models of human diseases conference (2010) tetrahymena model by dr. r. pearlman
DESCRIPTION
The Ciliate Protozoan Tetrahymena thermophila as an important animal model organism Dr. R.E. Pearlman, York University Models of Human Diseases Conference June 29, 2010TRANSCRIPT
The Ciliate ProtozoanTetrahymena thermophila as an important animal model organism
R.E. PearlmanYork University
Models of Human DiseasesJune 29, 2010
Tetrahymena thermophila
Tetrahymena thermophila
Turkewitz AP, Orias E, Kapler G. Trends Genet. 2002;18(1):35-40.
Why Study Tetrahymena?
Powerful eukaryotic model organism that is complementary to yeast having much “animal-like” biologyNon-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 yeastMolecular and genetic tools available for manipulation of organismGenome is completely sequenced
• 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
• 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
Tetrahymena: Nuclear DimorphismMacronucleus vs. Micronucleus
Micronucleus: Transcriptionally inactive diploid germ-line nucleus analogue
Macronucleus: Transcriptionally active polyploid somatic nucleus
Conjugation in Tetrahymena: an inducible and synchronous example of nuclear development
MIC
MAC
apoptoticdegradation
mitosis,nuclear development
meiosis,cross-fertilization
mitosis
amitosis
Vegetativegrowth
Conjugation
Tetrahymena thermophilaTetrahymena thermophila Unicellular eukaryoteUnicellular eukaryote Nuclear dimorphismNuclear dimorphism Micronucleus (Mic) – Micronucleus (Mic) –
germline, diploid; mostly germline, diploid; mostly transcriptionally silenttranscriptionally silent
Macronucleus (Mac) – Macronucleus (Mac) – somatic, polyploid; major somatic, polyploid; major site of gene expressionsite of gene expression 104-Mb genome 104-Mb genome
sequenced - ~24,000 sequenced - ~24,000 predicted genespredicted genes
Life cyclesLife cycles Asexual – growthAsexual – growth Sexual – conjugationSexual – conjugation
Selker 2003Parental expression
Zygotic expression
Dr. Marie-Hélène Bré 2005http://www.nikonsmallworld.com/
gallery
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.
1 2 3
1: Asf1 50mM NaCl, 0.2% NP402: same + sonication3: Asf1 300mM NaCl, 0.2% NP40
27kD
20kD
15kD
35kD
55kD
70kD
100kD
130kD
250kD
Invitrogen 4-20% gradient gel, silver stain
importin
Programmed Genomic Rearrangements occurduring Macronuclear development:
Published Sept. 20th 2002, Cell
Flag-HA-Twi1p
Twi1p associated proteins
Isolation of Interacting Proteins
Fig. 10
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.
97
66
45
31
21
14
WT
Dss
1-TA
P
Affinity Purification of the Proteasome with Dss1is Conserved in Human, Yeast and Tetrahymena
Krogan, Keogh, Fillingham et al. (2004) Mol. Cell
Affinity purification of protein complexes is possible in Tetrahymena
Rpn2Rpn1
Rpn3Rpt2Rpn5/Rpt5Rpt1/Rpt3/Pwp1Rpn6, Rpt6, Rpt4
Rpn7, Rpn8, Rpn9, Rpn11
Rpn12
OA
CR
UM
BB
CR
MOA
TetCP107-GFP
Major Ciliary Disorders AreMajor Ciliary Disorders Are::
•Primary Ciliary DyskinesiaPrimary Ciliary Dyskinesia
•Polycystic Kidney DiseasePolycystic Kidney Disease
•Bardet-Biedl SyndromeBardet-Biedl Syndrome
Acknowledgements:Canadian Institutes of Health ResearchNatural Sciences and Engineering Research CouncilOntario Research and Development Challenge FundMDS SciexGenome Canada (Protist EST Project)National Science FoundationNational Institutes of Health
Collaborators:
York Jeff Fillingham Dalhousie TIGR (JCVI)
Jeff Smith Mike Gray Jonathan EisenJyoti Garg David Spencer Robert CoyneDaryl 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