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Explanation of molecular
genetic tools dependent on unique Tetrahymena features |
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Table 1 |
Telomeres and telomerase
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Table 2 |
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Table 3 |
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Table 4 |
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Table 5 |
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Table 6 |
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Table 7 |
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Table 8 |
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Table 9 |
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Table 10 |
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Table 11 |
9 |
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Table 12 |
9 |
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Table 13 |
10 |
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Table 14 |
10 |
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Table 15 |
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Table 16 |
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Table 17 |
13 |
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Table 18 |
13 |
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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.
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 |
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 |
- 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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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.
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
·
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.