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Marisa D Ruehle, Eduardo Orias, Chad G Pearson, Tetrahymena as a Unicellular Model Eukaryote: Genetic and Genomic Tools, Genetics, Volume 203, Issue 2, 1 June 2016, Pages 649–665, https://doi.org/10.1534/genetics.114.169748
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Abstract
Tetrahymena thermophila is a ciliate model organism whose study has led to important discoveries and insights into both conserved and divergent biological processes. In this review, we describe the tools for the use of Tetrahymena as a model eukaryote, including an overview of its life cycle, orientation to its evolutionary roots, and methodological approaches to forward and reverse genetics. Recent genomic tools have expanded Tetrahymena’s utility as a genetic model system. With the unique advantages that Tetrahymena provide, we argue that it will continue to be a model organism of choice.
GENETIC model systems have a long-standing history as important tools to discover novel genes and processes in cell and developmental biology. The ciliate Tetrahymena thermophila is a model system that combines the power of forward and reverse genetics with a suite of useful biochemical and cell biological attributes. Moreover, Tetrahymena are evolutionarily divergent from the commonly studied organisms in the opisthokont lineage, permitting examination of both unique and universally conserved biological processes. Here we highlight the advantages of Tetrahymena as a model system, such as its unique and easily manipulated life cycle, that have contributed to important discoveries. The growing suite of molecular-genetic and genomic tools described here provides a system to couple gene discovery to mechanistic dissections of gene function in the cell.
T. thermophila (Figure 1) is a ciliate model organism whose study has led to fundamental biological insights covering the central dogma and beyond. Indeed, this single-celled, motile eukaryote provides the tools and techniques not only for novel gene discovery, but also for unveiling the important molecular mechanisms behind those genes’ functions. As such, Tetrahymena’s utility as a genetic model organism is revealed by its short life cycle, easy and cost-effective laboratory handling, and its accessibility to both forward and reverse genetics. Despite its affectionate reference as “pond scum” (Blackburn 2010), the beauty of Tetrahymena as a genetic model organism is displayed in many lights.
Tetrahymena has a long and distinguished history in the discovery of broad biological paradigms (Figure 2), beginning with the discovery of the first microtubule motor, dynein (Gibbons and Rowe 1965). Others include the Nobel Prize winning discoveries of catalytic RNA (Kruger et al. 1982) and telomere structure and telomerase (Greider and Blackburn 1985). The first histone-modifying enzyme (histone acetyl transferase) and its role as a transcription factor were discovered in Tetrahymena (Brownell et al. 1996), which gave birth to the “histone code” and the field of epigenetic control of gene expression by chromatin modification. The role of small interfering RNAs in heterochromatin formation and the massive, programmed excision of transposon-related DNA from the somatic genome (Mochizuki et al. 2002; Taverna et al. 2002) is another major Tetrahymena contribution. These fundamental discoveries in Tetrahymena have helped usher in the modern era of molecular and cellular biology. Many of the reasons why Tetrahymena was an advantageous system for these groundbreaking discoveries are the same that make Tetrahymena useful today and in the future, augmented by the ever-expanding toolkit available to Tetrahymena researchers.
In this review, we discuss major biological questions to which T. thermophila is amenable and the genetic tools available to answer them. It begins with the general biology of Tetrahymena and its unique advantages as an experimental model system. We then describe both forward and reverse genetic strategies in Tetrahymena to facilitate gene discovery and to interrogate the mechanistic underpinnings of those genes. Ultimately, we seek to engage future researchers by describing the wealth of experimental advantages (both historical and modern) that Tetrahymena can provide and preview its promising future.
Tetrahymena Biology
Tetrahymena are unicellular, ciliated eukaryotes that live in fresh water over a wide range of conditions. In the wild, Tetrahymena feed on bacteria, but laboratory strains typically live as axenic cultures in nutrient-rich media (derived from tissue extracts) or chemically defined media. Populations grow quickly, with cells dividing every 2–3 hr under optimal conditions. Each cell is large, 30–50 μm in length, making them ideal for light and electron microscopy-based investigations.
Tetrahymena, like all ciliates, are nuclear dimorphic, which means that the germline genome and the somatic genome exist within two separate nuclei in one cell: the micronucleus (MIC) and the macronucleus (MAC), respectively (Table 1, Table 2, and Figure 1). The diploid MIC genome consists of five pairs of chromosomes, whereas the MAC genome consists of ∼200 different chromosomes, each maintained at a ploidy of ∼45 during the G1 phase of the cell cycle. The MIC is transcriptionally silent, and all gene expression is driven from the MAC DNA. Despite the fact that Tetrahymena are unicellular organisms, this germline/soma separation is reminiscent of metazoans where distinct germ cells and somatic cells are maintained. Germline/soma separation is extremely rare among unicellular eukaryotes, though it is also found in certain Foraminifera (in the Rhizarian evolutionary group).
Tetrahymena thermophila glossary
Term . | Definition . |
---|---|
Chromosomal breakage site (CBS) | MIC chromosome location occupied by the highly conserved 15-bp chromosome breakage sequence, which is the necessary and sufficient DNA element for programmed chromosome fragmentation during MAC development. |
Cytogamy | A mating strategy in which gamete pronucleus exchange between conjugants is blocked; the two sister gamete pronuclei in each conjugant instead fuse to one another (double self-fertilization), generating whole-genome homozygous fertilization nuclei. The postzygotic events are normal. |
Fertilization nucleus | The nucleus that forms as a result of gamete pronuclear fusion during conjugation. In Figure 3F the fertilization nuclei are depicted as half white, half black small circles in each conjugant cell. Also referred to as the zygote nucleus. |
Genomic exclusion | A self-fertilization mating strategy to generate a whole-genome homozygote that uses a cross to a strain with a defective MIC (star strain). |
Heterokaryon | A cell in which the MIC and MAC genotypes are different. |
Homokaryon | A cell in which the MIC and MAC genotypes are the same. |
Karyonides | The four cells formed by the first fission of the two exconjugants of a pair, represented in Figure 3J. Each karyonide acquires an independently differentiated new MAC. |
MAC | Macronucleus. |
MAC anlagen | The newly developing MACs mitotically derived from zygote nucleus during the postzygotic steps of conjugation. |
MAC KO | Gene knockout in the macronucleus. |
MAC maturation or development | The process, during conjugation, whereby an initially diploid MAC anlage completes its structural and functional differentiation from the MIC. |
Meiotic segregant | A sexual progeny cell whose MIC and MAC are derived from meiotic products of a double heterozygote during conjugation; its genotype is classified as parental or recombinant. |
MIC | Micronucleus. |
MIC KO | Gene knockout in the micronucleus. |
Nullisomic | A cell whose MIC lacks (is null for) both copies of an entire chromosome or chromosome arm. Such strains show no phenotype when they are “covered” by a diploid MAC. |
Phenotypic assortment | The phenomenon in which a MAC initially heterozygous for an allelic variant becomes homozygous through successive rounds of amitosis during vegetative multiplication. |
Postzygotic mitoses | The two divisions of the fertilization nucleus that generate the two MIC and MAC anlagen during conjugation. |
Prezygotic mitosis | Division of the surviving haploid meiotic product that generates the migratory and stationary gamete pronuclei during conjugation. |
Segmental deletion | Loss of a large MIC chromosome segment. Deletion homozygotes show no phenotype when they are covered by a diploid MAC. |
Star strain | A cell having a severely defective MIC, which fails to generate any meiotic products during conjugation. |
Uniparental cytogamy (UPC) | A mating strategy to generate a whole-genome homozygote by inducing self-fertilization in a cross between a normal and a star strain. The exconjugant derived from the star strain dies. |
Term . | Definition . |
---|---|
Chromosomal breakage site (CBS) | MIC chromosome location occupied by the highly conserved 15-bp chromosome breakage sequence, which is the necessary and sufficient DNA element for programmed chromosome fragmentation during MAC development. |
Cytogamy | A mating strategy in which gamete pronucleus exchange between conjugants is blocked; the two sister gamete pronuclei in each conjugant instead fuse to one another (double self-fertilization), generating whole-genome homozygous fertilization nuclei. The postzygotic events are normal. |
Fertilization nucleus | The nucleus that forms as a result of gamete pronuclear fusion during conjugation. In Figure 3F the fertilization nuclei are depicted as half white, half black small circles in each conjugant cell. Also referred to as the zygote nucleus. |
Genomic exclusion | A self-fertilization mating strategy to generate a whole-genome homozygote that uses a cross to a strain with a defective MIC (star strain). |
Heterokaryon | A cell in which the MIC and MAC genotypes are different. |
Homokaryon | A cell in which the MIC and MAC genotypes are the same. |
Karyonides | The four cells formed by the first fission of the two exconjugants of a pair, represented in Figure 3J. Each karyonide acquires an independently differentiated new MAC. |
MAC | Macronucleus. |
MAC anlagen | The newly developing MACs mitotically derived from zygote nucleus during the postzygotic steps of conjugation. |
MAC KO | Gene knockout in the macronucleus. |
MAC maturation or development | The process, during conjugation, whereby an initially diploid MAC anlage completes its structural and functional differentiation from the MIC. |
Meiotic segregant | A sexual progeny cell whose MIC and MAC are derived from meiotic products of a double heterozygote during conjugation; its genotype is classified as parental or recombinant. |
MIC | Micronucleus. |
MIC KO | Gene knockout in the micronucleus. |
Nullisomic | A cell whose MIC lacks (is null for) both copies of an entire chromosome or chromosome arm. Such strains show no phenotype when they are “covered” by a diploid MAC. |
Phenotypic assortment | The phenomenon in which a MAC initially heterozygous for an allelic variant becomes homozygous through successive rounds of amitosis during vegetative multiplication. |
Postzygotic mitoses | The two divisions of the fertilization nucleus that generate the two MIC and MAC anlagen during conjugation. |
Prezygotic mitosis | Division of the surviving haploid meiotic product that generates the migratory and stationary gamete pronuclei during conjugation. |
Segmental deletion | Loss of a large MIC chromosome segment. Deletion homozygotes show no phenotype when they are covered by a diploid MAC. |
Star strain | A cell having a severely defective MIC, which fails to generate any meiotic products during conjugation. |
Uniparental cytogamy (UPC) | A mating strategy to generate a whole-genome homozygote by inducing self-fertilization in a cross between a normal and a star strain. The exconjugant derived from the star strain dies. |
Term . | Definition . |
---|---|
Chromosomal breakage site (CBS) | MIC chromosome location occupied by the highly conserved 15-bp chromosome breakage sequence, which is the necessary and sufficient DNA element for programmed chromosome fragmentation during MAC development. |
Cytogamy | A mating strategy in which gamete pronucleus exchange between conjugants is blocked; the two sister gamete pronuclei in each conjugant instead fuse to one another (double self-fertilization), generating whole-genome homozygous fertilization nuclei. The postzygotic events are normal. |
Fertilization nucleus | The nucleus that forms as a result of gamete pronuclear fusion during conjugation. In Figure 3F the fertilization nuclei are depicted as half white, half black small circles in each conjugant cell. Also referred to as the zygote nucleus. |
Genomic exclusion | A self-fertilization mating strategy to generate a whole-genome homozygote that uses a cross to a strain with a defective MIC (star strain). |
Heterokaryon | A cell in which the MIC and MAC genotypes are different. |
Homokaryon | A cell in which the MIC and MAC genotypes are the same. |
Karyonides | The four cells formed by the first fission of the two exconjugants of a pair, represented in Figure 3J. Each karyonide acquires an independently differentiated new MAC. |
MAC | Macronucleus. |
MAC anlagen | The newly developing MACs mitotically derived from zygote nucleus during the postzygotic steps of conjugation. |
MAC KO | Gene knockout in the macronucleus. |
MAC maturation or development | The process, during conjugation, whereby an initially diploid MAC anlage completes its structural and functional differentiation from the MIC. |
Meiotic segregant | A sexual progeny cell whose MIC and MAC are derived from meiotic products of a double heterozygote during conjugation; its genotype is classified as parental or recombinant. |
MIC | Micronucleus. |
MIC KO | Gene knockout in the micronucleus. |
Nullisomic | A cell whose MIC lacks (is null for) both copies of an entire chromosome or chromosome arm. Such strains show no phenotype when they are “covered” by a diploid MAC. |
Phenotypic assortment | The phenomenon in which a MAC initially heterozygous for an allelic variant becomes homozygous through successive rounds of amitosis during vegetative multiplication. |
Postzygotic mitoses | The two divisions of the fertilization nucleus that generate the two MIC and MAC anlagen during conjugation. |
Prezygotic mitosis | Division of the surviving haploid meiotic product that generates the migratory and stationary gamete pronuclei during conjugation. |
Segmental deletion | Loss of a large MIC chromosome segment. Deletion homozygotes show no phenotype when they are covered by a diploid MAC. |
Star strain | A cell having a severely defective MIC, which fails to generate any meiotic products during conjugation. |
Uniparental cytogamy (UPC) | A mating strategy to generate a whole-genome homozygote by inducing self-fertilization in a cross between a normal and a star strain. The exconjugant derived from the star strain dies. |
Term . | Definition . |
---|---|
Chromosomal breakage site (CBS) | MIC chromosome location occupied by the highly conserved 15-bp chromosome breakage sequence, which is the necessary and sufficient DNA element for programmed chromosome fragmentation during MAC development. |
Cytogamy | A mating strategy in which gamete pronucleus exchange between conjugants is blocked; the two sister gamete pronuclei in each conjugant instead fuse to one another (double self-fertilization), generating whole-genome homozygous fertilization nuclei. The postzygotic events are normal. |
Fertilization nucleus | The nucleus that forms as a result of gamete pronuclear fusion during conjugation. In Figure 3F the fertilization nuclei are depicted as half white, half black small circles in each conjugant cell. Also referred to as the zygote nucleus. |
Genomic exclusion | A self-fertilization mating strategy to generate a whole-genome homozygote that uses a cross to a strain with a defective MIC (star strain). |
Heterokaryon | A cell in which the MIC and MAC genotypes are different. |
Homokaryon | A cell in which the MIC and MAC genotypes are the same. |
Karyonides | The four cells formed by the first fission of the two exconjugants of a pair, represented in Figure 3J. Each karyonide acquires an independently differentiated new MAC. |
MAC | Macronucleus. |
MAC anlagen | The newly developing MACs mitotically derived from zygote nucleus during the postzygotic steps of conjugation. |
MAC KO | Gene knockout in the macronucleus. |
MAC maturation or development | The process, during conjugation, whereby an initially diploid MAC anlage completes its structural and functional differentiation from the MIC. |
Meiotic segregant | A sexual progeny cell whose MIC and MAC are derived from meiotic products of a double heterozygote during conjugation; its genotype is classified as parental or recombinant. |
MIC | Micronucleus. |
MIC KO | Gene knockout in the micronucleus. |
Nullisomic | A cell whose MIC lacks (is null for) both copies of an entire chromosome or chromosome arm. Such strains show no phenotype when they are “covered” by a diploid MAC. |
Phenotypic assortment | The phenomenon in which a MAC initially heterozygous for an allelic variant becomes homozygous through successive rounds of amitosis during vegetative multiplication. |
Postzygotic mitoses | The two divisions of the fertilization nucleus that generate the two MIC and MAC anlagen during conjugation. |
Prezygotic mitosis | Division of the surviving haploid meiotic product that generates the migratory and stationary gamete pronuclei during conjugation. |
Segmental deletion | Loss of a large MIC chromosome segment. Deletion homozygotes show no phenotype when they are covered by a diploid MAC. |
Star strain | A cell having a severely defective MIC, which fails to generate any meiotic products during conjugation. |
Uniparental cytogamy (UPC) | A mating strategy to generate a whole-genome homozygote by inducing self-fertilization in a cross between a normal and a star strain. The exconjugant derived from the star strain dies. |
Genome statistics
To replicate these distinct genomes and to maintain genetic diversity, the T. thermophila life cycle employs both asexual, vegetative cell division and sexual reorganization through a process called conjugation. During vegetative division, the MAC divides amitotically (random chromosome segregation), the MIC divides mitotically, and binary cell fission produces two daughter cells. However, under conditions of starvation, pairs of sexually mature cells with different mating types undergo conjugation. T. thermophila cells can have one of seven different mating types, and each mating type can conjugate with another mating type, but not with itself (Elliott and Gruchy 1952; Nanney and Caughey 1953). Conjugation involves highly conserved eukaryotic processes: meiosis, gametogenesis, and gamete nucleus fusion to form the fertilization (or “zygote”) nuclei of progeny cells (Figure 3). The fertilization nucleus divides mitotically and then differentiates into a new MIC as well as a new MAC, and the parental MAC is eliminated. Conjugation has important consequences: Mendelian genetic inheritance and increasing genotypic diversity. The genome’s maintenance and expression depend on widely conserved mechanisms and pathways across all lineages of life, making Tetrahymena a useful organism in which to study these universal processes.
Evolutionary context
As ciliates, Tetrahymena are a part of the Alveolate group of the Stramenopile–Alveolate–Rhizarian (SAR) lineage (Figure 4). Dinoflagellates and apicomplexans are also a part of the Alveolate group, but are more closely related to each other than to the ciliates. Thus, there is a large evolutionary distance from ciliates to their sister Alveolate groups, and an even greater distance to other commonly used eukaryotic model organisms which, like humans, are a part of the opisthokont group (yeast, worms, flies, mice, and many others). There is a noticeable dearth of model organisms outside of the opisthokont group, making research of nonopisthokonts such as Tetrahymena an attractive means to explore the remarkable diversity of eukaryotic cell biology (Lynch et al. 2014). Nevertheless, Tetrahymena utilize many universally conserved eukaryotic processes, making them also useful for illuminating these conserved features (Briguglio and Turkewitz 2014; Lynch et al. 2014).
Tetrahymena’s closest model organism relative (though not its closest relative outright) is Paramecium, which, along with Tetrahymena, is a part of the Oligohymenophorea class (Figure 4, bottom). However, based on deviations in small subunit ribosomal RNA (rRNA) sequences between Paramecium and Tetrahymena, the two likely shared a common ancestor several hundred million years ago and their evolutionary distance exceeds that between rat and brine shrimp (Greenwood et al. 1991; Frankel 2000). Thus, while much of the biology of Tetrahymena has been illuminated by studies in Paramecium and vice versa, differences between them are expected and often observed (Frankel 2000).
T. thermophila belongs to a group of Tetrahymena species that are morphologically indistinguishable from one another. These species were originally lumped together as a single species called T. pyriformis. The discovery of Tetrahymena mating types allowed dissection of the group based on sexual isolation and led to the recognition of T. thermophila and other sibling species as distinct Tetrahymena species. Along the way, T. thermophila was first called T. pyriformis variety 1 and later T. pyriformis syngen 1, before receiving its current name (Nanney and McCoy 1976). Isogenic, inbred lines of T. thermophila have been derived (Allen 1967). By convention, most genetic analysis and genome sequencing has used T. thermophila inbred strain B cells, while most genetic mapping has utilized DNA polymorphisms between inbred strains B and C3.
The sibling Tetrahymena species found in the original T. pyriformis group provide an invaluable resource for comparative genomic analyses, as evolutionary DNA sequence conservation is a useful predictor of biological function under natural selection (Romero and Blackburn 1991; Coyne and Yao 1996). It is particularly useful for the detection of lineage-specific genes of unknown function and to identify conserved DNA and protein sequences within genes.
Lineage-specific gene family expansions are interesting properties of any sequenced genome, as they can provide clues to the importance of particular cellular processes. In T. thermophila, gene family expansion has occurred mainly by localized gene duplication (Eisen et al. 2006). This is in contrast to Paramecium tetraurelia where two recent whole-genome duplications (WGDs) occurred after the divergence from the Tetrahymenine ciliates contributing to its gene family expansion (Aury et al. 2006). It has been suggested that an older WGD, clearly detectable in the Paramecium genome occurred prior to the divergence of Paramecium and Tetrahymena (Aury et al. 2006). However, no remaining evidence of a WGD in the Tetrahymena genome has yet been found. Major gene family expansions in the Tetrahymena genome involve genes whose products function in “sensing and responding to environmental cues (e.g. protein kinases, membrane transporters), mobilizing resources from the environment (e.g. membrane transporters and traffic components, proteases), and maintaining complex cell structure and movement (e.g. microtubule components, motors, and regulators, membrane traffic components)” (Eisen et al. 2006). The function of closely related gene family members can be difficult to dissect experimentally because of redundancy or significant functional similarity.
Ciliates have evolved lineage-specific changes in the nuclear genetic code. Tetrahymena uses only a single codon (UGA) to terminate MAC-encoded protein synthesis. The other stop codons in the universal genetic code, UAA and UAG, encode glutamine amino acids, along with the canonical glutamine codons, CAA and CAG. While the variant genetic code poses no special problem when expressing heterologous genes in Tetrahymena, the expression of Tetrahymena protein coding genes in heterologous systems requires first mutating UAA and UAG codons to either CAA or CAG. This hurdle, as well as that of optimizing codon usage (described later), is generally addressed by gene synthesis.
Two nuclei in one cell
Tetrahymena cells have a MIC that houses the germline genome, and a MAC that contains the somatic genome. Although the separation of germline and soma sounds familiar to us metazoans, the presence of two different nuclear genomes in the same cell is foreign. How does the MAC develop from the MIC, how are they similar or different, and what experimental advantages does nuclear dimorphism confer?
The diploid MIC genome consists of five pairs of chromosomes and its size is estimated to total ∼220 Mb of DNA (Table 2) (Yao and Gorovsky 1974). When the newly forming MAC (MAC anlagen) develops during conjugation (Figure 3, H and I) these 10 chromosomes are fragmented at specific sites called chromosomal breakage sequences (CBSs) and they lose some internal sequences (Yao et al. 1987), collectively referred to as internal eliminated sequences (IESs). Fragmentation and DNA elimination generates many new, smaller chromosomes. The CBS is a conserved 15-bp motif, which is necessary and sufficient for DNA breakage (Yao et al. 1987). Telomeric repeat addition occurs at the ends of the new chromosomes. The locations of IESs are marked through an elegant small RNA-dependent pathway, which ensures that these segments of DNA are not incorporated into the MAC genome (Mochizuki et al. 2002; Duharcourt et al. 2009; Fass et al. 2011; Schoeberl et al. 2012). Most of the eliminated sequences are repetitive elements likely derived from transposons (genomic parasites that randomly insert in the genome, causing deleterious genomic instability if not silenced). Thus, eliminating these sequences from the MAC protects the expressed genome of the organism. The newly generated MAC chromosomes are then endoreplicated (replicated multiple times without nuclear division), leading to the production of ∼45 copies of each chromosome per MAC. A unique and important exception to this copy number is the chromosome that encodes the 35S rRNA precursor gene (Tetrahymena homolog of the 45S rRNA precursor gene in other eukaryotes). This is the smallest MAC chromosome (∼20 kb) and is present at ∼9000 copies per MAC.
Mating type determination in the sexual progeny occurs during MAC development (Figure 3, H and I). It occurs in each of the four genetically identical developing MACs of a conjugating pair independently of one another or of the parental mating types. This is because mating type determination is based on stochastically selected, alternative DNA rearrangements at the mating type locus of the developing MAC (Cervantes et al. 2013). This process is unrelated to chromosome fragmentation or IES excision. A cell’s mating type is faithfully inherited during vegetative division. Progeny cells can mate only after a period of sexual immaturity that lasts 40–70 cell divisions after conjugation. The molecular basis of this maturation is unknown.
Two genetic systems in one cell
The organization and behavior of the germline vs. the somatic nuclei define two types of genetic systems at work in the life of every cell. The diploid MIC lineage undergoes meiosis and fertilization to bestow Mendelian genetic inheritance across sexual generations. On the other hand, during vegetative cell division, the MAC, with fragmented chromosomes that undergo amitotic distribution, generates a distinct genetic phenomenon called phenotypic assortment. This is akin to the genetics of high copy number bacterial plasmids. The interplay of these two genetic systems generates natural versatility that can be exploited to great experimental advantage in the laboratory. Because the less familiar genetic phenomena associated with amitotic division are critical to mutational and other genetic approaches, they are examined in more detail in the next section.
Amitosis and phenotypic assortment
During vegetative S phase, MAC DNA is replicated once (Andersen and Zeuthen 1971) and then is distributed to daughter MACs by amitosis. In this process, the MAC chromosomes are not equally segregated to daughter cells; rather chromosome segregation is stochastic. Familiar features of mitosis in animals and plants, such as bipolar spindle formation, extreme chromosome condensation, and microtubule attachment to centromeres, are all missing during MAC division; thus the kinetochore-based, equal chromatid separation in normal mitosis does not play a role in MAC division (Flickinger 1965; Cervantes et al. 2006). Microtubules are nonetheless required during amitosis to elongate the MAC before it divides (Williams and Williams 1976; Kushida et al. 2011). The molecular mechanisms that govern amitosis remain to be elucidated.
Because amitosis does not involve the high-fidelity, equal segregation of sister chromatids into daughter cells as in mitosis, a phenomenon called phenotypic assortment is observed during Tetrahymena vegetative growth (Figure 5A) (Allen and Nanney 1958; Doerder et al. 1975; Orias and Flacks 1975; Nanney and Preparata 1979). Phenotypic assortment is the ability of a heterozygous MAC to come to homozygosity or lead to fixation of either allele. This occurs over multiple rounds of amitosis as a result of unequal chromosome segregation during MAC division (Figure 5B). Daughter cells stochastically receive a greater or lesser number of copies of an allele, and MACs ultimately become pure for either allele during subsequent cell divisions (Orias and Flacks 1975; Nanney and Preparata 1979; Merriam and Bruns 1988). The steady-state rate of assortment is mathematically represented as 1 / (2n − 1) per fission (Schensted 1958), where n is the number of chromosome copies just after MAC division (45 in this case). In the wild, this phenomenon permits rapid somatic adaptation to selective pressures, and in the lab, it is useful for isolating mutations or phenotypes of interest. Despite the random assortment of chromosomes in the MAC, a mechanism to control chromosome copy number has been inferred (Doerder and DeBault 1978), although its molecular basis remains unknown.
The mating type locus is already present in multiple copies at the time of mating type determination in the developing MAC. Therefore, more than one mating type can be expressed in a progeny cell line immediately after conjugation. This condition is typically resolved by the time the mixed mating type progeny cells reach sexual maturity, when most cells have become fixed for a single mating type by phenotypic assortment. Sexually mature cells whose MAC remain mixed are called “selfers,” because they can pair with other members of the same clone when starved. If undetected, selfers can present a challenge to downstream experiments that require mating. If it is absolutely necessary to work with a selfer line, additional vegetative cell divisions permit the completion of phenotypic assortment and allow mating type to become fixed in a subclonal population of these cells.
The remarkable genetic features made possible by germline/soma differentiation in Tetrahymena provide a variety of experimental advantages. First, recessive mutations within a heterogeneous MAC can ultimately come to complete expression because phenotypic assortment will enhance the number of recessive mutant alleles and reduce the number of dominant wild-type alleles in a subset of the progeny. Second, whole-genome homozygotes can be generated quickly and easily by self-fertilization methods. As a consequence, recessive mutations present in the MIC can be isolated as efficiently as if the germline were haploid and are expressed in the MAC without assortment. Third, production of heterokaryon cells (cells whose MIC and MAC genotypes are different; Figure 6) allow lethal mutations to be maintained in the MIC during vegetative growth until induction of mating. Similarly, nullisomics and segmental deletion homozygotes can be maintained as heterokaryons, which allow for the physical mapping of mutations, DNA polymorphisms, or MIC-limited DNA elements, such as CBSs. These are advantages that Tetrahymena researchers commonly exploit to identify and study genes of interest.
Why Use Tetrahymena as a Genetic Model Organism?
Model systems are typically chosen based on their utility in identifying new avenues of research or in contributing to the mechanistic understanding of important biological problems. Tetrahymena have the advantages of a “do-it-all” model system; this is especially true as a genetic model system. Tetrahymena grow fast (∼2- to 3-hr doubling time), can undergo large scale and synchronous matings, genetic markers have been mapped, and its two nuclear genomes sequenced. The germline MIC exhibits Mendelian genetics, and drug resistance markers exist for positive selection. This suite of genetic advantages yields an effective model system to complement its powerful biochemical and cytological attributes. Indeed, both forward and reverse genetic tools are well-developed in Tetrahymena. Furthermore, the National Tetrahymena Stock Center, located at Cornell University, maintains and readily makes available many cell lines as well as plasmids, protocols, and other resources to facilitate these studies.
Forward and reverse genetics
Forward genetic strategies have been useful for generating Tetrahymena mutants. Mutagenesis screens with accompanying phenotypic screens have identified defects in biological processes including cortical patterning, metabolism, motility, cell cycle progression, RNA positioning, drug resistance, phagocytosis, and exocytosis (Bruns and Sanford 1978; Ahmed et al. 1998; Bowman et al. 2005). Such mutations can then be mapped to the genome. It was previously difficult to identify the mutations associated with a phenotype due to difficulties in producing genome-wide libraries to rescue mutant phenotypes. However, next generation sequencing (NGS) is now an effective tool to rapidly identify mutations in Tetrahymena and in other genomes (Galati et al. 2014; Schneeberger 2014). The development of NGS to identify mutations created in Tetrahymena forward genetic screens greatly increases the power of this system for new mechanistic research.
To study the functions of known genes, Tetrahymena is also amenable to reverse genetic strategies by the introduction of precisely targeted and heritable gene knockouts (KOs) and disruptions. Moreover, potentially deleterious genomic knockouts and gene mutations can be introduced into and propagated in the germline while maintaining a normal MAC genome. Thus, cells may propagate mutations in essential genes without associated phenotypes. To then express the mutant phenotype, genetically identical cells are mated to one another and all progeny will express the mutation of interest. These strategies allow for sophisticated reverse genetic studies.
Advantages to repetitive elements
An important advantage to Tetrahymena is their large numbers of repeating structural elements, such as the repeating cortical architecture composed of polarized individual ciliary units that are essential for cellular motility (Figure 1). This is a defining feature of ciliate cellular organization. Motility is a simple readout for experiments relating to cortical architecture, requiring minimal technological tools. Additionally, clathrin-dependent endocytosis is organized in an array at the cell cortex and is readily studied in Tetrahymena (Elde et al. 2005). The relatively large Tetrahymena cells (∼20 × 50 μm) with repeating elements enables rapid and high-quality cytological work using both light and electron microscopy and provides copious material for biochemical and structural studies of these repeating elements.
The Tetrahymena community has developed molecular tools and microscopy techniques to visualize Tetrahymena’s diverse cellular processes. Protein tags allow for biochemical analyses and live and fixed cell visualization of protein localization and cellular structure. The regular, crystal-like repetition of structural elements enables signal amplification in these localization studies, resulting in rapid and high-resolution detection of cortical architecture using light and electron microscopy.
In silico toolbox
Informatics tools exist to stimulate the study of the Tetrahymena genome and its expression (Table 3). These include the MIC and MAC genome sequences from the Broad Institute and The Institute for Genome Research (TIGR: now the J. Craig Venter Institute), respectively, a list of annotated genes, transcriptome-wide RNA sequencing (RNA-seq) data at different life cycle stages (http://tfgd.ihb.ac.cn/), comparative genomes of organisms closely related to T. thermophila, and a community database (TGD wiki; ciliate.org) that compiles these data into a user-friendly, searchable format. Researchers new to the field will appreciate the community-annotated genome page where genes are assigned with Gene Ontology (GO) terms, relevant literature references, and links to GenBank entries. The site also maintains a “Textpresso for Tetrahymena” function for full text searches. The combination of genomic resources with the genetic power of the organism and its accompanying molecular and biochemical tools, primes Tetrahymena for elucidating additional fundamental biological processes.
In silico toolbox and other resources
Resource . | Source . | Location . | Reference . |
---|---|---|---|
Genomics resources | |||
Genome browser | TGD: Tetrahymena genome database | http://www.Ciliate.org | Stover et al. 2006 |
NCBI genome | http://www.ncbi.nlm.nih.gov/genome/222 | Eisen et al. 2006 | |
Functional genomics | TetraFGD: Tetrahymena Functional Genomics Database | http://tfgd.ihb.ac.cn/ | Xiong et al. 2013 |
Experimental resources | |||
Strains | Tetrahymena stock center | https://tetrahymena.vet.cornell.edu/ | Cassidy-Hanley 2012 |
Methods resources | |||
Asai D. J. and J. D. Forney (eds.) 1999 Tetrahymena thermophila. Methods in Cell Biology, vol. 62 | |||
Collins K. (ed.) 2012 Tetrahymena thermophila, 1st edition. Methods in Cell Biology, vol. 109 | |||
Teaching resources | |||
ASSET teaching resource | ASSET: Advancing secondary science education thru Tetrahymena | https://tetrahymenaasset.vet.cornell.edu/ | Smith et al. 2012 |
Tetrahymena SUPRDB | SUPRDB: Student/Unpublished Results | http://suprdb.org/ | Wiley and Stover 2014 |
Ciliate genomics Consortium | Community/Wiley | http://faculty.jsd.claremont.edu/ewiley/ | |
Biotechnology resources | |||
Cilian | http://www.cilian.de/ (website in German) | ||
Tetragenetics | http://www.tetragenetics.com/ | ||
Tetratox | http://www.vet.utk.edu/TETRATOX/index.php | Schultz 1997 | |
Additional resources | |||
Tetrahymena history | http://www.life.illinois.edu/nanney/index.html | ||
Community listserv | https://listserv.uga.edu/cgi-bin/wa?A0=ciliatemolbio-l |
Resource . | Source . | Location . | Reference . |
---|---|---|---|
Genomics resources | |||
Genome browser | TGD: Tetrahymena genome database | http://www.Ciliate.org | Stover et al. 2006 |
NCBI genome | http://www.ncbi.nlm.nih.gov/genome/222 | Eisen et al. 2006 | |
Functional genomics | TetraFGD: Tetrahymena Functional Genomics Database | http://tfgd.ihb.ac.cn/ | Xiong et al. 2013 |
Experimental resources | |||
Strains | Tetrahymena stock center | https://tetrahymena.vet.cornell.edu/ | Cassidy-Hanley 2012 |
Methods resources | |||
Asai D. J. and J. D. Forney (eds.) 1999 Tetrahymena thermophila. Methods in Cell Biology, vol. 62 | |||
Collins K. (ed.) 2012 Tetrahymena thermophila, 1st edition. Methods in Cell Biology, vol. 109 | |||
Teaching resources | |||
ASSET teaching resource | ASSET: Advancing secondary science education thru Tetrahymena | https://tetrahymenaasset.vet.cornell.edu/ | Smith et al. 2012 |
Tetrahymena SUPRDB | SUPRDB: Student/Unpublished Results | http://suprdb.org/ | Wiley and Stover 2014 |
Ciliate genomics Consortium | Community/Wiley | http://faculty.jsd.claremont.edu/ewiley/ | |
Biotechnology resources | |||
Cilian | http://www.cilian.de/ (website in German) | ||
Tetragenetics | http://www.tetragenetics.com/ | ||
Tetratox | http://www.vet.utk.edu/TETRATOX/index.php | Schultz 1997 | |
Additional resources | |||
Tetrahymena history | http://www.life.illinois.edu/nanney/index.html | ||
Community listserv | https://listserv.uga.edu/cgi-bin/wa?A0=ciliatemolbio-l |
Resource . | Source . | Location . | Reference . |
---|---|---|---|
Genomics resources | |||
Genome browser | TGD: Tetrahymena genome database | http://www.Ciliate.org | Stover et al. 2006 |
NCBI genome | http://www.ncbi.nlm.nih.gov/genome/222 | Eisen et al. 2006 | |
Functional genomics | TetraFGD: Tetrahymena Functional Genomics Database | http://tfgd.ihb.ac.cn/ | Xiong et al. 2013 |
Experimental resources | |||
Strains | Tetrahymena stock center | https://tetrahymena.vet.cornell.edu/ | Cassidy-Hanley 2012 |
Methods resources | |||
Asai D. J. and J. D. Forney (eds.) 1999 Tetrahymena thermophila. Methods in Cell Biology, vol. 62 | |||
Collins K. (ed.) 2012 Tetrahymena thermophila, 1st edition. Methods in Cell Biology, vol. 109 | |||
Teaching resources | |||
ASSET teaching resource | ASSET: Advancing secondary science education thru Tetrahymena | https://tetrahymenaasset.vet.cornell.edu/ | Smith et al. 2012 |
Tetrahymena SUPRDB | SUPRDB: Student/Unpublished Results | http://suprdb.org/ | Wiley and Stover 2014 |
Ciliate genomics Consortium | Community/Wiley | http://faculty.jsd.claremont.edu/ewiley/ | |
Biotechnology resources | |||
Cilian | http://www.cilian.de/ (website in German) | ||
Tetragenetics | http://www.tetragenetics.com/ | ||
Tetratox | http://www.vet.utk.edu/TETRATOX/index.php | Schultz 1997 | |
Additional resources | |||
Tetrahymena history | http://www.life.illinois.edu/nanney/index.html | ||
Community listserv | https://listserv.uga.edu/cgi-bin/wa?A0=ciliatemolbio-l |
Resource . | Source . | Location . | Reference . |
---|---|---|---|
Genomics resources | |||
Genome browser | TGD: Tetrahymena genome database | http://www.Ciliate.org | Stover et al. 2006 |
NCBI genome | http://www.ncbi.nlm.nih.gov/genome/222 | Eisen et al. 2006 | |
Functional genomics | TetraFGD: Tetrahymena Functional Genomics Database | http://tfgd.ihb.ac.cn/ | Xiong et al. 2013 |
Experimental resources | |||
Strains | Tetrahymena stock center | https://tetrahymena.vet.cornell.edu/ | Cassidy-Hanley 2012 |
Methods resources | |||
Asai D. J. and J. D. Forney (eds.) 1999 Tetrahymena thermophila. Methods in Cell Biology, vol. 62 | |||
Collins K. (ed.) 2012 Tetrahymena thermophila, 1st edition. Methods in Cell Biology, vol. 109 | |||
Teaching resources | |||
ASSET teaching resource | ASSET: Advancing secondary science education thru Tetrahymena | https://tetrahymenaasset.vet.cornell.edu/ | Smith et al. 2012 |
Tetrahymena SUPRDB | SUPRDB: Student/Unpublished Results | http://suprdb.org/ | Wiley and Stover 2014 |
Ciliate genomics Consortium | Community/Wiley | http://faculty.jsd.claremont.edu/ewiley/ | |
Biotechnology resources | |||
Cilian | http://www.cilian.de/ (website in German) | ||
Tetragenetics | http://www.tetragenetics.com/ | ||
Tetratox | http://www.vet.utk.edu/TETRATOX/index.php | Schultz 1997 | |
Additional resources | |||
Tetrahymena history | http://www.life.illinois.edu/nanney/index.html | ||
Community listserv | https://listserv.uga.edu/cgi-bin/wa?A0=ciliatemolbio-l |
Genetic Studies in Tetrahymena
Forward genetic studies identify both important biological processes and the genes associated with them (Forsburg 2001; Patton and Zon 2001; Page and Grossniklaus 2002; Casselton and Zolan 2002; Jorgensen and Mango 2002; St Johnston 2002; Shuman and Silhavy 2003; Kile and Hilton 2005; Candela and Hake 2008). These approaches have been combined with reverse genetics methods to investigate details of how the discovered genes function. In Tetrahymena, this powerful approach has illuminated the biology of cilia, vesicular trafficking and exocytosis, telomeres, RNA enzymes, and chromatin, to name a few examples (Figure 2). Many Tetrahymena genes are conserved with humans; indeed humans share more orthologs with Tetrahymena than with other unicellular eukaryotes with a closer phylogenetic relationship (Eisen et al. 2006), making these methods a powerful way to study ciliate and human biology alike.
Forward genetics: unbiased gene discovery
Forward genetic screens employ random mutagenesis of the genome, followed by screening for a mutant phenotype of interest and then identifying the mutated gene(s). The method makes no preconceptions about what genes can be identified. Below we describe the general strategies and workflow for forward genetics approaches in Tetrahymena.
Mutagenesis:
Random mutations are induced by a brief exposure to a chemical mutagen (methylmethane sulfonate or nitrosoguanidine) during vegetative growth (Pennock 2000). Random mutagenesis leads to dominant and recessive mutations in the somatic and/or germline genomes. Only mutations present in the MAC lead to immediately observable phenotypes because the MIC is transcriptionally inactive. Germline mutations only come to expression after the mutagenized cells are crossed, and the progeny MACs carry the mutation. Using a mating strategy that generates homozygous progeny (such as uniparental cytogamy; Figure 7B) even recessive mutations can be phenotypically observed after a single cross (Karrer 2000).
Mutant isolation:
New mutations in the MAC are immediately expressed and their phenotypes become observable when the number of chromosomal copies carrying the mutation increases sufficiently by phenotypic assortment. However, these mutations are lost upon mating when the old MAC is destroyed, which makes MAC mutations less accessible to traditional genetic strategies. MIC mutations, on the other hand, can be propagated and used for traditional Mendelian genetic experiments. However, expression of the new mutation occurs only after it is transmitted to a new MAC following mating.
The isolation of recessive MIC mutations creates a special problem because a simple cross of the mutagenized cell to allow expression of the mutation in progeny MACs will result in heterozygous progeny showing the wild-type phenotype. This problem is elegantly resolved by inducing self-fertilization, where mutations are made homozygous for the mutant allele in both the MIC and MAC genomes allowing for expression of the mutant phenotype. Two approaches are commonly used to obtain Tetrahymena whole-genome homozygotes: genomic exclusion and uniparental cytogamy (UPC) (Figure 7) (Allen 1967; Cole and Bruns 1992). UPC is the most efficient way to accomplish self-fertilization for mutant isolation purposes. Recessive mutations are thus isolated as efficiently as in other microbes with a haploid genome. The progeny are then screened (or in favorable cases selected) for the desired mutant phenotype.
Screening mutants:
Phenotypic screening of mutants entails the rapid assessment of cells for classes of phenotypes (Forsburg 2001; Patton and Zon 2001; Casselton and Zolan 2002; Jorgensen and Mango 2002; Page and Grossniklaus 2002; St Johnston 2002; Shuman and Silhavy 2003; Kile and Hilton 2005; Candela and Hake 2008). These simple assays are designed to sort through hundreds to thousands of mutant cells. Creative strategies exist (or may need to be developed) to identify a mutant with a phenotype of interest. Notably, swimming assays for cilia defects, fluorescent detection of secreted proteases for exocytosis defects, and density gradients for feeding defects have been successfully employed (Nilsson and van Deurs 1983; Hünseler et al. 1987; Pennock et al. 1988; Tiedtke and Rasmussen 1988).
Sorting the mutant collection:
Identical mutant phenotypes can obviously result from mutations in more than one gene. To tease apart biological pathways following a screen, Tetrahymena is amenable to complementation tests to identify whether two independent mutations are in the same or different genes (Frankel et al. 1976; Gutiérrez and Orias 1992). In the simplest scenario, homozygous cell lines of two mutants of independent origin are mated to one another and phenotypes of the progeny are determined. Rescue of the wild-type phenotype is expected if the mutations are in different genes. More complex genetic interactions like intragenic complementation and nonallelic, noncomplementation are also accessible to these analyses. Complementation studies thus establish the minimum predicted number of genes involved in a pathway and potential genetic interactions.
Identifying mutations by NGS:
In the era of high-throughput NGS, comparative genomics is used to map mutations at nucleotide-level resolution in a “mapping-by-sequencing” fashion (Schneeberger 2014). This has been successful in various model organisms, including the ciliates Tetrahymena and Paramecium (Sarin et al. 2008; Blumenstiel et al. 2009; Birkeland et al. 2010; Cuperus et al. 2010; Galati et al. 2014; Marker et al. 2014; A. Turkewitz and J. Gaertig, personal communication). To map by sequencing, the mutant is backcrossed to the wild-type parent and a panel of homozygous F2 meiotic segregants is obtained by UPC. Alternatively, F1 progeny of different mating types can be crossed to one another and F2’s displaying the mutant phenotype can be selected (Galati et al. 2014; A. Turkewitz, personal communication). A pool of whole-cell DNA extracted from mutant F2’s and whole-cell DNA extracted from the wild-type parental line are sequenced. Sequencing reads are aligned to the MIC and MAC reference genomes, which are fully assembled and coaligned and variants identified. Candidate mutations are those that are found in 100% of both MAC and MIC reads.
The candidate mutations are prioritized, using one or more functional criteria: Is the mutation within an annotated gene sequence? Does the candidate gene have a function previously linked to the mutant phenotype in Tetrahymena or other organisms? Candidates are also tested by cloning and Sanger sequencing. The final candidate is confirmed by expression of a wild-type copy of the candidate gene to rescue the mutant phenotype.
Auxiliary tools: mapping mutations to chromosome segments:
Prior to the advent of NGS, the physical location of mutations within the genome was determined using deletion mapping. This remains a useful complementary approach to narrow candidate mutations from mapping-by-sequencing experiments. In deletion mapping, the physical location of mutations within the genome is determined by crossing a homozygous mutant to a nullisomic cell line (Figure 6) (Gutiérrez and Orias 1992). Nullisomic cell lines are heterokaryons with a functional MAC, but lack both MIC copies of a chromosome or a chromosome arm. Crossing a mutant to a series of nullisomic lines that collectively span the entire germline genome allows one to rapidly identify the chromosome location of the mutation. Upon mating of a homozygous mutant with a nullisomic strain, the fertilization nuclei will be monosomic, i.e., they will have only one copy of the chromosome (arm) missing in the nullisomic. If the mutation is recessive and maps to that chromosome (arm), then there will be no wild-type allele to rescue the mutant phenotype and the progeny will display the mutant phenotype; otherwise the progeny phenotype will be wild type. Nullisomic mapping eliminates the vast majority of false candidates in NGS experiments, since only variants in the MAC contigs that map to the MIC chromosome arm determined to harbor the mutation need to be considered. Segmental deletion homozygotes allow even finer mapping by the same method. Dominant mutations can also be deletion mapped in a similar manner. However, because all progeny will display the dominant mutant phenotype, cells must be assorted for many generations. If the mutation lies within the chromosome region that is missing in the nullisomic parent, the location will be recognized because the progeny will not have a wild-type allele and the wild-type phenotype will never be recovered after assortment.
If still necessary, a candidate mutation’s location can be mapped at higher resolution using classical genetic meiotic recombination linkage analysis, regardless of whether the mutation is dominant or recessive. DNA polymorphisms called randomly amplified polymorphic DNAs (RAPDs) are commonly used for this analysis. Differences in RAPD locations within inbred strains B and C3 have been mapped in the Tetrahymena MIC genome. Additionally, nearly every MAC chromosome has a mapped DNA polymorphism (Lynch et al. 1995; Brickner et al. 1996; Wickert and Orias 2000). After crossing the mutant (usually generated in inbred strain B) with a wild-type cell (usually strain C3) the DNA of every F2 homozygous segregant (mutant and wild type) is tested by PCR amplification to identify whether the RAPDs are from strain C3 or B. For efficiency, this testing is limited to the MAC chromosomes that were derived from the MIC chromosome arm or smaller segment previously determined to carry the mutation. The proximity of RAPD loci to the mutation of interest is determined by linkage analysis, where the recombination frequency is proportional to the distance between the two loci, allowing identification of a MAC chromosome segment containing the mutation of interest.
Molecular approaches to forward genetics:
As an alternative to random mutagenesis in forward genetics experiments, complementary DNA (cDNA) library screens have also led to the unbiased discovery of proteins involved in a variety of processes in Tetrahymena and other organisms. For example, GFP-fusion libraries have been constructed where cDNAs are fused in frame with the coding sequence of GFP (Sawin and Nurse 1996; Fujii et al. 1999; Rolls et al. 1999; Ding et al. 2000; Misawa et al. 2000; Escobar et al. 2003; Yao et al. 2007). Upon transformation of these constructs, the localization of GFP within the cell identifies the localization of the protein encoded by the cDNA. The plasmids can then be recovered and sequenced to identify the genes that encode proteins that localize to regions of interest. Variations on this experiment include using different promoters that have different cell cycle and developmental timing of expression to analyze protein localization at specific stages of the cell cycle or during conjugation (Yao et al. 2007).
Tetrahymena cDNA libraries also have been utilized to produce “antisense ribosomes” (Sweeney et al. 1996). Antisense ribosomes are ribosomes that display the reverse complement of a portion of a messenger RNA (mRNA) of interest (or, in this case, an entire cDNA library) inserted at a harmless position in the large rRNA subunit. The target mRNA is translationally repressed. This is an effective method to knock down a library of genes, which can be followed by phenotypic screening and identification of the genes by sequencing the recovered antisense ribosomal DNA (rDNA) vectors. This method was used to identify genes involved in secretion (Chilcoat et al. 2001). This study highlights the first example of a Tetrahymena gene identified and cloned solely based on its mutant phenotype.
Reverse genetics: analysis of identified genes
Once identified, gene functions can be further illuminated using a suite of modern molecular tools in Tetrahymena. Here, we describe a variety of methods for gene perturbation and study.
Gene perturbation: heritable transgenics:
DNA can be stably targeted and integrated in the somatic or germline genomes of Tetrahymena using homologous recombination. The DNA cassettes described below enable gene knockouts, knockins, and mutations.
Transformation of DNA into Tetrahymena is performed using microinjection, electroporation, or biolistics. Transformations require little DNA (microgram quantities) and positive transformants are acquired in <1 week. Transformation of DNA into the MAC or the MIC depends on the method used; in microinjections, needles can specifically deliver DNA to the MAC, whereas electroporation and biolistics rely on the precise timing of the transformation during the cell cycle or during conjugation. Recovery of MIC transformants requires mating, while MAC transformants can be selected directly. Transformations using biolistics are simple and efficient, making it the method of choice. This is especially important for low-efficiency transformations that target MIC DNA integration (Cassidy-Hanley et al. 1997; Hai and Gorovsky 1997).
DNA is typically introduced into Tetrahymena in one of two forms: as rDNA vectors that are autonomously replicating, extrachromosomal MAC DNA, or by integration into and precise replacement of a targeted chromosomal segment by homologous recombination. The rDNA vectors are high copy vectors (∼9,000 copies) containing a drug resistance marker and are used for exogenous protein overexpression. Gene perturbations, however, are typically introduced into the Tetrahymena genome by homologous recombination. In this process, linear DNA sequences containing a drug resistance marker flanked by homologous regions to a gene of interest are used to specifically target and replace that gene (Yao and Yao 1991; Hai et al. 2000). These knockout constructs confer resistance to cycloheximide, paromomycin, or blasticidine. Upon transformation into Tetrahymena cells, the knockout construct replaces the endogenous gene by exact homologous recombination (Yao and Yao 1991; Kahn et al. 1993).
For somatic MAC transformations, the DNA cassette inserts into only one or a few copies of the targeted locus while the remaining copies possess a wild-type gene (Hai et al. 2000). Once transformed, phenotypic assortment is used to generate MACs pure for somatic MAC knockouts or knockdowns containing a drug resistance gene (Figure 5B). Stepwise increases in drug concentration are used to select for cells that stochastically increase the gene copies containing the knockout cassette during division while killing those that do not. For nonessential genes, homozygous MAC genomic knockouts are obtained. Essential genes can also be selected through phenotypic assortment and driven toward knockdown, although incompletely because the loss of all copies of the wild-type allele is lethal. This produces a knockdown genotype and is a major advantage of Tetrahymena since this is not easily achieved in other organisms. When desired, homozygous KOs for essential genes are generated by MIC transformation, as described below.
Germline MIC mutations are produced by transforming mating cells during meiosis, when recombination is highest in the MIC genome (Cassidy-Hanley et al. 1997). This introduces at least one copy of the drug resistance gene into the diploid MIC genome, producing a heterozygote that expresses it in the newly developed MAC, and can be selected for during subsequent vegetative growth. Confirmed MIC integrants are made homozygous heterokaryons using round I of genomic exclusion (Figure 7A). The knockout is thus propagated in the MIC without a phenotype. To reveal the phenotype, two such homozygous heterokaryons of different mating types are mated to produce progeny that are homozygous MAC genomic knockouts (round II of genomic exclusion). An advantage to this strategy is that lethal mutations can be propagated in phenotypically wild-type cells and then knockout conditions can be induced by mating in synchronous and large populations of cells for phenotypic analyses.
Recently, a rapid method for MAC gene knockouts has been developed that does not depend on homologous recombination (Hayashi and Mochizuki 2015; Noto et al. 2015). Codeletion (CoDel) harnesses the endogenous mechanism for IES DNA elimination during MAC development to remove ∼1 kb of a gene of interest. While this method leads to only a knockdown of essential genes, it is a highly effective knockout strategy for nonessential genes. The target sequence for knockout is flanked by known IES sequences in a high copy number rDNA extrachromosomal vector that is introduced into conjugating cells undergoing MAC differentiation. The IES sequences trigger the production of small RNAs that are complementary to both the IES sequences and to the target sequence they flank. These small RNAs can then simultaneously target most of the copies of the endogenous MAC locus of the gene of interest and induce its elimination during MAC development. Two independent targets can be included in the same CoDel vector. This promising method allows for fast, targeted gene disruption in Tetrahymena.
Gene perturbation: RNA-based knockdowns:
Several RNA-based perturbation strategies are accessible in Tetrahymena. Antisense ribosomes were first reported as an effective tool for reducing the expression of genes (Sweeney et al. 1996), as described above for cDNA libraries. Rather than using a library of antisense cDNAs, an antisense DNA fragment to the 5′ untranslated region of a specific mRNA is inserted into the rRNA gene. The antisense RNA is displayed on every ribosome and inhibits translation of the gene.
As discussed previously, Tetrahymena RNA interference (RNAi) pathways control programmed DNA elimination from the MAC genome during MAC development (Chalker et al. 2013). Likewise, related RNAi machinery can be hijacked throughout the cell cycle and development to induce gene silencing. RNA hairpin constructs can be expressed by strong transcriptional promoters (Howard-Till and Yao 2006; Beales et al. 2007; Awan et al. 2009; Howard-Till et al. 2011, 2013). The RNA hairpins then become processed into 23- and 24-nucleotide sequences to induce gene silencing by mRNA degradation.
Exogenous gene expression:
The introduction of DNA for controlled protein expression in Tetrahymena cells is utilized to rescue mutant phenotypes, express mutant forms of a gene or exogenously tagged protein, and large-scale protein production. Such DNA cassettes are introduced as either rDNA integrating constructs for high copy number insertion and protein expression, or they can be integrated at specific genomic loci by homologous recombination. These systems allow for fine-tuned exogenous expression of proteins, but as a general consideration, all exogenous sequences including tags and fusions may need to be codon optimized for Tetrahymena.
Exogenous expression of Tetrahymena genes is controlled by either constitutive or inducible promoters. In Tetrahymena, the major constitutive promoter utilized is the histone H4 promoter (Kahn et al. 1993). Different promoters can be used for selective cell cycle- and developmental-specific gene expression. Additionally, strongly inducible promoters provide direct control over the temporal expression and the relative protein levels. This is typically controlled by using various metallothionein (MTT) gene promoters, which can be induced by the addition of cadmium, mercury, copper, or zinc. The MTT1 promoter, induced by cadmium chloride (CdCl2) (Shang et al. 2002), is the most commonly used; however, copper inducible versions also exist (Boldrin et al. 2008). In addition, the Tetrahymena Hsp70-2 promoter is highly inducible by a short heat shock (Yu et al. 2012). As the repertoire of inducible promoters expands (including tetracycline inducible systems), their utility in exogenous gene expression will become more accessible.
Protein tagging:
A diverse collection of reagents for protein tagging are available and freely shared by the Tetrahymena research community. These reagents facilitate the generation of constructs for protein localization using light and electron microscopy, biochemical purifications, and protein dynamics studies. Many of these reagents and Tetrahymena strains are available through the National Tetrahymena Stock Center. Codon optimized EGFP and mCherry constructs are available for live and fixed cell imaging of protein localization in Tetrahymena cells using both fluorescence localization and immuno-EM (IEM) to visualize the localization of proteins (Kataoka et al. 2010; Stemm-Wolf et al. 2013). Several of the localization tags are also amenable to biochemical purifications with localization and purification (LAP) tags that have been adapted from mammalian LAP tags and then codon optimized for Tetrahymena (Rigaut et al. 1999; Cheeseman and Desai 2005; Pearson et al. 2009; Winey et al. 2012). Additional purification strategies have been developed apart from the LAP tag (Couvillion and Collins 2012). These tags provide the ability to couple powerful in vivo cell biology experiments with the detail gleaned from reductionist biochemical approaches.
Conclusions and Perspectives
Tetrahymena’s evolutionary divergence from the more commonly studied model organisms, while retaining most of the cell biology inherited from the last eukaryotic common ancestor, is a major advantage as a model system. Within the context of its unusual life cycle, genetic and molecular manipulations that are impossible in other organisms enable Tetrahymena to reveal universally conserved cellular processes. Harnessing this unique power continues to underlie Tetrahymena’s utility to researchers working on diverse scientific problems.
What does the future hold for Tetrahymena research? Certainly, advances will be built upon existing methods, and the quest for more information, higher resolution, and less ambiguous results is never ending. The Tetrahymena research community strives to improve molecular tools such as RNAi knockdown strategies, to develop clustered regularly interspaced short palindromic repeat (CRISPR) technology amenable to MIC genome editing, and to expand the repertoire of inducible promoter systems. Additionally, optogenetic and dimerization targeting tools will be used to induce protein localization or gene expression upon light and chemical exposure. Along with the development of quantitative fixed and live cell imaging methods (a significant hurdle for a motile organism), these efforts will expand the reverse genetics tools in the Tetrahymena toolbox. In forward genetics approaches, initiating synthetic genetic array (SGA) analyses similar to those that were successfully performed in other model organisms (for review see Dixon et al. 2009) will be invaluable to identify genes that contribute to similar processes and will build systems-level genetic networks. Additionally, Tetrahymena researchers will benefit from improved next generation sequencing methods and their accompanying computational tools. Because many of these technologies have not necessarily been developed for Tetrahymena, adapting them to the organism will be a major focus, and one that is quite promising and reachable.
Although Tetrahymena was established as an advantageous model organism in the early 1920s (Collins and Gorovsky 2005), its utility to researchers has only increased over the last century. Tetrahymena continues to be a vehicle for groundbreaking discoveries in structural, molecular, and cellular biology because of its ability to be genetically manipulated, biochemically deconstructed, and visually inspected. Together, these unique advantages give us an unprecedented view into the inner workings of the ciliate’s complex life, and it, in turn, has taught us about the mechanisms that govern our own. And, the end is not in sight; as more genetics and genomics tools emerge, we see Tetrahymena continuing to keep its place as one of the fastest, most accessible, and relevant model organisms to address crucial biological questions that must be answered at the lightning speed of science today.
Acknowledgments
The authors thank Domenico “Nick” Galati, Brian Bayless, Aaron Turkewitz, and Alex Stemm-Wolf for critical reading and comments, and A. Turkewitz and J. Gaertig for communicating unpublished results. C.G.P. is funded by the National Institutes of Health–National Institute of General Medical Sciences GM099820, Pew Biomedical Scholars Program, and the Boettcher Foundation. E.O. is funded by a University of California Santa Barbara Academic Senate Research Award.
Footnotes
Communicating editor: O. Hobert