Presentation by Jonathan Eisen in February 2003 to NSF Microbial Genomes Workshop on the Tetrahymena genome project

1. Tetrahymena thermophila
genome project
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2. 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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3. Nice Places Tetrahymena is not From
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4. Nasty Things that are Not Tetrahymena
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5. 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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9. 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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10. 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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11. 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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12. Fig 8-33 Telomerase carries a short RNA molecule that acts as a template for the
addition of the complementary DNA sequence at the 3’ end of the double
helix. In the ciliate Tetrahymena, the DNA sequence added is TTGGGG
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13. Self-Splicing of
Tetrahymena rRNA
(group I intron)
• 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
TIGR Fig 14.45, Weaver

14. 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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15. 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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16. Genome Processing Between
Micro and Macronucleus
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17. 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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18. 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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19. 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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20. 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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21. Tetrahymena Genome Project
• 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)
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22. 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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23. 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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