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Models of Human Diseases Conference (2010) Tetrahymena model by Dr. R. Pearlman


The Ciliate Protozoan Tetrahymena thermophila as an important animal model organism …

The Ciliate Protozoan Tetrahymena thermophila as an important animal model organism
Dr. R.E. Pearlman, York University
Models of Human Diseases Conference
June 29, 2010

Published in Education
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  • Figure 4. Immunofluorescence localization image of TCP107:GFP transformants. The ciliary rows (CR) and oral apparatus (OA) with basal bodies (BB) are distinctly visible by the green signal of GFP fusion protein expression. The oral apparatus structure of membranelles, undulating membrane and an oral crescent is distinguishable.
  • Aberrant motile and nonmotile cilia functions are known to result in astonishing number of different illnesses. Numerous disorders associated with basal body or cilia dysfunction are including but not limited to Kartagener syndrome also known as Primary Ciliary Dyskinesia and Polycystic kidney disease, which are both associated with immobile cilia. Primary Ciliary Dyskinesia is a rare autosomal recessive genetic disorder caused by a defect in the action of cilia lining the respiratory tract. Specifically, it is a defect in dynein protein arms within the ciliary structure. It is characterized by the triad of sinusitis, bronchitis, and situs inversus, the condition in which the normal left–right symmetry of the internal organs is disrupted . Polycystic Kidney Disease - A rare inherited condition in which the kidney are composed of multiple cysts. Kidney cysts are associated with an increased incidence of cerebral aneurysm. Symptoms usually appear later (if they do at all) and include blood in the urine, flank pain, excessive urination at night and abdominal pain. Individuals may also have elevated blood pressure. Chronic (end-stage renal disease) renal failure is the most common result in the 5th to 6th decades of life. = Kidney failure 3. More recently studied Bardet-Biedl Syndrome - Inherited rare disorder. Hereditory disease From defects in basal bodies. Characteristics of this syndrome include mental retardation, degeneration of the retinas of the eyes, kidney abnormalities, delayed sexual development or underdeveloped reproductive organs, obesity, and/or abnormal fingers and toes. Basal body – A structure at the base of a cilium or flagellum; consists of nine triplet microtubules arranged in a circle with no central microtubule.


  • 1. The Ciliate Protozoan Tetrahymena thermophila as an important animal model organism R.E. Pearlman York University Models of Human Diseases June 29, 2010
  • 2. Tetrahymena thermophila
  • 3. Tetrahymena thermophila Turkewitz AP, Orias E, Kapler G. Trends Genet . 2002;18(1):35-40.
  • 4.  
  • 5. Why Study Tetrahymena?
    • Powerful eukaryotic model organism that is complementary to yeast having much “animal-like” biology
    • Non-universal genetic code-TGA only stop codon
    • -TAA and TGA encode Q
    • Possesses biological processes present in metazoans but not in yeast:
      • Phagocytosis
      • Ciliary motility
      • Regulated secretion
    • Has about 6x more proteins than yeast
    • Molecular and genetic tools available for manipulation of organism
    • Genome is completely sequenced
  • 6. • Grows fast (2.5hr doubling time) • Grows in axenic medium • freezing in liquid nitrogen allows for facile long-term maintenance of strains • Genetic mapping of both the micronucleus and the macronucleus is in progress • Micronuclear (germ-line) and macronuclear (somatic) transformation are possible • Can perform gene knockouts by exact gene replacement (homologous recombination) • GFP and other tagged vectors permit rapid and high resolution localization of gene products Tetrahymena as a Model Organism
  • 7.
    • • First descriptions of self-splicing introns and catalytic RNA (Cech, 1990)
    • • First description of telomerase (Greider, 1995)
    • • First linkage of nuclear histone acetylation and transcriptional activation
    • • First in vivo function for histone H1 (Shen, 1995)
    • • First in vivo function for phosphorylation of histones H1 / H3
    • • First in vivo function for a tubulin post-translational modification (Xia, 2000)
    • Role of small RNAs and histone modifications in developmentally regulated genome reorganization and heterochromatin formation (Mochizuki et al., 2002; Taverna et al., 2002); RNAi mediated irreversible gene silencing
    Discoveries Made Using Tetrahymena
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  • 12. Tetrahymena: Nuclear Dimorphism Macronucleus vs. Micronucleus Micronucleus:  Transcriptionally inactive  diploid  germ-line nucleus analogue Macronucleus:  Transcriptionally active  polyploid  somatic nucleus
  • 13. Conjugation in Tetrahymena : an inducible and synchronous example of nuclear development MIC MAC apoptotic degradation mitosis, nuclear development meiosis, cross-fertilization mitosis amitosis Vegetative growth Conjugation
  • 14. Tetrahymena thermophila
    • Unicellular eukaryote
    • Nuclear dimorphism
    • Micronucleus (Mic) – germline, diploid; mostly transcriptionally silent
    • Macronucleus (Mac) – somatic, polyploid; major site of gene expression
      • 104-Mb genome sequenced - ~24,000 predicted genes
    • Life cycles
    • Asexual – growth
    • Sexual – conjugation
    Selker 2003 Parental expression Zygotic expression Dr. Marie-Hélène Bré 2005
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  • 17. Orthologs across selected eukaryotic genomes. A Venn diagram showing orthologs shared among human, yeast, Plasmodium falciparum and Tetrahymena thermophila . Lineage specific gene duplications in each of the organisms were identified and treated as one single gene (or super-ortholog) for later comparisons. Pairwise mutual best-hits by Blastp were then identified as putative orthologs.
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  • 22. 1 2 3 1: Asf1 50mM NaCl, 0.2% NP40 2: same + sonication 3: Asf1 300mM NaCl, 0.2% NP40 27kD 20kD 15kD 35kD 55kD 70kD 100kD 130kD 250kD Invitrogen 4-20% gradient gel, silver stain importin
  • 23. Programmed Genomic Rearrangements occur during Macronuclear development :
  • 24. Published Sept. 20 th 2002, Cell
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  • 28. Flag-HA-Twi1p Twi1p associated proteins Isolation of Interacting Proteins Fig. 10
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  • 32. Tetrahymena Organelle Proteomics (Pearlman Lab) Smith JC , Northey JG , Garg J , Pearlman RE , Siu KW ., J Proteome Res. 2005 May-Jun; 4 (3):909-19. Robust method for proteome analysis by MS/MS using an entire translated genome: demonstration on the ciliome of Tetrahymena thermophila. Bowman GR , Smith DG , Michael Siu KW , Pearlman RE , Turkewitz AP . J Eukaryot Microbiol. 2005 Jul-Aug; 52 (4):291-7. Genomic and proteomic evidence for a second family of dense core granule cargo proteins in Tetrahymena thermophila. Jacobs ME , DeSouza LV , Samaranayake H , Pearlman RE , Siu KW , Klobutcher LA . Eukaryot Cell. 2006 Dec; 5 (12):1990-2000. Epub 2006 Sep 29. The Tetrahymena thermophila phagosome proteome. Smith DGS, Gawryluk RMR, Spencer DS, Pearlman RE, Siu KWM, Gray MW. J. Mol. Biol. 2007 374 :837-63. Exploring the mitochondrial proteome of the ciliated protozoon Tetrahymena thermophila : direct analysis by tandem mass spectrometry.
  • 33. Affinity Purification of the Proteasome with Dss1 is Conserved in Human, Yeast and Tetrahymena 97 66 45 31 21 14 WT Dss1-TAP Krogan, Keogh, Fillingham et al. (2004) Mol. Cell Affinity purification of protein complexes is possible in Tetrahymena Rpn2 Rpn1 Rpn3 Rpt2 Rpn5/Rpt5 Rpt1/Rpt3/Pwp1 Rpn6, Rpt6, Rpt4 Rpn7, Rpn8, Rpn9, Rpn11 Rpn12
  • 34. UM BB CR M OA TetCP107-GFP OA CR
  • 35.
    • Major Ciliary Disorders Are :
    • Primary Ciliary Dyskinesia
    • Polycystic Kidney Disease
    • Bardet-Biedl Syndrome
  • 36. Acknowledgements: Canadian Institutes of Health Research Natural Sciences and Engineering Research Council Ontario Research and Development Challenge Fund MDS Sciex Genome Canada (Protist EST Project) National Science Foundation National Institutes of Health
  • 37. Collaborators: York Jeff Fillingham Dalhousie TIGR (JCVI) Jeff Smith Mike Gray Jonathan Eisen Jyoti Garg David Spencer Robert Coyne Daryl Smith Ryan Gawryluk Jane Carlton Hongyan Li Bill Majoros Konstantin Savitsky Quinghu Ren Leroi deSouza Joana Silva Michael Siu UCSB Martin Wu Ed Orias Dongying Wu OTHER Eileen Hamilton Mochizuki Lab-IMP, Vienna Gaertig Lab-UGA Wylie Lab-Claremont Colleges Gorovsky Lab-U Rochester