoan Tetrahymena thermophila encodes two distinct nucleus-specific linker histones, macronuclear Hho1 and micronuclear Mlh1. We used a comparative proteomics approach to identify the Hho1 and Mlh1 protein-protein interaction networks in Tetrahymena during growth, starvation, and sexual development. Affinity purification followed by mass spectrometry analysis of the Hho1 and Mlh1 proteins revealed a non-overlapping set of co-purifying proteins suggesting that Tetrahymena nucleus-specific linker histones are subject to distinct regulatory pathways. Furthermore, we found that linker histones interact with distinct proteins under the different stages of the Tetrahymena life cycle. Hho1 and Mlh1 co-purified with several Tetrahymena-specific as well as conserved interacting partners involved in chromatin structure and function and other important cellular pathways. Our results suggest that nucleus-specific linker histones might be subject to nucleus-specific regulatory pathways and are dynamically regulated under different stages of the Tetrahymena life cycle.York University Librarie

    RACS: Rapid Analysis of ChIP-Seq data for contig based genomes

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    Background: Chromatin immunoprecipitation coupled to next generation sequencing (ChIP-Seq) is a widely used technique to investigate the function of chromatin-related proteins in a genome-wide manner. ChIP-Seq generates large quantities of data which can be difficult to process and analyse, particularly for organisms with contig based genomes. Contig-based genomes often have poor annotations for cis-elements, for example enhancers, that are important for gene expression. Poorly annotated genomes make a comprehensive analysis of ChIP-Seq data difficult and as such standardized analysis pipelines are lacking. Methods: We report a computational pipeline that utilizes traditional High-Performance Computing techniques and open source tools for processing and analysing data obtained from ChIP-Seq. We applied our computational pipeline "Rapid Analysis of ChIP-Seq data" (RACS) to ChIP-Seq data that was generated in the model organism Tetrahymena thermophila, an example of an organism with a genome that is available in contigs. Results: To test the performance and efficiency of RACs, we performed control ChIP-Seq experiments allowing us to rapidly eliminate false positives when analyzing our previously published data set. Our pipeline segregates the found read accumulations between genic and intergenic regions and is highly efficient for rapid downstream analyses. Conclusions: Altogether, the computational pipeline presented in this report is an efficient and highly reliable tool to analyze genome-wide ChIP-Seq data generated in model organisms with contig-based genomes. RACS is an open source computational pipeline available to download from: https://bitbucket.org/mjponce/racs --or-- https://gitrepos.scinet.utoronto.ca/public/?a=summary&p=RACSComment: Submitted to BMC Bioinformatics. Computational pipeline available at https://bitbucket.org/mjponce/rac

    Transformation of \u3cem\u3eTetrahymena thermophila\u3c/em\u3e with Hypermethylated rRNA Genes

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    The extrachromosomal rRNA genes (rDNA) of Tetrahymena thermophila contain 0.4% N6-methyladenine. C3 strain rDNA was isolated, hypermethylated in vitro, and microinjected into B strain host cells. Clonal cell lines were established, and transformants were selected on the basis of resistance to paromomycin, conferred by the injected rDNA. The effects of methylation by three enzymes which methylate the sequence 5\u27-NAT-3\u27, the dam, EcoRI, and ClaI methylases, were tested. Hypermethylation of the injected rDNA had no effect on transformation efficiency relative to mock-methylated controls. The injected C3 strain rDNA efficiently replaced host rDNA as the major constituent of the population of rDNA molecules. Hypermethylation of the injected DNA was not maintained through 20 to 25 cell generations

    A novel malaria vaccine candidate antigen expressed in Tetrahymena thermophila

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    Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens

    Germ Line-Specific DNA Sequences are Present on All Five Micronuclear Chromosomes in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

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    The development of the macronucleus from the zygotic micronucleus in the ciliated protozoan Tetrahymena spp. involves the elimination of specific DNA sequences (M. C. Yao and M. Gorovsky, Chromosoma 48:1-18 1974). The present study demonstrates that micronucleus-specific DNA is present on all five of the micronuclear chromosomes. Fragments of micronuclear DNA from Tetrahymena thermophila were cloned in the plasmid vector pBR322. A procedure was developed to examine the organization of the cloned sequences in micro- and macronuclear DNA without nick translating each individual probe. Twenty-three percent of randomly selected DNA sequences examined by this method were micronucleus (germ line) specific. They were all members of families of repeated sequences. Hybridization of six micronucleus-specific DNA sequences to micronuclear DNA from nullisomic strains of T. thermophila, which are lacking one or more pairs of chromosomes in the micronucleus, suggested that these sequences are present on several chromosomes. One micronucleus-specific sequence was shown by in situ hybridization to be present on all five of the micronuclear chromosomes

    La captaciĂłn de colesterol por Tetrahymena thermophila se realiza principalmente por fagocitosis

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    The free-living ciliate Tetrahymena thermophila is a unicellular model organism in which landmark biological processes have been discovered, such as the first description of telomerase activity and the molecular structure of telomeres, the mechanism of self-splicing RNA and ribozymes, the function of histone acetylation in transcription regulation and a number of pioneer experiments on the interference (RNAi) mechanism for programmed genome rearrangements, among others...Fil: Elguero, MarĂ­a Eugenia. Consejo Nacional de Investigaciones CientĂ­ficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de NanobiotecnologĂ­a. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂ­mica. Instituto de NanobiotecnologĂ­a; ArgentinaFil: SĂĄnchez Granel, MarĂ­a Luz. Consejo Nacional de Investigaciones CientĂ­ficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de NanobiotecnologĂ­a. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂ­mica. Instituto de NanobiotecnologĂ­a; ArgentinaFil: Montes, MarĂ­a Guadalupe. Consejo Nacional de Investigaciones CientĂ­ficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de NanobiotecnologĂ­a. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂ­mica. Instituto de NanobiotecnologĂ­a; ArgentinaFil: Cid, NicolĂĄs Gonzalo. Consejo Nacional de Investigaciones CientĂ­ficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de NanobiotecnologĂ­a. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂ­mica. Instituto de NanobiotecnologĂ­a; ArgentinaFil: Favale, Nicolas Octavio. Consejo Nacional de Investigaciones CientĂ­ficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de QuĂ­mica y FĂ­sico-QuĂ­mica BiolĂłgicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y BioquĂ­mica. Instituto de QuĂ­mica y FĂ­sico-QuĂ­mica BiolĂłgicas; ArgentinaFil: Nudel, Berta Clara. Consejo Nacional de Investigaciones CientĂ­ficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de NanobiotecnologĂ­a. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂ­mica. Instituto de NanobiotecnologĂ­a; ArgentinaFil: Nusblat, Alejandro David. Consejo Nacional de Investigaciones CientĂ­ficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de NanobiotecnologĂ­a. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂ­mica. Instituto de NanobiotecnologĂ­a; Argentin
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