tetrahymena as a unicellular model eukaryote: genetic and … · 2016-05-30 · dissections of gene...

REVIEW

GENETIC TOOLBOX

Tetrahymena as a Unicellular Model Eukaryote:Genetic and Genomic Tools

Marisa D. Ruehle,*,1 Eduardo Orias,† and Chad G. Pearson**Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, 80045, and

†Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, California 93106

ABSTRACT Tetrahymena thermophila is a ciliate model organism whose study has led to important discoveries and insights into bothconserved 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 reversegenetics. Recent genomic tools have expanded Tetrahymena’s utility as a genetic model system. With the unique advantages thatTetrahymena provide, we argue that it will continue to be a model organism of choice.

KEYWORDS Tetrahymena thermophila; ciliates; model organism; genetics; amitosis; somatic polyploidy

GENETIC model systems have a long-standing history asimportant tools to discover novel genes and processes in

cell and developmental biology. The ciliate Tetrahymena ther-mophila is a model system that combines the power of for-ward and reverse genetics with a suite of useful biochemicaland cell biological attributes. Moreover, Tetrahymena areevolutionarily divergent from the commonly studied organ-isms in the opisthokont lineage, permitting examination ofboth unique and universally conserved biological processes.Here we highlight the advantages of Tetrahymena as a modelsystem, such as its unique and easily manipulated life cycle,that have contributed to important discoveries. The growingsuite of molecular-genetic and genomic tools described hereprovides a system to couple gene discovery to mechanisticdissections of gene function in the cell.

T. thermophila (Figure 1) is a ciliate model organismwhose study has led to fundamental biological insights cov-ering the central dogma and beyond. Indeed, this single-celled, motile eukaryote provides the tools and techniquesnot only for novel gene discovery, but also for unveiling theimportant molecular mechanisms behind those genes’ func-tions. As such, Tetrahymena’s utility as a genetic modelorganism is revealed by its short life cycle, easy and

cost-effective laboratory handling, and its accessibility to bothforward and reverse genetics. Despite its affectionate referenceas “pond scum” (Blackburn 2010), the beauty of Tetrahymenaas a genetic model organism is displayed in many lights.

Tetrahymena has a long and distinguished history in thediscovery of broad biological paradigms (Figure 2), begin-ning with the discovery of the first microtubule motor, dynein(Gibbons and Rowe 1965). Others include the Nobel Prizewinning discoveries of catalytic RNA (Kruger et al. 1982) andtelomere structure and telomerase (Greider and Blackburn1985). The first histone-modifying enzyme (histone acetyltransferase) and its role as a transcription factor were discov-ered in Tetrahymena (Brownell et al. 1996), which gave birthto the “histone code” and the field of epigenetic control ofgene expression by chromatin modification. The role of smallinterfering RNAs in heterochromatin formation and the mas-sive, programmed excision of transposon-related DNA fromthe somatic genome (Mochizuki et al. 2002; Taverna et al.2002) is another major Tetrahymena contribution. These fun-damental discoveries in Tetrahymena have helped usher inthemodern era of molecular and cellular biology. Many of thereasons why Tetrahymena was an advantageous system forthese groundbreaking discoveries are the same that makeTetrahymena useful today and in the future, augmentedby the ever-expanding toolkit available to Tetrahymenaresearchers.

In this review, we discuss major biological questions towhich T. thermophila is amenable and the genetic tools avail-able to answer them. It begins with the general biology of

Copyright © 2016 by the Genetics Society of Americadoi: 10.1534/genetics.114.169748Manuscript received March 1, 2016; accepted for publication April 8, 2016.1Corresponding author: Department of Cell and Developmental Biology, University ofColorado Denver School of Medicine, 12801 E. 17th Ave., Mail Stop 8108, Aurora,CO 80045. E-mail: [email protected]

Genetics, Vol. 203, 649–665 June 2016 649

mailto:[email protected]

Tetrahymena and its unique advantages as an experimentalmodel system. We then describe both forward and reversegenetic strategies in Tetrahymena to facilitate gene discoveryand to interrogate the mechanistic underpinnings of thosegenes. Ultimately, we seek to engage future researchers bydescribing the wealth of experimental advantages (both his-torical and modern) that Tetrahymena can provide and pre-view its promising future.

Tetrahymena Biology

Tetrahymena are unicellular, ciliated eukaryotes that live infresh water over a wide range of conditions. In the wild,Tetrahymena feed on bacteria, but laboratory strains typicallylive as axenic cultures in nutrient-rich media (derived fromtissue extracts) or chemically defined media. Populationsgrow quickly, with cells dividing every 2–3 hr under optimalconditions. Each cell is large, 30–50 mm in length, mak-ing them ideal for light and electron microscopy-basedinvestigations.

Tetrahymena, like all ciliates, are nuclear dimorphic,which means that the germline genome and the somatic ge-nome exist within two separate nuclei in one cell: the micro-nucleus (MIC) and the macronucleus (MAC), respectively(Table 1, Table 2, and Figure 1). The diploid MIC genomeconsists of five pairs of chromosomes, whereas the MAC

genome consists of�200 different chromosomes, each main-tained at a ploidy of�45 during the G1 phase of the cell cycle.The MIC is transcriptionally silent, and all gene expression isdriven from the MAC DNA. Despite the fact that Tetrahymenaare unicellular organisms, this germline/soma separation isreminiscent of metazoans where distinct germ cells and so-matic cells are maintained. Germline/soma separation is ex-tremely rare among unicellular eukaryotes, though it is alsofound in certain Foraminifera (in the Rhizarian evolutionarygroup).

To replicate these distinct genomes and to maintain ge-netic diversity, the T. thermophila life cycle employs both asex-ual, vegetative cell division and sexual reorganization througha process called conjugation. During vegetative division, theMAC divides amitotically (random chromosome segregation),the MIC divides mitotically, and binary cell fission producestwo daughter cells. However, under conditions of starvation,pairs of sexually mature cells with different mating types un-dergo conjugation. T. thermophila cells can have one of sevendifferent mating types, and each mating type can conjugatewith another mating type, but not with itself (Elliott andGruchy 1952; Nanney and Caughey 1953). Conjugation in-volves highly conserved eukaryotic processes: meiosis, game-togenesis, and gamete nucleus fusion to form the fertilization(or “zygote”) nuclei of progeny cells (Figure 3). The fertiliza-tion nucleus divides mitotically and then differentiates into anew MIC as well as a new MAC, and the parental MAC iseliminated. Conjugation has important consequences: Mende-lian genetic inheritance and increasing genotypic diversity. Thegenome’s maintenance and expression depend on widely con-served mechanisms and pathways across all lineages of life,making Tetrahymena a useful organism inwhich to study theseuniversal processes.

Evolutionary context

As ciliates, Tetrahymena are a part of the Alveolate group ofthe Stramenopile–Alveolate–Rhizarian (SAR) lineage (Fig-ure 4). Dinoflagellates and apicomplexans are also a part ofthe Alveolate group, but are more closely related to eachother than to the ciliates. Thus, there is a large evolutionarydistance from ciliates to their sister Alveolate groups, and aneven greater distance to other commonly used eukaryoticmodelorganisms which, like humans, are a part of the opisthokontgroup (yeast, worms, flies, mice, and many others). There is anoticeable dearth ofmodel organisms outside of the opisthokontgroup, making research of nonopisthokonts such as Tetrahy-mena an attractive means to explore the remarkable diversityof eukaryotic cell biology (Lynch et al. 2014). Nevertheless,Tetrahymena utilize many universally conserved eukaryoticprocesses, making them also useful for illuminating theseconserved features (Briguglio and Turkewitz 2014; Lynchet al. 2014).

Tetrahymena’s closest model organism relative (thoughnot its closest relative outright) is Paramecium, which, alongwith Tetrahymena, is a part of the Oligohymenophorea class(Figure 4, bottom). However, based on deviations in small

Figure 1 Tetrahymena cell image, illustrating its crystal-like organizationof ciliary units. A single Tetrahymena cell labeled for basal bodies (a-centrin,red) (Stemm-Wolf et al. 2005), kinetodesmal fibers (5D8, green) (Jerka-Dziadosz et al. 1995), and DNA (Hoerscht-33342, blue). Note that basalbodies are packed together in the four ciliary “membranelles” of the oralapparatus, for which the genus is named. Bar, 10 mm.

650 M. D. Ruehle, E. Orias, and C. G. Pearson

subunit ribosomal RNA (rRNA) sequences between Parame-cium and Tetrahymena, the two likely shared a commonancestor several hundred million years ago and their evolu-tionary distance exceeds that between rat and brine shrimp(Greenwood et al. 1991; Frankel 2000). Thus, while much ofthe biology of Tetrahymena has been illuminated by studiesin Paramecium and vice versa, differences between them areexpected and often observed (Frankel 2000).

T. thermophila belongs to a group of Tetrahymena speciesthat are morphologically indistinguishable from one another.These species were originally lumped together as a singlespecies called T. pyriformis. The discovery of Tetrahymenamating types allowed dissection of the group based on sexualisolation and led to the recognition of T. thermophila andother sibling species as distinct Tetrahymena species. Alongthe way, T. thermophilawas first called T. pyriformis variety 1

and later T. pyriformis syngen 1, before receiving its currentname (Nanney and McCoy 1976). Isogenic, inbred lines ofT. thermophila have been derived (Allen 1967). By conven-tion, most genetic analysis and genome sequencing has usedT. thermophila inbred strain B cells, while most genetic map-ping has utilized DNA polymorphisms between inbred strainsB and C3.

The sibling Tetrahymena species found in the originalT. pyriformis group provide an invaluable resource for com-parative genomic analyses, as evolutionary DNA sequenceconservation is a useful predictor of biological function undernatural selection (Romero and Blackburn 1991; Coyne andYao 1996). It is particularly useful for the detection of lineage-specific genes of unknown function and to identify conservedDNA and protein sequences within genes.

Lineage-specific gene family expansions are interestingproperties of any sequenced genome, as they can provideclues to the importance of particular cellular processes. InT. thermophila, gene family expansion has occurred mainlyby localized gene duplication (Eisen et al. 2006). This is incontrast to Paramecium tetraurelia where two recent whole-genome duplications (WGDs) occurred after the divergencefrom the Tetrahymenine ciliates contributing to its gene fam-ily expansion (Aury et al. 2006). It has been suggested that anolder WGD, clearly detectable in the Paramecium genomeoccurred prior to the divergence of Paramecium and Tetrahy-mena (Aury et al. 2006). However, no remaining evidence ofa WGD in the Tetrahymena genome has yet been found. Ma-jor gene family expansions in the Tetrahymena genome in-volve genes whose products function in “sensing andresponding to environmental cues (e.g. protein kinases,membrane transporters), mobilizing resources from the en-vironment (e.g. membrane transporters and traffic compo-nents, proteases), and maintaining complex cell structureand movement (e.g. microtubule components, motors, andregulators, membrane traffic components)” (Eisen et al.2006). The function of closely related gene family memberscan be difficult to dissect experimentally because of redun-dancy or significant functional similarity.

Ciliates have evolved lineage-specific changes in the nu-clear genetic code. Tetrahymena uses only a single codon(UGA) to terminate MAC-encoded protein synthesis. Theother stop codons in the universal genetic code, UAA andUAG, encode glutamine amino acids, along with the canon-ical glutamine codons, CAA and CAG. While the variant ge-netic code poses no special problem when expressingheterologous genes in Tetrahymena, the expression of Tetra-hymena protein coding genes in heterologous systems re-quires first mutating UAA and UAG codons to either CAA orCAG. 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 ge-nome, and a MAC that contains the somatic genome. Al-though the separation of germline and soma sounds

Figure 2 Tetrahymena areas of research. Fields of study that Tetrahy-mena has impacted.

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familiar to us metazoans, the presence of two different nu-clear genomes in the same cell is foreign. How does the MACdevelop from the MIC, how are they similar or different, andwhat experimental advantages does nuclear dimorphismconfer?

The diploid MIC genome consists of five pairs of chromo-somes and its size is estimated to total �220 Mb of DNA(Table 2) (Yao andGorovsky 1974).When the newly formingMAC (MAC anlagen) develops during conjugation (Figure 3,H and I) these 10 chromosomes are fragmented at specificsites called chromosomal breakage sequences (CBSs) andthey lose some internal sequences (Yao et al. 1987), collec-tively 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 thenew chromosomes. The locations of IESs are marked throughan elegant small RNA-dependent pathway, which ensuresthat 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 theeliminated sequences are repetitive elements likely derivedfrom transposons (genomic parasites that randomly insert inthe genome, causing deleterious genomic instability if notsilenced). Thus, eliminating these sequences from the MACprotects the expressed genome of the organism. The newlygenerated MAC chromosomes are then endoreplicated (rep-licated multiple times without nuclear division), leading tothe production of �45 copies of each chromosome per MAC.A unique and important exception to this copy number is thechromosome that encodes the 35S rRNA precursor gene(Tetrahymena homolog of the 45S rRNA precursor gene inother eukaryotes). This is the smallest MAC chromosome(�20 kb) and is present at �9000 copies per MAC.

Mating type determination in the sexual progeny occursduring MAC development (Figure 3, H and I). It occurs ineach of the four genetically identical developing MACs ofa conjugating pair independently of one another or ofthe parental mating types. This is because mating type

Table 1 Tetrahymena thermophila glossary

Term Definition

Chromosomal breakage site (CBS) MIC chromosome location occupied by the highly conserved 15-bp chromosome breakage sequence, which isthe necessary and sufficient DNA element for programmed chromosome fragmentation during MACdevelopment.

Cytogamy A mating strategy in which gamete pronucleus exchange between conjugants is blocked; the two sister gametepronuclei in each conjugant instead fuse to one another (double self-fertilization), generating whole-genomehomozygous 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 fertilizationnuclei are depicted as half white, half black small circles in each conjugant cell. Also referred to as the zygotenucleus.

Genomic exclusion A self-fertilization mating strategy to generate a whole-genome homozygote that uses a cross to a strain with adefective 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.


determination is based on stochastically selected, alternativeDNA rearrangements at the mating type locus of the devel-oping MAC (Cervantes et al. 2013). This process is unrelatedto chromosome fragmentation or IES excision. A cell’s mat-ing type is faithfully inherited during vegetative division. Prog-eny cells can mate only after a period of sexual immaturitythat lasts 40–70 cell divisions after conjugation. The molec-ular basis of this maturation is unknown.

Two genetic systems in one cell

The organization and behavior of the germline vs. the somaticnuclei define two types of genetic systems at work in the life ofevery cell. The diploid MIC lineage undergoes meiosis and fer-tilization to bestowMendelian genetic inheritance across sexualgenerations. On the other hand, during vegetative cell division,theMAC, with fragmented chromosomes that undergo amitoticdistribution, generates a distinct genetic phenomenon calledphenotypic assortment. This is akin to the genetics of high copynumber bacterial plasmids. The interplay of these two geneticsystems generates natural versatility that can be exploited togreat experimental advantage in the laboratory. Becausethe less familiar genetic phenomena associated with amitoticdivision 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 daugh-ter MACs by amitosis. In this process, the MAC chromosomesare not equally segregated to daughter cells; rather chromo-some segregation is stochastic. Familiar features of mitosis inanimals and plants, such as bipolar spindle formation, extremechromosome condensation, and microtubule attachment tocentromeres, are all missing during MAC division; thus thekinetochore-based, equal chromatid separation in normal mito-sis does not play a role in MAC division (Flickinger 1965;Cervantes et al. 2006). Microtubules are nonetheless requiredduring amitosis to elongate theMAC before it divides (WilliamsandWilliams 1976; Kushida et al. 2011). Themolecular mech-anisms that govern amitosis remain to be elucidated.

Because amitosis does not involve the high-fidelity, equalsegregation of sister chromatids into daughter cells as in mito-sis, aphenomenoncalledphenotypicassortment isobserveddur-ing Tetrahymena vegetative growth (Figure 5A) (Allen andNanney 1958; Doerder et al. 1975; Orias and Flacks 1975;Nanney and Preparata 1979). Phenotypic assortment is the abil-ity of a heterozygous MAC to come to homozygosity or lead tofixation of either allele. This occurs over multiple rounds of

amitosis as a result of unequal chromosome segregation duringMAC division (Figure 5B). Daughter cells stochastically receivea greater or lesser number of copies of an allele, and MACsultimately become pure for either allele during subsequent celldivisions (Orias and Flacks 1975; Nanney and Preparata 1979;Merriam and Bruns 1988). The steady-state rate of assort-ment is mathematically represented as 1 / (2n 2 1) perfission (Schensted 1958), where n is the number of chromo-some copies just after MAC division (45 in this case). In thewild, this phenomenon permits rapid somatic adaptation toselective pressures, and in the lab, it is useful for isolatingmutations or phenotypes of interest. Despite the random as-sortment of chromosomes in the MAC, a mechanism to controlchromosome copy number has been inferred (Doerder andDeBault 1978), although itsmolecular basis remains unknown.

Themating type locus is already present in multiple copiesat the time of mating type determination in the developingMAC. Therefore, more than onemating type can be expressedin a progeny cell line immediately after conjugation. Thiscondition is typically resolved by the time the mixed matingtype progeny cells reach sexual maturity, when most cellshave become fixed for a single mating type by phenotypicassortment. Sexually mature cells whose MAC remain mixedare called “selfers,” because they can pair with other mem-bers of the same clone when starved. If undetected, selferscan present a challenge to downstream experiments that re-quire mating. If it is absolutely necessary to work with a selferline, additional vegetative cell divisions permit the comple-tion of phenotypic assortment and allow mating type to be-come fixed in a subclonal population of these cells.

The remarkablegenetic featuresmadepossible by germline/soma differentiation in Tetrahymena provide a variety of ex-perimental advantages. First, recessivemutations within a het-erogeneous MAC can ultimately come to complete expressionbecause phenotypic assortment will enhance the number ofrecessive mutant alleles and reduce the number of dominantwild-type alleles in a subset of the progeny. Second, whole-genome homozygotes can be generated quickly and easily byself-fertilization methods. As a consequence, recessive muta-tions present in the MIC can be isolated as efficiently as if thegermline were haploid and are expressed in the MAC withoutassortment. Third, production of heterokaryon cells (cellswhose MIC and MAC genotypes are different; Figure 6) allowlethalmutations to bemaintained in theMIC during vegetativegrowth until induction of mating. Similarly, nullisomics andsegmental deletion homozygotes can bemaintained as hetero-karyons, which allow for the physical mapping of mutations,DNA polymorphisms, or MIC-limited DNA elements, such asCBSs. These are advantages that Tetrahymena researcherscommonly exploit to identify and study genes of interest.

Why Use Tetrahymena as a Genetic ModelOrganism?

Model systems are typically chosen based on their utility inidentifying new avenues of research or in contributing to

Table 2 Genome statistics

No. of chromosomes Ploidy Size (Mb)

MIC 5 2 220a

MAC �200 �45 �105a A more accurate estimate will become available upon the pending publication ofthe MIC genome sequencing project.


the mechanistic understanding of important biologicalproblems. Tetrahymena have the advantages of a “do-it-all”model system; this is especially true as a genetic modelsystem. Tetrahymena grow fast (�2- to 3-hr doublingtime), can undergo large scale and synchronous matings,genetic markers have been mapped, and its two nuclear

genomes sequenced. The germline MIC exhibits Mendeliangenetics, and drug resistance markers exist for positiveselection. This suite of genetic advantages yields an effec-tive model system to complement its powerful biochemicaland cytological attributes. Indeed, both forward and re-verse genetic tools are well-developed in Tetrahymena.

Figure 3 Tetrahymena conjugation. The life cycle of Tetrahymena includes a sexual phase called conjugation. Cells are represented as ovals with amicronucleus (small circle) and a macronucleus (large circle) whose DNA content (ploidy) is indicated. Images of cells at corresponding steps are shownon the periphery of the illustration and are lettered accordingly. To begin the sexual phase of the life cycle, two cells of different mating types (A),homozygous for black and white alleles, respectively, undergo pairing (B). Completion of two rounds of meiosis (C) leads to the production of four haploidproducts (half circles, indicated as 1n). One of these meiotic products is positioned at the anterior cytoplasm of each conjugant, while the remaining threeare targeted for elimination (red outline) at the conjugant’s posterior end. Subsequently, mitosis of the surviving meiotic product generates two gametepronuclei (D). Each migratory pronucleus is transferred to the opposite conjugant in a process called pronuclear exchange (E). The incoming migratorypronuclei fuse with the stationary pronuclei (pronuclear fusion), restoring the diploid character of the MIC and thus generating the fertilization (or zygote)nucleus (F). Thus, each fertilization nucleus gets one haploid genome from each parent (black and white semicircles). The fertilization nucleus undergoes twopostzygotic mitoses (G), leading to the production of four genetically identical diploid nuclei in each conjugant. (H) The two anterior nuclei (MAC anlagen)develop into new MACs, while the two posterior nuclei become the new MICs. New MAC maturation involves ploidy increase and programmed DNArearrangement (see text). The parental MACs are eliminated apoptotically (red outline) and contribute no DNA to the progeny. (I) The exconjugant cellsseparate and one of the two MICs is eliminated. When the exconjugants divide, one of the two fully developed MACs and a mitotic copy of the survivingMIC in each exconjugant are segregated to their daughter cells, called karyonides because each one gets an independently developed MAC (J). Havingcompleted the conjugation process, these cells must sexually mature through vegetative cell division before they can conjugate.


Furthermore, the National Tetrahymena Stock Center, lo-cated at Cornell University, maintains and readily makesavailable many cell lines as well as plasmids, protocols, andother resources to facilitate these studies.

Forward and reverse genetics

Forward genetic strategies have been useful for generatingTetrahymena mutants. Mutagenesis screens with accom-panying phenotypic screens have identified defects inbiological processes including cortical patterning, metab-olism, motility, cell cycle progression, RNA positioning,drug resistance, phagocytosis, and exocytosis (Bruns andSanford 1978; Ahmed et al. 1998; Bowman et al. 2005).Such mutations can then be mapped to the genome. It waspreviously difficult to identify the mutations associated witha phenotype due to difficulties in producing genome-wide libraries to rescue mutant phenotypes. However,next generation sequencing (NGS) is now an effectivetool to rapidly identify mutations in Tetrahymena andin other genomes (Galati et al. 2014; Schneeberger2014). The development of NGS to identify mutations cre-ated in Tetrahymena forward genetic screens greatly in-creases the power of this system for new mechanisticresearch.

To study the functions of knowngenes,Tetrahymena is alsoamenable to reverse genetic strategies by the introduction ofprecisely targeted and heritable gene knockouts (KOs) anddisruptions. Moreover, potentially deleterious genomicknockouts and gene mutations can be introduced into andpropagated in the germline while maintaining a normal MACgenome. Thus, cells may propagate mutations in essential

genes without associated phenotypes. To then express themutant phenotype, genetically identical cells are mated toone another and all progeny will express the mutation ofinterest. These strategies allow for sophisticated reverse ge-netic studies.

Advantages to repetitive elements

An important advantage to Tetrahymena is their large num-bers of repeating structural elements, such as the repeatingcortical architecture composed of polarized individual cili-ary 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 cor-tical architecture, requiring minimal technological tools.Additionally, clathrin-dependent endocytosis is organizedin an array at the cell cortex and is readily studied inTetrahymena (Elde et al. 2005). The relatively largeTetrahymena cells (�20 3 50 mm) with repeating elementsenables rapid and high-quality cytological work using bothlight and electron microscopy and provides copious materialfor biochemical and structural studies of these repeatingelements.

The Tetrahymena community has developed moleculartools and microscopy techniques to visualize Tetrahymena’sdiverse cellular processes. Protein tags allow for biochemicalanalyses and live and fixed cell visualization of protein local-ization and cellular structure. The regular, crystal-like repe-tition of structural elements enables signal amplificationin these localization studies, resulting in rapid and high-resolution detection of cortical architecture using light andelectron microscopy.

Figure 4 Evolutionary relationship of Tetrahy-mena to other eukaryotes. (Top) Eukaryotic su-pergroups and their subgroups. Adapted fromLynch et al. (2014). (Bottom) Expanded tree ofthe Alveolates showing the lineage leading toTetrahymena (red lines) (Cavalier-Smith 1993).Line lengths do not represent evolutionarydistances.


In silico toolbox

Informatics tools exist to stimulate the study of the Tetrahy-mena genome and its expression (Table 3). These include theMIC and MAC genome sequences from the Broad Instituteand The Institute for Genome Research (TIGR: now theJ. Craig Venter Institute), respectively, a list of annotatedgenes, transcriptome-wide RNA sequencing (RNA-seq) dataat different life cycle stages (http://tfgd.ihb.ac.cn/), compar-ative 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 GeneOntology (GO) terms, relevant literature references, and linksto GenBank entries. The site also maintains a “Textpressofor Tetrahymena” function for full text searches. The combi-nation of genomic resources with the genetic power of theorganism and its accompanying molecular and biochemicaltools, primes Tetrahymena for elucidating additional funda-mental biological processes.

Figure 5 Amitotic macronuclear division and phenotypic assortment. (A) Phenotypic assortment: During vegetative growth, the MIC undergoesmitotic division, whereas the MAC divides amitotically, meaning that chromosome copies are randomly distributed to the daughter macro-nuclei. When the MAC is heterozygous, this leads to unequal segregation of allelic genetic information, a phenomenon called phenotypicassortment, illustrated by the all-white or all-black MACs, the two alternative endpoints of the assortment process. The mitotically dividing MICremains heterozygous. (B) Amitosis leads to phenotypic assortment: A vegetatively growing cell whose MAC is represented in (a) one of thechromosome copies has a mutant allele, represented by a star. (For simplicity of illustration, only one MAC chromosome is shown, and the G1ploidy is 4 instead of 45 copies.) One possible sequence of events is illustrated. (b) The number of copies has doubled during S phase. The twomutant copies can be distributed in one of two equally probable ways: one mutant copy goes to each daughter MAC (no change, not shown),or both mutant copies go to the same daughter (c), so that the other daughter becomes fixed for the wild-type allele. Additional cell cycles (dand e) generate MACs fixed for the mutant allele (f). Once a cell with a pure MAC is generated, every MAC in the resulting subclone remainsfixed. Thus, the fraction of cells with pure MACs continuously increases in the clone. At steady state, the probability that a cell with a mixedMAC will generate a daughter with a pure MAC is 1 / (2n 2 1), where n is the ploidy at G1 stage, or 0.011/fission for n = 45. The initial alleleratio in the progenitor cell’s MAC (a) is called the input ratio. The output ratio is the ultimate ratio of cells whose MACs have become pure foreither allele after many fissions. In the absence of selection, the output ratio should be identical to the input ratio. Heterozygous MACsproduced by crossing, as in Figure 3, will generally have an �1:1 input ratio. In contrast, when MAC transformation is carried out, the initialtransformant is likely to have an input ratio highly biased against the transforming allele, which is compensated for by introducing selection forthe transformed allele.


http://tfgd.ihb.ac.cn/

Genetic Studies in Tetrahymena

Forward genetic studies identify both important biologicalprocesses and the genes associated with them (Forsburg2001; Patton and Zon 2001; Page and Grossniklaus 2002;Casselton and Zolan 2002; Jorgensen and Mango 2002; StJohnston 2002; Shuman and Silhavy 2003; Kile and Hilton2005; Candela and Hake 2008). These approaches have beencombined with reverse genetics methods to investigate de-tails 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). ManyTetrahymena genes are conserved with humans; indeed hu-mans share more orthologs with Tetrahymena than withother unicellular eukaryotes with a closer phylogenetic re-lationship (Eisen et al. 2006), making these methods a pow-erful way to study ciliate and human biology alike.

Forward genetics: unbiased gene discovery

Forward genetic screens employ random mutagenesis of thegenome, followed by screening for a mutant phenotype ofinterest and then identifying the mutated gene(s). Themethod makes no preconceptions about what genes can beidentified. Belowwedescribe thegeneral strategies andwork-flow for forward genetics approaches in Tetrahymena.

Mutagenesis: Random mutations are induced by a briefexposure to a chemical mutagen (methylmethane sulfonateor nitrosoguanidine) during vegetative growth (Pennock2000). Randommutagenesis leads to dominant and recessivemutations in the somatic and/or germline genomes. Onlymutations present in the MAC lead to immediately observ-able phenotypes because the MIC is transcriptionally inac-tive. Germline mutations only come to expression after themutagenized cells are crossed, and the progeny MACs carrythe mutation. Using a mating strategy that generates homo-zygous progeny (such as uniparental cytogamy; Figure 7B)even recessive mutations can be phenotypically observed af-ter a single cross (Karrer 2000).

Mutant isolation: New mutations in the MAC are immedi-ately expressed and their phenotypes become observablewhen thenumberof chromosomal copies carrying themutationincreasessufficientlybyphenotypicassortment.However, thesemutations are lost uponmatingwhen theoldMAC is destroyed,which makes MAC mutations less accessible to traditional ge-netic strategies. MIC mutations, on the other hand, can bepropagated and used for traditional Mendelian genetic exper-iments. However, expression of the new mutation occurs onlyafter it is transmitted to a new MAC following mating.

The isolation of recessive MIC mutations creates a specialproblem because a simple cross of the mutagenized cell to

Figure 6 Useful heterokaryons. (A) Drug resistance heterokaryon: often used to eliminate unmated parental cells (drug sensitive) and recover only trueprogeny (drug resistant) in a mass cross. (B) Heterokaryon for a lethal mutation: used to propagate a homozygous lethal mutation in the MIC, while“covered” by a wild-type MAC. The phenotype is expressed when two such cell lines are crossed to one another. (C) Nullisomic heterokaryons: thelethal MIC genotype is the absence of both copies of one MIC chromosome or chromosome arm (arrow); such heterokaryons are useful for geneticallymapping mutations and DNA polymorphisms to chromosome locations. (D) Gene knockout: the lethal mutation is a deleterious or lethal gene KO, inwhich the coding sequence of the KO’s gene has been replaced by a cassette expressing drug resistance (to paromomycin, related to neomycin). The KOcomes to expression in the progeny when such heterokaryons are crossed while the neo cassette allows elimination of wild-type parental anduntransformed progeny cells.


allow expression of the mutation in progeny MACs will resultin heterozygous progeny showing the wild-type pheno-type. This problem is elegantly resolved by inducing self-fertilization, where mutations are made homozygous forthemutant allele in both theMIC andMAC genomes allowingfor expression of the mutant phenotype. Two approaches arecommonly used to obtain Tetrahymenawhole-genome homo-zygotes: genomic exclusion and uniparental cytogamy (UPC)(Figure 7) (Allen 1967; Cole and Bruns 1992). UPC is themost efficient way to accomplish self-fertilization for mutantisolation purposes. Recessive mutations are thus isolated asefficiently as in other microbes with a haploid genome. Theprogeny are then screened (or in favorable cases selected) forthe desired mutant phenotype.

Screening mutants: Phenotypic screening of mutants en-tails the rapid assessment of cells for classes of phenotypes(Forsburg 2001; Patton and Zon 2001; Casselton and Zolan2002; Jorgensen and Mango 2002; Page and Grossniklaus2002; St Johnston 2002; Shuman and Silhavy 2003; Kileand Hilton 2005; Candela and Hake 2008). These simpleassays are designed to sort through hundreds to thousandsof mutant cells. Creative strategies exist (or may need to bedeveloped) to identify a mutant with a phenotype of inter-est. Notably, swimming assays for cilia defects, fluorescentdetection of secreted proteases for exocytosis defects, and

density gradients for feeding defects have been successfullyemployed (Nilsson and van Deurs 1983; Hünseler et al.1987; Pennock et al. 1988; Tiedtke and Rasmussen 1988).

Sorting the mutant collection: Identical mutant phenotypescan obviously result frommutations inmore than one gene. Totease apart biological pathways following a screen, Tetrahy-mena is amenable to complementation tests to identifywhether two independent mutations are in the same or differ-ent genes (Frankel et al. 1976; Gutiérrez and Orias 1992). Inthe simplest scenario, homozygous cell lines of two mutantsof independent origin are mated to one another and pheno-types of the progeny are determined. Rescue of the wild-typephenotype is expected if the mutations are in different genes.More complex genetic interactions like intragenic complemen-tation and nonallelic, noncomplementation are also accessibleto these analyses. Complementation studies thus establishthe minimum predicted number of genes involved in a path-way and potential genetic interactions.

Identifying mutations by NGS: In the era of high-throughputNGS, comparative genomics is used to map mutations atnucleotide-level resolution in a “mapping-by-sequencing”fashion (Schneeberger 2014). This has been successful invarious model organisms, including the ciliates Tetrahymenaand Paramecium (Sarin et al. 2008; Blumenstiel et al. 2009;

Table 3 In silico toolbox and other resources

Resource Source Location Reference

Genomics resourcesGenome browser TGD: Tetrahymena genome

databasehttp://www.Ciliate.org Stover et al. 2006

NCBI genome http://www.ncbi.nlm.nih.gov/genome/222 Eisen et al. 2006Functional genomics TetraFGD: Tetrahymena Functional

Genomics Databasehttp://tfgd.ihb.ac.cn/ Xiong et al. 2013

Experimental resourcesStrains Tetrahymena stock center https://tetrahymena.vet.cornell.edu/ Cassidy-Hanley 2012

Methods resourcesAsai 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 resourcesASSET teaching resource ASSET: Advancing secondary

science education thruTetrahymena

https://tetrahymenaasset.vet.cornell.edu/ Smith et al. 2012

Tetrahymena SUPRDB SUPRDB: Student/UnpublishedResults

http://suprdb.org/ Wiley and Stover 2014

Ciliate genomics Consortium Community/Wiley http://faculty.jsd.claremont.edu/ewiley/

Biotechnology resourcesCilian http://www.cilian.de/ (website in German)Tetragenetics http://www.tetragenetics.com/Tetratox http://www.vet.utk.edu/TETRATOX/index.php Schultz 1997

Additional resourcesTetrahymena history http://www.life.illinois.edu/nanney/index.htmlCommunity listserv https://listserv.uga.edu/cgi-bin/wa?A0=ciliatemolbio-l


http://www.Ciliate.org

http://www.ncbi.nlm.nih.gov/genome/222

http://tfgd.ihb.ac.cn/

https://tetrahymena.vet.cornell.edu/

https://tetrahymenaasset.vet.cornell.edu/

http://suprdb.org/

http://faculty.jsd.claremont.edu/ewiley/

http://www.cilian.de/

http://www.tetragenetics.com/

http://www.vet.utk.edu/TETRATOX/index.php

http://www.life.illinois.edu/nanney/index.html

https://listserv.uga.edu/cgi-bin/wa?A0=ciliatemolbio-l

Birkeland et al. 2010; Cuperus et al. 2010; Galati et al. 2014;Marker et al. 2014; A. Turkewitz and J. Gaertig, personal com-munication). Tomap by sequencing, the mutant is backcrossedto the wild-type parent and a panel of homozygous F2 meioticsegregants is obtained by UPC. Alternatively, F1 progeny ofdifferent mating types can be crossed to one another and F2’sdisplaying the mutant phenotype can be selected (Galati et al.2014; A. Turkewitz, personal communication). A pool ofwhole-cell DNA extracted from mutant F2’s and whole-cell DNAextracted from the wild-type parental line are sequenced. Se-quencing reads are aligned to the MIC and MAC referencegenomes,which are fully assembled and coaligned and variantsidentified. Candidate mutations are those that are found in100% of both MAC and MIC reads.

The candidatemutations are prioritized, using one ormorefunctional criteria: Is the mutation within an annotated genesequence?Does the candidate genehavea functionpreviously

linked to the mutant phenotype in Tetrahymena or other or-ganisms? Candidates are also tested by cloning and Sangersequencing. The final candidate is confirmed by expressionof a wild-type copy of the candidate gene to rescue the mu-tant phenotype.

Auxiliary tools: mapping mutations to chromosome segments:Prior to the advent of NGS, the physical location of mutationswithin the genome was determined using deletion mapping.This remains a useful complementary approach to narrowcandidate mutations from mapping-by-sequencing experi-ments. In deletionmapping, the physical location of mutationswithin the genome is determined by crossing a homozygousmutant to a nullisomic cell line (Figure 6) (Gutiérrez andOrias1992). Nullisomic cell lines are heterokaryons with a func-tional MAC, but lack both MIC copies of a chromosome or achromosome arm. Crossing a mutant to a series of nullisomic

Figure 7 Genetic approaches to generate whole-genomehomozygotes from a heterozygote. Symbols are as in Fig-ure 3. Asterisk denotes a mutation or allele of interest,such as a drug resistance gene. (A) Genomic exclusion.A cell containing a heterozygous MIC (represented byblack and white) for a mutation in the MIC (asterisk) iscrossed to a cell line with a defective MIC—called a “star”strain—which is incapable of contributing germline DNAto sexual progeny (round I). At meiosis, the remnant of itsMIC disintegrates without cytological trace (stage b). TheMIC of the nonstar mate undergoes normal meiosis, ga-metogenic mitosis, and gamete pronucleus transfer(stages c and d). The gamete pronuclei diploidize andthe exconjugants separate without undergoingpostzygotic nuclear differentiation (stage e). The twoexconjugants are heterokaryons: their MICs are bothwhole-genome homozygotes derived from the single, dip-loidized meiotic product, and their MACs are those of thetwo parental cells that remain unchanged. The two typesof whole-genome homozygotes are obtained in 1:1 ratio(only the white, mutated is shown). If two exconjugantsfrom the same round 1 pair are crossed to one another(round II; stage f), whole-genome homozygous homo-karyon progeny are obtained (stage g). (B) UPC. In thismethod, a cell heterozygous for a mutation is crossed toa star cell (stages a and b are identical to stages a and b ingenomic exclusion), but gamete pronucleus transfer isblocked by a suitably timed hyperosmotic shock (betweenstages c and d). As a consequence, the two gamete pro-nuclei fuse to one another, generating a diploid, whole-genome homozygous zygote nucleus (stage d). Unlikeround I of the genomic exclusion cross, postzygotic nu-clear differentiation and old MAC destruction occur nor-mally. The star mate dies for lack of a MIC and MAC (stagee). The resulting cell line has MICs and MACs that arewhole-genome homozygous for the haploid genome ofthe surviving meiotic product at stage b (homokaryons).UPC is the method of choice to isolate mutants homozy-gous for recessive mutations after mutagenesis.


lines that collectively span the entire germline genome allowsone to rapidly identify the chromosome location of the muta-tion. Upon mating of a homozygous mutant with a nullisomicstrain, the fertilization nuclei will be monosomic, i.e., they willhave only one copy of the chromosome (arm) missing in thenullisomic. If the mutation is recessive and maps to that chro-mosome (arm), then there will be nowild-type allele to rescuethemutant phenotype and the progenywill display themutantphenotype; otherwise the progeny phenotype will be wildtype. Nullisomic mapping eliminates the vast majority of falsecandidates inNGS experiments, since only variants in theMACcontigs that map to the MIC chromosome arm determined toharbor the mutation need to be considered. Segmental dele-tion homozygotes allow even finer mapping by the samemethod. Dominant mutations can also be deletion mappedin a similar manner. However, because all progeny will displaythe dominant mutant phenotype, cells must be assorted formany generations. If themutation lies within the chromosomeregion that is missing in the nullisomic parent, the locationwillbe recognized because the progeny will not have a wild-typeallele and the wild-type phenotype will never be recoveredafter assortment.

If still necessary, a candidate mutation’s location can bemapped at higher resolution using classical genetic meioticrecombination linkage analysis, regardless of whether themutation is dominant or recessive. DNA polymorphismscalled randomly amplified polymorphic DNAs (RAPDs) arecommonly used for this analysis. Differences in RAPD loca-tions within inbred strains B and C3 have been mapped in theTetrahymena MIC genome. Additionally, nearly every MACchromosome has a mapped DNA polymorphism (Lynchet al. 1995; Brickner et al. 1996; Wickert and Orias 2000).After crossing the mutant (usually generated in inbred strainB) with a wild-type cell (usually strain C3) the DNA of everyF2 homozygous segregant (mutant andwild type) is tested byPCR amplification to identify whether the RAPDs are fromstrain C3 or B. For efficiency, this testing is limited to theMACchromosomes that were derived from the MIC chromosomearm or smaller segment previously determined to carry themutation. The proximity of RAPD loci to the mutation of in-terest is determined by linkage analysis, where the recombi-nation frequency is proportional to the distance between thetwo loci, allowing identification of a MAC chromosome seg-ment containing the mutation of interest.

Molecular approaches to forward genetics: As an alterna-tive to randommutagenesis in forward genetics experiments,complementary DNA (cDNA) library screens have also led tothe unbiased discovery of proteins involved in a variety ofprocesses in Tetrahymena and other organisms. For example,GFP-fusion libraries have been constructed where cDNAs arefused in frame with the coding sequence of GFP (Sawin andNurse 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 localiza-tion of GFP within the cell identifies the localization of the

protein encoded by the cDNA. The plasmids can then be re-covered and sequenced to identify the genes that encodeproteins that localize to regions of interest. Variations on thisexperiment include using different promoters that have dif-ferent cell cycle and developmental timing of expression toanalyze protein localization at specific stages of the cell cycleor during conjugation (Yao et al. 2007).

Tetrahymena cDNA libraries also have been utilized to pro-duce “antisense ribosomes” (Sweeney et al. 1996). Antisenseribosomes are ribosomes that display the reverse comple-ment of a portion of a messenger RNA (mRNA) of interest(or, in this case, an entire cDNA library) inserted at a harmlessposition in the large rRNA subunit. The target mRNA is trans-lationally repressed. This is an effective method to knockdown a library of genes, which can be followed by phenotypicscreening and identification of the genes by sequencing therecovered antisense ribosomal DNA (rDNA) vectors. Thismethod was used to identify genes involved in secretion(Chilcoat et al. 2001). This study highlights the first exampleof a Tetrahymena gene identified and cloned solely based onits mutant phenotype.

Reverse genetics: analysis of identified genes

Once identified, gene functions can be further illuminatedusing a suite ofmodernmolecular tools inTetrahymena. Here,we describe a variety of methods for gene perturbation andstudy.

Gene perturbation: heritable transgenics: DNA can be sta-bly targeted and integrated in the somatic or germline ge-nomes of Tetrahymena using homologous recombination.The DNA cassettes described below enable gene knockouts,knockins, and mutations.

Transformation of DNA into Tetrahymena is performedusing microinjection, electroporation, or biolistics. Transfor-mations require little DNA (microgram quantities) and posi-tive transformants are acquired in ,1 week. Transformationof DNA into the MAC or the MIC depends on the methodused; in microinjections, needles can specifically deliverDNA to the MAC, whereas electroporation and biolistics relyon the precise timing of the transformation during the cellcycle or during conjugation. Recovery of MIC transformantsrequires mating, while MAC transformants can be selecteddirectly. Transformations using biolistics are simple and effi-cient, making it the method of choice. This is especially im-portant for low-efficiency transformations that target MICDNA integration (Cassidy-Hanley et al. 1997; Hai andGorovsky 1997).

DNA is typically introduced into Tetrahymena in one oftwo forms: as rDNA vectors that are autonomously replicat-ing, extrachromosomal MAC DNA, or by integration into andprecise replacement of a targeted chromosomal segment byhomologous recombination. The rDNA vectors are high copyvectors (�9,000 copies) containing a drug resistance markerand are used for exogenous protein overexpression. Geneperturbations, however, are typically introduced into the


Tetrahymena genome by homologous recombination. In thisprocess, linear DNA sequences containing a drug resistancemarker flanked by homologous regions to a gene of interestare used to specifically target and replace that gene (Yao andYao 1991; Hai et al. 2000). These knockout constructs conferresistance to cycloheximide, paromomycin, or blasticidine.Upon transformation into Tetrahymena cells, the knockoutconstruct replaces the endogenous gene by exact homolo-gous recombination (Yao and Yao 1991; Kahn et al. 1993).

For somatic MAC transformations, the DNA cassette in-serts into only one or a few copies of the targeted locus whilethe remaining copies possess a wild-type gene (Hai et al.2000). Once transformed, phenotypic assortment is used togenerate MACs pure for somatic MAC knockouts or knock-downs containing a drug resistance gene (Figure 5B). Step-wise increases in drug concentration are used to select forcells that stochastically increase the gene copies containingthe knockout cassette during division while killing those thatdo not. For nonessential genes, homozygous MAC genomicknockouts are obtained. Essential genes can also be selectedthrough phenotypic assortment and driven toward knock-down, although incompletely because the loss of all copiesof the wild-type allele is lethal. This produces a knockdowngenotype and is a major advantage of Tetrahymena since thisis not easily achieved in other organisms. When desired, ho-mozygous KOs for essential genes are generated by MICtransformation, as described below.

Germline MIC mutations are produced by transformingmating cells duringmeiosis, when recombination is highest inthe MIC genome (Cassidy-Hanley et al. 1997). This intro-duces at least one copy of the drug resistance gene into thediploid MIC genome, producing a heterozygote that ex-presses it in the newly developed MAC, and can be selectedfor during subsequent vegetative growth. Confirmed MICintegrants are made homozygous heterokaryons using roundI of genomic exclusion (Figure 7A). The knockout is thuspropagated in the MIC without a phenotype. To reveal thephenotype, two such homozygous heterokaryons of differentmating types are mated to produce progeny that are homo-zygous MAC genomic knockouts (round II of genomic exclu-sion). An advantage to this strategy is that lethal mutationscan be propagated in phenotypically wild-type cells and thenknockout conditions can be induced by mating in synchro-nous and large populations of cells for phenotypic analyses.

Recently, a rapidmethod forMACgene knockouts has beendeveloped that does not depend on homologous recombina-tion (Hayashi andMochizuki 2015;Noto et al.2015). Codeletion(CoDel) harnesses the endogenous mechanism for IES DNAelimination during MAC development to remove �1 kb of agene of interest. While this method leads to only a knock-down of essential genes, it is a highly effective knockoutstrategy for nonessential genes. The target sequence forknockout is flanked by known IES sequences in a high copynumber rDNA extrachromosomal vector that is introducedinto conjugating cells undergoing MAC differentiation. TheIES sequences trigger the production of small RNAs that are

complementary to both the IES sequences and to the targetsequence they flank. These small RNAs can then simulta-neously target most of the copies of the endogenous MAClocus of the gene of interest and induce its elimination duringMAC development. Two independent targets can be includedin the same CoDel vector. This promising method allows forfast, 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 toolfor reducing the expression of genes (Sweeney et al. 1996), asdescribed above for cDNA libraries. Rather than using a li-brary of antisense cDNAs, an antisense DNA fragment to the59 untranslated region of a specific mRNA is inserted into therRNA gene. The antisense RNA is displayed on every ribo-some and inhibits translation of the gene.

As discussed previously, Tetrahymena RNA interference(RNAi) pathways control programmedDNA elimination fromthe MAC genome during MAC development (Chalker et al.2013). Likewise, related RNAi machinery can be hijackedthroughout the cell cycle and development to induce genesilencing. RNA hairpin constructs can be expressed by strongtranscriptional promoters (Howard-Till and Yao 2006; Bealeset 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 mRNAdegradation.

Exogenous gene expression: The introduction of DNA forcontrolled protein expression in Tetrahymena cells is utilizedto rescue mutant phenotypes, express mutant forms of a geneor exogenously tagged protein, and large-scale protein pro-duction. Such DNA cassettes are introduced as either rDNAintegrating constructs for high copy number insertion andprotein expression, or they can be integrated at specific ge-nomic loci by homologous recombination. These systems al-low for fine-tuned exogenous expression of proteins, but as ageneral consideration, all exogenous sequences includingtags and fusions may need to be codon optimized forTetrahymena.

Exogenous expression of Tetrahymena genes is controlledby either constitutive or inducible promoters. In Tetrahymena,the major constitutive promoter utilized is the histone H4 pro-moter (Kahn et al. 1993). Different promoters can be used forselective cell cycle- and developmental-specific gene expres-sion. Additionally, strongly inducible promoters provide directcontrol over the temporal expression and the relative protein lev-els. This is typically controlled by using various metallothionein(MTT) gene promoters, which can be induced by the additionof cadmium, mercury, copper, or zinc. The MTT1 promoter,induced by cadmium chloride (CdCl2) (Shang et al. 2002), isthemost commonly used; however, copper inducible versionsalso exist (Boldrin et al. 2008). In addition, the TetrahymenaHsp70-2 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 util-ity in exogenous gene expression will become more accessible.

Protein tagging: A diverse collection of reagents for proteintagging are available and freely shared by the Tetrahymenaresearch community. These reagents facilitate the generationof constructs for protein localization using light and electronmicroscopy, biochemical purifications, and protein dynamicsstudies. Many of these reagents and Tetrahymena strains areavailable through the National Tetrahymena Stock Center.Codon optimized EGFP and mCherry constructs are availablefor live and fixed cell imaging of protein localization inTetrahymena cells using both fluorescence localization andimmuno-EM (IEM) to visualize the localization of proteins(Kataoka et al. 2010; Stemm-Wolf et al. 2013). Several ofthe localization tags are also amenable to biochemical puri-fications with localization and purification (LAP) tags thathave been adapted from mammalian LAP tags and thencodon optimized for Tetrahymena (Rigaut et al. 1999;Cheeseman and Desai 2005; Pearson et al. 2009; Wineyet al. 2012). Additional purification strategies have been de-veloped apart from the LAP tag (Couvillion and Collins2012). These tags provide the ability to couple powerfulin vivo cell biology experiments with the detail gleaned fromreductionist biochemical approaches.

Conclusions and Perspectives

Tetrahymena’s evolutionary divergence from the more com-monly studied model organisms, while retaining most of thecell biology inherited from the last eukaryotic common ances-tor, is amajor advantage as amodel system.Within the contextof its unusual life cycle, genetic and molecular manipulationsthat are impossible in other organisms enable Tetrahymena toreveal universally conserved cellular processes. Harnessingthis unique power continues to underlie Tetrahymena’s utilityto researchers working on diverse scientific problems.

What does the future hold for Tetrahymena research? Cer-tainly, advances will be built upon existing methods, and thequest for more information, higher resolution, and less am-biguous results is never ending. The Tetrahymena researchcommunity strives to improve molecular tools such as RNAiknockdown strategies, to develop clustered regularly inter-spaced short palindromic repeat (CRISPR) technology ame-nable to MIC genome editing, and to expand the repertoire ofinducible promoter systems. Additionally, optogenetic anddimerization targeting tools will be used to induce proteinlocalization or gene expression upon light and chemical ex-posure. Along with the development of quantitative fixed andlive cell imaging methods (a significant hurdle for a motileorganism), these efforts will expand the reverse geneticstools in the Tetrahymena toolbox. In forward genetics ap-proaches, initiating synthetic genetic array (SGA) analysessimilar to those that were successfully performed in othermodel organisms (for review see Dixon et al. 2009) will beinvaluable to identify genes that contribute to similar

processes and will build systems-level genetic networks. Ad-ditionally, Tetrahymena researchers will benefit from im-proved next generation sequencing methods and theiraccompanying computational tools. Because many of thesetechnologies have not necessarily been developed for Tetra-hymena, adapting them to the organismwill be amajor focus,and one that is quite promising and reachable.

Although Tetrahymena was established as an advanta-geous model organism in the early 1920s (Collins andGorovsky 2005), its utility to researchers has only increasedover the last century. Tetrahymena continues to be a vehiclefor groundbreaking discoveries in structural, molecular, andcellular biology because of its ability to be genetically manip-ulated, biochemically deconstructed, and visually inspected.Together, these unique advantages give us an unprecedentedview into the inner workings of the ciliate’s complex life, andit, in turn, has taught us about the mechanisms that governour own. And, the end is not in sight; as more genetics andgenomics tools emerge, we see Tetrahymena continuing tokeep its place as one of the fastest, most accessible, and rel-evant model organisms to address crucial biological ques-tions that must be answered at the lightning speed ofscience today.

Acknowledgments

The authors thank Domenico “Nick” Galati, Brian Bayless,Aaron Turkewitz, and Alex Stemm-Wolf for critical readingand comments, and A. Turkewitz and J. Gaertig for commu-nicating unpublished results. C.G.P. is funded by the Na-tional Institutes of Health–National Institute of GeneralMedical Sciences GM099820, Pew Biomedical Scholars Pro-gram, and the Boettcher Foundation. E.O. is funded by aUniversity of California Santa Barbara Academic Senate Re-search Award.

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