Appendix for the Tetrahymena Concept Paper - November 5, 2001

 

Table of contents

 

Section

Topic

Page

1

Explanation of molecular genetic tools dependent on unique Tetrahymena features

1

2

Unique or special scientific contributions that would be enabled by the genome sequence

3

  Table 1

Telomeres and telomerase

3

  Table 2

Genome stability

3

  Table 3

Chromatin dynamics

4

  Table 4

Epigenetic inheritance

5

  Table 5

Apoptosis

5

  Table 6

Transcription and translation

5

  Table 7

Microtubules and tubulins

6

  Table 8

Cytoskeletal motors

7

  Table 9

Regulated Secretion

7

  Table 10

Phagocytosis and bacterial pathogenesis

8

  Table 11

Signal Transduction

9

  Table 12

Cell cycle control

9

  Table 13

Development

10

  Table 14

Biotechnology

10

  Table 15

Environmental adaptation and monitoring

11

  Table 16

Eukaryotic Evolution

12

  Table 17

Science education

13

  Table 18

Additional intriguing phenomena, not yet investigated molecularly

13

3

Post-genomic resources preview

13

4

Acknowledgments

14

 

References : Separate document

 

------------------

 

1. Explanation of molecular genetic tools dependent on unique Tetrahymena features

·                     Heterokaryons are cell lines in which the MIC and MAC differ genotypically and they are readily constructed. They are especially useful for the germline maintenance of lethal mutations, losses of single or multiple (up to four of the existing five) chromosomes, and essential gene knockouts in the homozygous state. Unrestricted survival of such homozygous heterokaryons is ensured by endowing the cell with a wild type expressed nucleus (MAC). The cross of two heterokaryons with identical homozygous genotype to one another uncovers the mutant (or lethal) phenotype in every progeny. The stage and cell process at which the lethal mutation acts can be determined by cytological and/or molecular observations in synchronous conjugating cultures.

·                     Phenotypic assortment. The MAC genome is polyploid (~45 copies, except the more abundant rDNA minichromosome) and its chromosome copies are randomly distributed at MAC division [51; 43]. As a consequence, the ratio of alleles in the clonal descendants of a heterozygote varies continuously from pure dominant (45:0) to pure recessive (0:45) through every intermediate mix. Left to chance alone, the end result is the assortment to purity for either allele. Assortment to purity allows recessive gene expression without sexual reproduction, genetic mapping of loci to MAC chromosomes and construction of MIC:MAC relational genetic maps [43]. The dynamic phenotypic gradation is very useful for a) the maintenance of lethal mutations or gene knockouts of essential genes in the MAC by balanced selection and b) in vivo dose-response studies of the phenotype, e.g., as determined by a dominant negative protein.

·                     DNA-mediated transformation. Transformation is routinely accomplished by electroporation, biolistic bombardment or microinjection -- each with its own advantages [52-54]. Transforming DNA can be selectively targeted to the MIC, the differentiating MAC or the vegetative (mature) MAC. In the MAC, the few initial copies of the transgene allele are driven to fixation (complete replacement of the endogenous allele) in mass culture either by assortment and selection for drug resistance or by using minichromosome vectors with differential replication advantage. Integrative, high-copy-replicative and developmental processing vectors are each available. High-copy-number replicative vectors have been extensively used to study cis-acting signals for DNA rearrangement, chromosome maintenance and amplification, and for antisense ribosome and GFP-fusion screening (see below).  The high rates of transformation (over 104/microgram DNA) allow mass-screening and make cloning by complementation a feasible proposition.

·                     Precise homologous recombination. This is the exclusive mechanism of integration of linearized recombinant DNA constructs in Tetrahymena when they contain a few hundred base pairs of homology on either flank. Exact recombination gives complete control over the placement of the inserted gene, and thus facilitates interpretation of the resulting phenotypes and speeds up experimental progress. It allows highly specific gene replacement, disruption (knockout) and targeted ectopic insertion. Gene disruption is used to determine if a Tetrahymena gene is essential and to test the in vivo function of a gene in a specific cell process. Gene replacement is the foundation of in vivo structure/function studies and has been extensively and productively used to study, for example, the function of protein post-translational modification in Tetrahymena. Gene insertion has been used, for example, for the high level expression of foreign genes in Tetrahymena. Integration by exact homologous recombination provides also a particularly powerful tool for functional dissection within multigene families. For example, the homologous integration mechanism distinguishes between two Tetrahymena beta-tubulin genes that encode identical proteins and are 98% identical in their coding regions.

·                     Cotransformation. Transformation of Tetrahymena cells with a mixture of two constructs yields double transformants in much greater frequency than expected by chance alone -- a phenomenon known as cotransformation [55]. Each construct specifically integrates at its own, physically separate homologous locus.  Cotransformants can amount to 50% of the cells transformed by either construct. Desired cotransformants thus can be readily screened after mass selecting for a drug resistance construct. This approach confers great experimental versatility because it bypasses complications (technical or interpretational) due to the artificial placement of a drug-resistance gene adjacent to the replacement gene.

·                     Gene overexpression. Two independent approaches, readily used in combination, allow a high degree of gene overexpression in Tetrahymena: 1) high-copy-number vectors, based on the rDNA minichromosome replicon, which allow 200-fold gene amplification relative to the single genomic copy DNA [53] and 2) a recently developed inducible metallothionein (MTT) promoter which allows controlled, cadmium-dependent regulation of expression over at least a 1000-fold dynamic range [56]. This is a useful tool for studying protein interactions by overexpression suppression of a mutant phenotype, or for probing gene function.

·                     Ribosomal antisense repression. This is a novel, robust and stably inherited method of antisense RNA-mediated gene-specific translational repression. Every ribosome in the cell is engineered to display an antisense sequence from the 5'-untranslated region of a particular gene. This is accomplished by inserting the antisense segment at a cloning site in the 28S rRNA gene that does not compromise rRNA function [57]. Repression of the targeted gene is stably maintained and is inherited by the clonal descendants of the transformed cell.

·                     Ribosomal antisense mutagenesis and direct phenotype-based cloning. This is a novel extension of ribosomal antisense repression for efficient "forward genetics" [58]. By exposing wild type cells to MAC transformation with a ribosomal antisense library, mutant cells randomly and heritably repressed for expression of virtually any gene can be screened or mass selected for a desired phenotype. The affected gene is then easily cloned by two successive PCR reactions.

·                     Random mutagenesis of a particular gene. A general method involves first making homozygous heterokaryons for the gene of interest disrupted in the micronuclear (silent) genome. The cloned wild type allele is randomly mutagenized in vitro. It is then targeted as a cotransforming linearized molecule to differentiating MACs that are homozygous for the disruption -- obtained by crossing two heterokaryons with identical disruption genotype. If the disruption is lethal, then there is absolute selection for progeny rescue even by point mutations that confer a deleterious phenotype. This method combines the power of a targeted study of one particular gene with the absence of preconceptions about its function that is inherent in a random genetic mutagenic screen.

·                     Protein tagging. Epitope and ligand affinity tagging technology is well developed in Tetrahymena. A variety of epitopes have been used to hereditarily tag Tetrahymena proteins and visualize their cellular localization [59], including the tandem affinity purification (TAP) tag developed for -- and currently revolutionizing -- yeast proteomics. Tetrahymena is also highly amenable to massive GFP-labeling of specific proteins [59]. GFP-tagged cDNA libraries inserted into a standard high-copy developmental processing vector have revealed localizations of cytoskeletal and other proteins in vegetative cells  [60], and a wide array of nuclear proteins in conjugating cells [59]. Extension of this strategy has enormous potential.

 


2. Unique or special scientific contributions that would be enabled by the genome sequence

 

Table 1) Telomeres and telomerase

 

Unique or special biological feature

Significance

Relevance to humans

Funding

Interest

- Abundance of telomeres

- Elucidation of structure and function of telomeres [14]

-  Discovery of telomerases and telomerase RNA (starts of a major research field) [15;16]

- Genome instability induced by telomere dysfunction promotes epithelial carcinogenesis, and thus the overwhelming majority of human cancers.

NIGMS

NSF

NCI

NIA

- Highly active telomerase

- Tetrahymena's is the only telomerase efficiently reconstituted in vitro from recombinant components [48]

- Structure and function of a telomerase, whose activity is more similar to human's than either is to yeast's [61;62]

- Sequence will allow quickly finding and investigating in Tetrahymena the function of telomerase components identified in other model systems and thus better understanding of the human telomerase [63]

- Opportunity for better therapeutics for diseases of telomerase insufficiency (age-dependent somatic cell proliferative deficiencies) and hyperactivation (cancer).

NIGMS

NSF

NIA

NCI

 

 

Table 2) Genome stability

 

Unique or special biological feature

Significance

Relevance to humans

Funding

Interest

- Some replicons occur in small, natural chromosomes readily accessible for in vivo and in vitro studies

- -Differential amplification of the rDNA minichromosome

High-resolution analysis and genetic dissection of Tetrahymena cis-acting replication determinants [64], which are more similar to human than to yeast, and regulatory proteins [65;66]

-Mechanisms of minichromosome formation and maintenance in drug-resistant cancer cells

NIGMS

NSF

NCI

Developmental formation of large palindromic DNA in the macronucleus

- Identification of cis-acting sequence determinants and proposed mechanism [67]

Amplified genes in human cancers are frequently found in palindromic form

NIGMS

NCI

- Random distribution of ~45 copies of each chromosome [51;43]

- Independent control of copy number of ~300 chromosomes [68]

Unique opportunity to elucidate novel mechanisms of chromosome homeostasis, particularly with regard to chromosome counting and programmed chromosome amplification

Many cancers result from chromosome instability, frequently accompanied by gene amplification in acentric chromosomal fragments.

NIGMS

NSF

Double-strand breaks efficiently repaired in concert with developmental chromosome fragmentation [69]

- Study of telomerase requirements for chromosome healing [18]

- Special opportunity to investigate the mechanism and evolution of chromosome and telomere biogenesis

- Mechanisms underlying genome rearrangements in cancer cells

- Chromosome healing dysfunction in aging and cancer

- Improved methods for the creation of artificial chromosomes.

NIGMS

NSF NCI

NIA

~6,000 distinct developmentally programmed, site-specific deletion events per haploid genome during a 2-hr period [70]

- No easily recognizable common DNA features within or flanking the deleted segments

- Role of chromodomain proteins and histone modification in programmed DNA rearrangement and degradation of excised DNA [71-74]

- Opportunity to study the enzymatic basis for genome rearrangements, which has been refractory to direct, biochemical approaches

Programmed DNA rearrangements associated with immune system evasion (e.g. trypanosomes, bacterial phase variation), spread of antibiotic resistance, immunoglobulin gene expression and diversity.

-Rearrangement gone awry associated with cancers (e.g., Burkitt's lymphoma)

NIGMS

NSF

NHLBI

NCI

NIAID

Mitosis is dispensable because the expressed nucleus is not mitotic; in other experimental model systems, severe mitotic defects would render the cells inviable

Mutational studies allowed the identification of genes that are important for mitotic chromosome distribution in living cells  (e.g., telomere-binding protein [75], dynein [76], RAD51 [77])

- Chromosome instability is associated with the majority of human cancers

- Age-dependent chromosome losses in the human germline

NIGMS

NSF

NCI

NIA

NICHD

 

Table 3) Chromatin dynamics

 

Unique or special biological feature

Significance

Relevance to humans

Funding

Interest

- Separation of replication, transcription and mitotic function to different nuclei in the same cytoplasm

- Histone genes are present in single or double copy only, making knockouts and site-directed mutagenesis uniquely possible.

- Dynamic reorganization of histone variants during conjugation can be studied biochemically and cytologically.  This includes regulated proteolytic processing of a core histone (micronuclear H3), an event so far unique to Tetrahymena [78]

- Discoveries of the functions of histone post-translational modifications:

- Histone acetylase as a transcriptional regulation factor -- led to a new paradigms of how histone modifications affect chromatin function (start of a major research field) [20]

- H1 phosphorylation is involved in transcriptional regulation [79] and functions by a novel "charge patch" mechanism [80]

- Acetylation of H2A.Z is essential and functions by creating a charge patch [81]

- H3 phosphorylation on S10 functions in chromosome segregation and condensation [82]

-H3 methylation on K9 functions in programmed DNA elimination [71]

-Human homologs of the mitotic H3 S10 kinase (aurora kinases) are overexpressed in a variety of human cancers.

-Other health-related signaling pathways also likely target chromatin (e.g. H3 K9 methylation widely associated with gene silencing; H3 K4 methylation and acetylation of various residues associated with gene activation).

NIGMS

NSF

NCI

 


Table 4) Epigenetic inheritance

 

Unique or special biological feature

Significance

Relevance to humans

Funding

Interest

State of DNA in parental macronucleus affects DNA rearrangement events in newly developing macronucleus in sequence-specific manner [83], although no actual exchange of DNA appears to occur.  Ciliates are so far unique in this form of epigenetic inheritance [84].

Epigenetically controlled excision from the expressed genome may have evolved as a novel defense against parasitic DNAs, functionally related to epigenetic gene regulation by DNA methylation and chromatin structure in other organisms [85].  In Tetrahymena, involvement of chromodomain proteins [72; 73] and bidirectional deletion element transcription [86] in excision suggests potential mechanistic similarities as well.

- Epigenetic inactivation of tumor suppressors has been implicated in human cancers [87; 88]. 

- Epigenetic modifications play prominent roles in normal development [89].

- Transgenes are subject to epigenetic inactivation, especially important in plant biotechnology [90]. 

NIGMS

NSF

NCI

NICHD

USDA

 

Table 5) Apoptosis

 

Unique or special biological feature

Significance

Relevance to humans

Funding

Interest

Degeneration of the old macronucleus during conjugation occurs by an apoptotic mechanism that is blocked by caspase inhibitors [91].

Opportunity for genetic and pharmacological investigations of apoptosis in a unicellular model organism.

Apoptosis or its suppression is implicated in numerous human diseases, including cancer, and neurodegenerative, autoimmune, infectious, cardiovascular, and aging-related diseases [92-97].

NIGMS, NSF, NCI, NIA, NICHD, NIAMS, NINDS, NIAID, NHLBI

 

Table 6) Transcription and translation

 

Unique or special biological feature

Significance

Relevance to humans

Funding

Interest

- Self-splicing intron in rDNA [19]

- Highest rate of rRNA synthesis in a eukaryotic nucleus

Discovery of ribozymes (start of a major research field) [19]

Opportunity for therapy by ribozyme-mediated mRNA targeting

NIGMS

NSF

- Single germline copy of 18S/4.5S/28S rRNA genes (a unique Tetrahymena feature) [98]

- Simple Mendelian genetic analysis of rDNA [99; 100]

- Genetic approaches to the in vivo study of rRNA structure/function, by replacing most or all of the ribosomes by in vitro engineered ones [101; 102]

Development of novel ribosome-based antisense technology [58]

NIGMS

NSF

- rRNA genes are amplified to ~20,000 copies per cell (1-2% of total cell DNA) in a minichromosome of their own

- rRNA genes are clustered in ~500 nucleoli that can be purified in a transcriptionally active state, facilitating proteomic approaches [103]

- Opportunity to study the function and interaction of ribosomal RNA genes with trans-acting factors identifiable by genomic/proteomic approaches.

 

Elucidation of important but little understood nucleolar biology

NIGMS

NSF

 

Table 7) Microtubules and tubulins

                                   

Unique or special biological feature

Significance

Relevance to humans

Funding

Interest

- In ciliates, microtubules are assembled into 17 types of distinct systems, including those shared with mammals, such as the basal bodies/centrioles, cilia and the mitotic spindle [104]

- Arrayed, two-dimensional, ciliary deployment, such as found in human respiratory tract and oviduct)

-Cilia regeneration system

- Insights into the mechanism of assembly of cilia [105]

- First evidence that assembly of cilia involves extracellular signaling i.e., it is under control of a Tetrahymena extracellular factor.

 [31]

 

Mammals have flagellated sperm cells and cilia on cells of respiratory, reproductive and neural tissues. In the respiratory tract, cilia are essential for maintaining proper mucociliary clearance. Vertebrate photoreceptors are modified sensory neurons where the light sensitive segment is a highly modified nonmotile cilium.

NHLBI

NICHD

NEI

- Few tubulin genes

- Alpha- and beta-tubulins undergo evolutionarly conserved post-translational modifications (uniquely absent in S. cerevisiae), including acetylation, polyglutamylation and polyglycylation, which increase the number of different functional isoforms.

- Polyglycylation is specific to axonemes and conserved between protists and humans.

- Discoveries that tubulin polyglycylation is involved in ciliary motility and cytokinesis in Tetrahymena  [106] and controls axonemal stability [107].

- Opportunity to analyze mechanisms underlying microtubule diversity

Tetrahymena is the only experimental system where it has been possible to directly test the functional consequence of post-translational tubulin modifications, by the combination of knockout, replacement and dominance approaches

NIGMS

NSF

The surface of ciliates have hundreds of basal bodies (absent in S. cerevisiae), with 9 microtubule triplets -- exactly like the centrioles of mammals and unlike the highly divergent singlet centrioles of invertebrate model species: C. elegans and Drosophila [108]

- Ciliates have homologs of delta-, epsilon- and eta-tubulins, tubulin-like proteins conserved in mammalian genomes but not found in the genomes of yeast, C. elegans or Drosophila

- In the ciliate Paramecium, and likely in Tetrahymena, gamma and eta-tubulins are required for the basal body duplication [109;110], while delta-tubulin is required for the assembly of the C-tubule of triplet microtubules [111]

- Unusual opportunity to investigate the mechanisms of centriole/basal body duplication and the function of unusual tubulins found in humans but lacking in other well-characterized model organisms

- Microtubule structures are required for organizing mitotic spindle and for the normal mitotic and meiotic segregation of chromosomes. Defects in these processes lead to spontaneous abortion, and birth defects.

-MTOC amplification is a hallmark of a variety of human cancers

NIGMS

NSF

NICHD

NCI

 

Table 8) Cytoskeletal motors

 

Unique or special biological feature

Significance

Relevance to humans

Funding

Interest

Tetrahymena expresses at least 14 dynein (ciliary and cytoplasmic) isoforms [112;113] found in humans and other metazoans [114], but not in yeast, the benchmark unicellular model organism

 

- Dynein, the minus-end directed microtubule-dependent motor protein was first purified from Tetrahymena cilia [8;9]

- Gene knockouts used to study the function of polypeptides in the ciliary dynein complex. [115;76]

- Investigations of the cellular localization and functional specialization of individual dynein isoforms [116; 117]

- Unique opportunity to replace wild type motor protein genes with engineered genes, analyze the effects of the modifications in the living cell, and then measure the in vitro activity of the recovered altered motor protein.

- Axonemal dynein of embryonic nodal cilia is required for establishment of the left-right asymmetry of the mammalian body plan  [118; 119]

- Cytoplasmic dynein-2 appears to be required for normal ciliogenesis in metazoa [120]

- Determining the mechanisms underlying the production and regulation of ciliary bending is important for understanding normal and abnormal ciliary activity affecting sperm motility, movement of the egg through the oviduct, mucus clearance from the bronchial/tracheal passages, left-right handedness of developing embryos, and activity of ciliated ependymal cells in the nervous system.

NICHD

NHLBI

NINDS

Tetrahymena expresses many kinesin genes conserved in humans and other metazoans

 

- Functional synergy between two different kinesin II heavy chains involved in ciliary outgrowth demonstrated by targeted double mutation [121]

Unusual opportunity to use gene knockouts to investigate cell motor function

Kinesins are possible targets for therapeutic agents for cancer.

NIGMS

NSF

NCI

- Actin/myosin-based motility systems are involved in phagocytosis and cytokinesis.

Enormous potential for the study of actin/myosin-based motor activity in a unicellular organism with such an elaborate cytoskeleton and advanced experimental tools.

Acto-myosin associated defects are implicated in a variety of human diseases, such as hypertrophic cardiomyopathy and muscular dystrophy.

NIGMS

NIAMS

 

Table 9) Regulated Secretion

Unique or special biological feature

Significance

Relevance to humans

Funding

Interest

- Highly developed regulated secretory pathways, involving dense-core granules that are strikingly similar to those that are hallmarks of endocrine, exocrine and neuronal tissues in multicellular organisms.

- Synchronous, induced release of thousands of secretory granules.

- The core machinery of the secretory pathway is shared between ciliates and other eukaryotes.

- Easily obtained mutants [122;123] used to identify discrete steps in the regulated secretion pathway, and to discover relationships between membrane fusion and transcription activation [124]

- A distinct membrane structure associated with vesicle docking in Paramecium  [125] and Tetrahymena has recently been associated with a new membrane fusion protein identified in mammalian cells [126]

The experimental accessibility of regulated secretion in Tetrahymena may provide the best system for molecular genetic analysis of its precise function and yield otherwise inaccessible insights into the biology of this process in human endocrine, exocrine and neuronal tissues  [25]

 

NIDDK

NINDS

 

Table 10) Phagocytosis and bacterial pathogenesis

 

Unique or special biological feature

Significance

Relevance to humans

Funding

Interest

- Very active (~1 per minute) and readily observable phagosome formation

- Phagosomes in different stages of maturation can be purified in much higher amounts than possible for mouse macrophage phagosomes [127]

- 2-D electrophoresis revealed ~250 Coomassie-Blue spots, in amounts sufficient for oligopeptide sequencing by tandem mass spectroscopy [128]

- Well characterized dynamics and cytology of phagocytosis

- Phagocytosis is not essential, facilitating mutant isolation.

- Phagosome biogenesis occurs at a single cellular site [129]

- 5 matches to mouse phagosome proteins found among ~300 ESTs (i.e., ~1% of Tetrahymena proteins). This provides opportunity to use high throughput proteomic methods to identify components of the maturing phagosome and understand much better the mechanism of phagocytosis

- Excellent model to further unravel the details of phagocytosis, a poorly known phenomenon that does not occur in the benchmark unicellular eukaryotic model organism, the yeast S. cerevisiae
-
Opportunity to study changes in the molecular composition of the phagosome during its maturation

Phagocytosis plays a critical role In humans:

- Ridding the body of its own dead cells during early development and in the adult organism [130]

- Renewal of retinal photoreceptor cells [131].

- Investigating phagocytosis is important for understanding tissue maintenance and immune defense in humans and for a better understanding of diseases caused by phagocytosis defects

NHLBI

NEI

NIAID

- Natural host for Legionella [132-134], a bacterium that gains human entry through macrophage phagosomes and causes Legionnaire's disease

- The phagocytic pathway is reprogrammed by Legionella in ways ultrastructurally similar to that of human lung macrophages [135]

- Legionella variants unable to infect human cells are also unable to infect Tetrahymena, implying a conserved mode of infection [133]

- Excellent opportunity to study both host and bacterial genetic factors involved in microbial pathogenicity.

- Excellent opportunity to develop better-informed strategies for preventing entry of pathogens that reprogram the phagocytic pathway.

- General usefulness of Tetrahymena as model for a natural reservoir for Legionella (and perhaps other intracellular pathogens)

- Phagocytosis is used by specialized blood cells as a first line of defense to rid the body of microbial pathogens and as a crucial step in stimulating the immune system to produce specific antibodies [136;137]

- The human macrophage phagosome is the primary invasion route of many other microbial pathogens, e.g., Salmonella, Mycobacterium, Bacillus anthracis (inhalation anthrax), and Leishmania; understanding phagocytosis is important for elucidating and combating their infection strategies [138]

NIAID

 

Table 11) Signal Transduction

 

Unique or special biological feature

Significance

Relevance to humans

Funding

Interest

- ATP is a chemorepellent in Tetrahymena and Paramecium

- Ciliates are the only eukaryotic unicells shown to have ATP receptors [139]

- ATP receptors in ciliates, human pain receptors and ciliated epithelium are conserved and may well be indicators of nearby cell lysis.

- The Tetrahymena ATP receptor cross-reacts with a peptide antibody to a vertebrate ATP receptor and is down-regulated by receptor-mediated endocytosis [140]

- Unique opportunities to study cellular sensory transduction, adaptation and signal transduction pathways by using both forward and reverse genetic approaches

- In ciliated epithelial cells in vertebrate lung, ATP sensing increases ciliary beat frequency and clears debris by increased mucociliary transport. ATP inhalers are useful in cystic fibrosis, asthma and other lung clearance problems.

- ATP receptors are vertebrate pain receptors (nociceptors) in some peripheral nerves [141]

- Tetrahymena is an excellent potential surrogate animal model to study pain reception and analgesia

NHLBI

NCRR

- Responds to morphine and the opioid b-endorphin affect phagocytosis in this protist [188].

- Possesses phospholipase (PL) A2, PLC, and PLD, prostaglandins [187] and enzymes for leukotriene biosynthesis (EST project)

- Morphine and the opioid b-endorphin affect phagocytosis in this protist; their activity is reversed by naloxone, a specific morphine antagonist.

Tetrahymena is a surrogate animal model to study inflammation, pain reception and analgesia

NHLBI

NIDA

 

Table 12) Cell cycle control

 

Unique or special biological feature

Significance

Relevance to humans

Funding

Interest

Three different CDK’s acting at different times in the cell cycle of Paramecium, two of them known to be associated with cyclins; similar complexity likely in Tetrahymena [142]

First demonstration of multiple CDK-cyclin pairs in a lower eukaryote

Unicellular model of complex cell cycle controls with excellent knockout possibilities.

NCI

 

-Experimental dissociability of macronuclear replication and cell division cycles [143]

-Dissociation of micro- and macronuclear S-phases in a common cytoplasm [144]

- Polyploidization of the expressed genome during the first postzygotic cell cycle [145]

Unusual features of cell cycle progression and checkpoint control offer opportunities to enrich understanding of these fundamental processes.

Control of cell cycle events is crucial to normal differentiation and proliferation.  Breakdown is a hallmark of cancer

NCI

Cell-cycle coupled regulation of assembly and disassembly of cytoskeletal proteins [146]

Novel targets linking cell cycle regulation to cortical patterning.

Cell differentiation, cancer

NCI

NICHD

Presence of piwi/argonaute homologs [29;32;147] which are involved in stem-cell self-maintenance in flies and humans (absent in yeast)

Opportunity to study mechanism and evolution of germline/soma differentiation

Diseases of stem cell maintenance, e.g., cancer, fertility and likely many more.

NCI

NICHD

 

Table 13) Development

 

Unique or special biological feature

Significance

Relevance to humans

Funding

Interest

- Ciliates comprise one of only two major groups of unicellular eukaryotes to have evolved separation between germline and soma, represented by the micro- and macronucleus. (For-aminifera is the other group)

- Germline/soma differentiation involves selective and simultaneous reprogramming of multiple nuclei within the same cytoplasm. 

- Silent micronucleus allows genetic and pharmacological perturbations that would be lethal in other unicells.

- Nuclear fate determinants experimentally shown to be localized within the cytoplasm/cell cortex [148-151]. 

- Nuclear placement is based on spindle orientation and length [152; 153]. 

- Genetic dissection has revealed a number of genes required for specific conjugation/nuclear differentiation events [154; 155]. 

- Genome sequencing would facilitate the cloning and analysis of those genes, the identification of fate determinants, and a better understanding of evolution of germline/soma differentiation.

 

Sequence homology may give clues to human dysfunctions of germline vs. soma differentiation

NSF

NHGRI

NICHD

Antero-posterior and circumferential axes of cell polarity, revealed by Tetrahymena mutants and unique ciliate cell manipulations; reversal of large-scale cellular handedness [156]; intracellular positional information [157]

Unusual opportunity to discover the molecular and cellular basis of phenomena that challenge current models of development and to extend current paradigms of cell pattern formation beyond differential gene expression to cytoplasmic sources of positional information and self-assembling architecture [26]

Better understanding of cellular basis of human developmental diseases

NICHD

 

Table 14) Biotechnology

 

Unique or special biological feature

Significance

Relevance to humans

Funding

Interest

- Cells export a variety of lysosomal hydrolases by constitutive exocytosis, including nucleases, glycosidases, proteinases and lipases/phospholipases [158]

- Hydrolases secreted by constitutive exocytosis are N-glycosylated by a pathway found also in human hepatocytes [159]

Opportunity to explore the phenomenon of the regulated fusion of lysosomes [160]

 

- Release of lysosomal enzymes, for unknown reasons, is exaggerated in many mammalian tumor cells [161]. Potential diagnostics for a wider range of cancer types

NCI

NIDDK

- Large-scale culture in bioreactors using inexpensive growth media [162]

- Abundant source of secreted phospholipase A1, a hydrolase currently not commercially available [163]

 

- Phospholipase A1 has been readily purified from spent culture medium used to grow wild type and hypersecretory strains of Tetrahymena [163;164]

- Tetrahymena phospholipase A1 completes the tool kit for lipid analyses and allows the synthesis of phospholipids with specific ligands in the flexible sn-1 position.

- Accomplishments highlight the enormous potential of Tetrahymena for biotechnology [165]

- Opportunity to target valuable proteins for high level constitutive secretion and easy purification

Pharma

Biotech

Particularly well-suited to the expression of surface membrane proteins:

- Abundant regulated expression of GPI-anchored surface membrane antigens;

- Abundant cilia provide high surface-to-volume ratios;

- High level expression of foreign genes under the control of the metallothionein promoter

 

- Membrane antigens from the parasitic ciliate, Ichthyophthirius multifiliis, ("Ich") have been expressed in T. thermophila --both at the cell surface and in secreted form -- at levels up to 1% of total cell protein (up to 100 mg/l of culture) [166-168]

- Channel catfish (the natural host of "Ich") are completely protected against an otherwise lethal parasite challenge when immunized with the live Tetrahymena vaccine vector [169]

- Useful reference point for investigating the function and evolution of surface antigens that play important role in host immune evasion by microbial pathogens

T. thermophila offers significant potential in the arena of biotechnology.

- Extremely attractive system for the expression of foreign proteins [170]

- Useful as a live vector for the delivery of recombinant subunit vaccines, e.g., in preventing economically important diseases in commercial aquaculture.

- Potential general vector for production of vaccines in other vertebrates

USDA

Biotech

Certain Tetrahymena species are deadly histophagous parasites of invertebrates hosts [171-173] for causative agents of medically and economically important diseases, e.g., malaria, schistosomiasis, onchocerchiasis

The Tetrahymena genome sequence would allow a search for genetic variations in the putative enzymatic pathways responsible for histophagy [174] and might lead to strategies of biological control of the malarial vector

Potential control of human infectious diseases

NIAID

USDA

The apicomplexans (which constitute the largest group of protozoan parasites and includes the malaria parasite) form a monophyletic assemblage with the ciliates [175]

- The circumsporozoite protein of P. falciparum (the malaria parasite) has now been expressed in T. thermophila and localizes to the cell surface [176]

- Excellent system for high-level expression of candidate vaccine antigens from parasites having direct relevance to human/animal health (most notably, malarial plasmodia).

NIAID

 

Table 15) Environmental adaptation and monitoring

 

Unique or special biological feature

Significance

Relevance to humans

Funding

Interest

- Axenic culture, synchronized cells, and short generation time allow sampling and processing of very homogenous eukaryotic cell populations

- Easy detection of growth inhibition by environmental toxicants [177]

- Acclimation to organic solvents is best described in bacteria and Tetrahymena [178;179]

- Use of Tetrahymena to determine Quantitative Structure-Activity Relationship (QSAR) for over 2,000 potentially toxic compounds released by industrial processes [177]

- Opportunity to serve as an efficient cellular biosensor, enhanceable by sequence-dependent expression-profiling technology

- Unique opportunity to study the mechanism of acclimation to organic solvents (and also to temperature), which are not well understood in any organism.

Protection of human/animal health and environment from toxic compounds generated industrially

- Surrogate for fish lethality-based tests [177]

NIEHS

EPA

DOE

NSF

Biotech

- Abundant source of stress proteins

- Highly inducible stress response to heavy metals leading to sequestration through bioaccumulation

- Possession of unusually Cys-rich conserved metallothioneins

- Specialized bioaccumulatory cell organelles

- Used as bioassay organism for heavy metal toxicity [180;181]

- Excellent unicellular animal model for investigating the biology of heavy metal stress

- Opportunity to genetically engineer robust metal resistance technology exportable to other organisms

- Opportunity to engineer Tetrahymena metallothioneins as molecular biosensors for heavy metal detection

Protection of human/animal health and environment from toxic compounds generated industrially

NIEHS

EPA

DOE

NSF

Biotech

- Efficient induction and recovery of recessive or dominant mutants; fast growth & phenotype testing of progeny

- Conserved animal biochemistry makes it likely that Tetrahymena metabolizes potential ecotoxicants in similar ways to humans and animals

Opportunity to serve as test system for ecotoxicity and genotoxicity assessment and monitoring

Protection of human/animal health and environment

NIEHS

DOE

EPA

NSF

Biotech

 

Table 16) Eukaryotic Evolution

 

Unique or special biological feature

Significance

Relevance to humans

Funding

Interest

Nonstandard nuclear genetic code UAR=gln. The same variant genetic code has arisen at least 7 times independently during evolution [182], including other ciliate lineages (e.g. hypotrichs) as well as diplomonads and algae

Wealth of genetic code variation has led to robust hypotheses regarding structure/function relationships in eukaryotic elongation factor eRF1 [183;184] which in Tetrahymena are amenable to experimental investigation

Some human pathogens have variant codes and antibiotics often target translation

NSF

NIGMS

NHGRI

Genealogical analyses of a limited number of protein coding genes from ciliates and other eukaryotes are discordant. 

Sequencing the Tetrahymena genome will be essential to gain an understanding of how ciliates, and ciliate genomes, are related to other eukaryotes, by comparing genealogies generated from multiple protein-coding genes and from combined data sets

Increase understanding of the evolutionary forces that have shaped eukaryotic genomes.

 

NSF

NIGMS

NHGRI

Discovery of possible relic chloroplast genes transferred to alveolate nucleus [185]

Ciliates may (surprisingly) have a photosynthetic ancestry and could even harbor a relic plastid, like that of Apicomplexa

Future therapies against related pathogenic alveolates (e.g. Plasmodium and Toxoplasma) could target those vestigial chloroplast proteins that exhibit higher sequence conservation

NSF

NIGMS

NHGRI

 


Table 17) Science education

Unique or special biological feature

Significance

Relevance to humans

Funding

Interest

- Very complete eukaryotic and animal genome

- Very high frequency of EST matches to human proteins

Excellent opportunity for:

- Undergraduate EST sequencing projects accompanied by blast searches for sequence similarity

- Student projects in information management and annotation, e.g., development and maintenance of searchable databases, cataloguing structural information, etc.

Fun and instructive way:

- To learn through the internet huge amounts, limited only by intellectual curiosity, of bioinformatics, cell biology, biochemistry, developmental biology, evolution, human ageing and genetic diseases

- To explore and appreciate different aspects of bioinformatics as important modern "ways of knowing" and their positive impact on various biological fields.

NSF

NHGRI

Genes can be readily predicted because introns are small & have distinct A+T composition, and no differential splicing or editing has been reported

Excellent opportunity for student projects to experimentally test gene predictions by PCR. Original research will often be involved here, as there will be literally thousands of genes to test.

Opportunity to experience the most elementary excitement of discovery through experiment (phenomena not previously reported in Tetrahymena or to complete or correct previous hypotheses

NSF

NHGRI

 

Table 18) Additional intriguing phenomena, not yet investigated molecularly. The following areas may well become accessible to investigation with the help of the genome sequence:

-          Signaling pathways: G-proteins, inositol phospholipid pathway components, protein kinases, adenylate cyclase/cAMP systems, guanylate cyclase/cGMP systems, calcium calmodulin systems, and nitric oxide systems.

-          Hormones and neurotransmitter activities: insulin, somatostatin, adrenocorticotrophin (ACTH), b endorphin, relaxin, vasotocin, calcitonin, serotonin, and adrenaline; physiological responses to insulin, histamine, and other mammalian signaling molecules.

Molecular investigations of these signaling observations have been hampered by the unavailability of the genome sequence of any free-living members of the Alveolate clade. The Tetrahymena genome sequence would quickly allow the identification of those phenomena that have a conserved basis. Experimental investigation could then contribute fundamental insights into their biological function and evolution and provide a unique opportunity to identify natural and synthetic pharmaceuticals that can act on these signal transduction systems.

 

3. Post-genomic resources preview

Additional sequence-dependent resources that rely on automation and high throughput technology will maximize the benefits of the Tetrahymena genomic sequence. While it is premature to seek funding for those resources now, it is important to list those concrete post-genomic needs already identified by the on-going work of research community.

·                     Maintenance of the Tetrahymena Database and expansion to include genomic data from other ciliates.

·                     Proteomics of purified cell organelles, with high priority for those where Tetrahymena can make a difference quickly: cilia and basal bodies (centriole homologs), phagosomes, secretory vesicles and nucleoli.

·                     Expression profiling: Microarrays or SAGE analysis -- to replace current laborious and intrinsically incomplete approaches based on differential display or random cloning from subtracted DNA libraries.

·                     Limited WGS sequencing of purified MIC DNA -- in order to map sites of developmental, immunoglobulin-gene-like, internal DNA deletion with respect to the finished MAC sequence.

·                     Systematic knockout and phenotype determination for known or predicted Tetrahymena genes, with highest priority for a) genes, identified by sequence similarity or proteomic approaches, involved in important mechanisms under active investigation and b) genes for proteins of unknown function but with disease potential sharing sequence similarity with humans and not yeast.

Genomic technology advances rapidly and better approaches may be available when the whole-genome sequencing project has been completed.

 

4. Acknowledgments

Steering Committee for the Tetrahymena Genome Project:

·                     Ed Orias, UC Santa Barbara, Coordinator

·                     David Asai, Purdue University

·                     Cliff Brunk, UCLA

·                     Peter Bruns, Howard Hughes Medical Institute

·                     Ted Clark, Cornell University

·                     Jean Cohen, CNRS, France

·                     Kathy Collins, UC Berkeley

·                     Jacek Gaertig, University of Georgia

·                     Marty Gorovsky, University of Rochester

·                     Carolyn Jahn, Northwestern University

·                     Jeff Kapler, Texas A&M University

·                     Kathy Karrer, Marquette University

·                     Larry Klobutcher, University of Connecticut

·                     Ching Kung, University of Wisconsin

·                     Laura Landweber, Princeton University

·                     Ron Pearlman, York University, Toronto, Canada

·                     Linda Sperling, CNRS, France

·                     Aaron Turkewitz, University of Chicago

 

Additional contributors to the concept paper:

·                     Thomas Cech, University of Colorado and Howard Hughes Medical Institute

·                     Eric Cole, St Olaf College

·                     Robert S. Coyne, University of Virginia Medical School

·                     Paul Doerder, Cleveland State University

·                     Joseph Frankel, University of Iowa

·                     Juan Carlos Gutierrez, Universidad Complutense, Madrid

·                     Todd Hennessey, SUNY Buffalo

·                     Piroska Huvos, Southern Illinois University

·                     Robert S. Jacobs, UC Santa Barbara

·                     Andrzej Kaczanowski, Warsaw University, Poland

·                     Laura A. Katz, Smith College

·                     Denis Lynn, University of Guelph, Canada

·                     Terry W.  Schultz, University of Tennessee

·                     Arno Tiedtke, Muenster University, Germany

·                     Emily Wiley, Mount Holyoke College

 

We are very grateful to Anthony Carter, NIGMS, for helping us understand the NMMC process; William Nierman (TIGR), Bruce Birren (Whitehead Institute), Maynard Olson (University of Washington), Bruce Roe (University of Oklahoma) and Nathan Bach (Integrated Genomics Inc.), who provided us with detailed estimates of sequencing, closure and annotation costs; and J. Michael Cherry (SGD and Stanford University), William Gelbart (Flybase and Harvard University) and Lincoln Stein (Wormbase and Cold Spring Harbor Laboratory), who helped us estimate database needs and costs.