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Tetrahymena thermophila genome project

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Genome Project Planning - coordinated by Ed Orias at UCSB

• 8/99 Workshop in Ciliate Genomics

• 10/99 First Meeting of Tetrahymena Genome Project Steering Committee

• 10/00 Second Meeting of Tetrahymena Genome Project Steering Committee

• 8/01 Third Meeting of Tetrahymena Genome Project Steering Committee

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Nice Places Tetrahymena is not From

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Nasty Things that are Not Tetrahymena

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What is Tetrahymena

• Fresh-water single-celled protozoan

• Member of the alveolate group - ciliate subgroup

• Swimming/mobile

• Free-living

• Grown in pure culture

• Dual nuclei - somatic and germ line

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Cilia Cover the Surface of Tetrahymena, Allowing It to Move and to Sweep Food to Its Gullet

Cilia powering cell movement

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Why Tetrahymena?

• Model alveolate and ciliate

• Genetic unicellular eukaryotic model

• Fundamental biology of eukaryotes

• Robust and novel molecular genetic tools

• Large research community

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Examples of Major Discoveries from Studies of Tetrahymena

• Dynein motors

• RNA mediated catalysis

• Telomeres and telomerase

• Histone acetylation regulation of transcription

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Fig 8-33 Telomerase carries a short RNA molecule that acts as a template for theaddition of the complementary DNA sequence at the 3’ end of the doublehelix. In the ciliate Tetrahymena, the DNA sequence added is TTGGGG

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Self-Splicing of Tetrahymena rRNA

(group I intron)

Fig 14.45, Weaver

• GTP attacks A at 5’ end of intron

• Exon 1 uses 3’OH to attack intron/exon 2 phosphodiester bond

• Splicing releases linear intron

• Intron loses nucleotides at 5’ end

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Tetrahymena as a Model Alveolate

• Ciliates are sister group for apicomplexans

• Model for studies of ciliates (e.g., Ich, Parmecium)

• Free-living, pure culture, non-pathogenic

• Heterologous expression of Alveolate genes

• Large research community (~100 groups)

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Tetrahymena Uses for Fundamental Biology of Eukaryotes

• Cell motility• Programmed DNA rearrangements• Regulated secretion• Phagocytosis• Tubulins• Histones• Telomeres maintenance and function• Many genes, processes and cellular components

not found in yeasts

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Genome Processing Between Micro and Macronucleus

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Macronuclear Genome

• Little repetitive DNA• 180 Mbp genome• Little evidence for large duplications• No centromeres• Few and small introns• No alternative splicing reported• Genes are lower At (63%) than rest of the genome

(83%)

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Tools in Tetrahymena

• Conventional genetic tools– Conjugation, Genetic crossing, Inducible self-fertilizatoin

• Advanced genetic tools– Transformation, Gene disruption, Gene replacement– Gene overexpression, Ribosome antisense repression

• Ease of use– Grows fast (1.5 h doubling) in pure culture– Large cell size– Large T° range for growth– Stoarge in liquid N2

– Large scale sub-cellular compartment fractionation

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Tetrahymena Genomic Resources

• Genetic maps (for mic and mac)

• Physical maps

• EST projects– Protist EST project in Canada - 50,000 ESTs– EST project at U. Chicago– Gene index at TIGR

• Genome wide libraries

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Other Ciliate Projects• Paramecium genomic survey (Dr. Linda Sperling,

Centre de Genetique Moleculaire, CNRS, France)• European rumen ciliate cDNA project (C. Jamie

Newbold, Rowett Research Institute, Aberdeen, UK)

• Oxytricha (Spirotrich ciliate) micronuclear BAC project (Laura Landweber, Princeton University);

• Ichthyophthirius EST sequencing proposal (Theodore G. Clark, Cornell University

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• Strain selection:– inbred strain B- strain of choice for molecular genetic work– Strain SB210

• Macronuclear DNA– Most of the repeats already removed– Mitochondrial DNA can be mostly removed– Working on removing rDNA

• Small and medium insert libraries made• Due to high AT content large inserts are difficult to construct• Proposals in review by NSF (3x) and NIGMS (5x plus

database)

Tetrahymena Genome Project

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Bioinformatics Plans

• Gene finding, gene indices, ESTs

• Functional annotation

• Orthologs with mammals/animals– Especially those not in yeast

• Orthologs with Apicomplexans

• Phylogeny

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Tetrahymena Genome Database

• Phenotypes associated with gene knockouts, replacements and other types of mutations.

• Gene regulation information from the literature.• Post-translational modifications.• Linkage & physical maps • DNA polymorphisms • Experimental protocols• Links to other sites

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