Donna Cassidy-Hanley

Long-term storage.

Publisher Summary This chapter presents a simple method for long-term storage of Tetrahymena thermophila , based on freezing starved cells. Starving cells before freezing greatly enhances the recovery of cells and eliminates the need for complicated freezing apparatuses or protocols. Tetrahymena thermophila and other ciliates require that growth be minimized or even eliminated during long-term storage: extended growth leads to strain degradation. Cultures of fresh exconjugants exhibit normal growth rates and can undergo normal conjugation, following a strain-specific period of immaturity. However, fertility begins to drop within a year or less of continuous growth in the laboratory; matings increasingly tend to undergo the abortive pathway called genomic exclusion. Cells still form pairs in these matings, but they fail to contribute any genome to the progeny. In addition, because they are not expressed and are therefore not selected against, micronuclear deletions accumulate with clonal age. The successful freezing of eukaryotic cells in liquid nitrogen is dependent on stringent control of a number of parameters, including: (1) the physiological state of the cells at the time of freezing; (2) the cryoprotectant and medium in which the cells are frozen; (3) the rate of cooling; (4) the temperature at which the cells are ultimately stored; and (5) the rate and temperature of thawing.

Biolistic transformation of macro- and micronuclei.

Publisher Summary This chapter presents methods for the biolistic transformation of both macro- and micronuclei of Tetrahymena thermophila . DNA-coated gold particles are fired into starved vegetative cells and, during late phases of conjugation, into mating cells to introduce gene sequences into the macronucleus. Early stages of mating are bombarded to add genes to the micronucleus. Methods for cell preparation and a detailed protocol for shooting and recovering transformed cells are also presented. Development of successful transformation techniques in any ciliate is complicated by the separation of somatic and germinal functions into two distinctly different nuclei: the polyploid macronucleus, which solely directs the cells phenotype, and the transcriptionally inert diploid micronucleus, which contributes the genotype to subsequent sexual generations. Each of these nuclei is a unique target for DNA transformation, requires different strategies for the introduction of genes, and responds in different ways following successful transformation.

Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure

BackgroundTetrahymena thermophila, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a germline micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymenas coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing.ResultsWe addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified.ConclusionWe report here significant progress in genome closure and reannotation of Tetrahymena thermophila. Our experience to date suggests that complete closure of the MAC genome is attainable. Using the new EST evidence, automated and manual curation has resulted in substantial improvements to the over 24,000 gene models, which will be valuable to researchers studying this model organism as well as for comparative genomics purposes.

Endosymbiotic Bacteria in the Parasitic Ciliate Ichthyophthirius multifiliis

ABSTRACT Endosymbiotic bacteria were identified in the parasitic ciliate Ichthyophthirius multifiliis, a common pathogen of freshwater fish. PCR amplification of DNA prepared from two isolates of I. multifiliis, using primers that bind conserved sequences in bacterial 16S rRNA genes, generated an ∼1,460-bp DNA product, which was cloned and sequenced. Sequence analysis demonstrated that 16S rRNA gene sequences from three classes of bacteria were present in the PCR product. These included Alphaproteobacteria (Rickettsiales), Sphingobacteria, and Flavobacterium columnare. DAPI (4′,6-diamidino-2-phenylindole) staining showed endosymbionts dispersed throughout the cytoplasm of trophonts and, in most, but not all theronts. Endosymbionts were observed by transmission electron microscopy in the cytoplasm, surrounded by a prominent, electron-translucent halo characteristic of Rickettsia. Fluorescence in situ hybridization demonstrated that bacteria from the Rickettsiales and Sphingobacteriales classes are endosymbionts of I. multifiliis, found in the cytoplasm, but not in the macronucleus or micronucleus. In contrast, F. columnare was not detected by fluorescence in situ hybridization. It likely adheres to I. multifiliis through association with cilia. The role that endosymbiotic bacteria play in the life history of I. multifiliis is not known.

Metallothionein Gene from Tetrahymena thermophila with a Copper-Inducible-Repressible Promoter

ABSTRACT We describe a novel metallothionein gene from Tetrahymena thermophila that has a strong copper-inducible promoter. This promoter can be turned on and off rapidly, making it a useful system for induction of ectopic gene expression in Tetrahymena and enhancing its applications in cell and molecular biology, as well as biotechnology.

Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome

The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymena’s germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum. DOI: http://dx.doi.org/10.7554/eLife.19090.001

Tetrahymena in the laboratory: strain resources, methods for culture, maintenance, and storage.

The ciliated protozoan Tetrahymena thermophila has been an important model system for biological research for many years. During that time, a variety of useful strains, including highly inbred stocks, a collection of diverse mutant strains, and wild cultivars from a variety of geographical locations have been identified. In addition, thanks to the efforts of many different laboratories, optimal conditions for growth, maintenance, and storage of Tetrahymena have been worked out. To facilitate the efficient use of Tetrahymena, especially by those new to the system, this chapter presents a brief description of many available Tetrahymena strains and lists possible resources for obtaining viable cultures of T. thermophila and other Tetrahymena species. Descriptions of commonly used media, methods for cell culture and maintenance, and protocols for short- and long-term storage are also presented.

A Simple, Efficient Technique for Freezing Tetrahymena thermophila

ABSTRACT. We have developed a simple, efficient procedure for the long term freezing of Tetrahymena thermophila in liquid nitrogen. This technique yields excellent recovery of viable cells with all strains tested and does not require the use of a controlled rate low temperature freezer. To optimize the freezing technique, we have examined the effects of varying a number of parameters, including the physiological state of the cells prior to freezing, the time of exposure to cryoprotectant, and the rate of freezing and thawing. the frequency of viable cell recovery following freezing using this technique has been tested for a variety of different cell lines.

Transcriptional profiling of stage specific gene expression in the parasitic ciliate Ichthyophthirius multifiliis

The parasitic ciliate, Ichthyophthirius multifiliis (Ich), is among the most important protozoan pathogens of freshwater fish. Ichthyophthirius cannot be grown in cell culture, and the development of effective prophylactic and therapeutic treatments has been hampered by a lack of information regarding genes involved in virulence, differentiation and growth. To help address this issue, we have generated EST libraries from the two major stages of the parasite life cycle that infect and develop within host tissues. A total of 25,084 ESTs were generated from non-normalized libraries prepared from polyA+ RNA of infective theronts and host-associated trophonts, respectively. Cluster analysis identified 5311 unique transcripts (UniScripts), of which 2091 were contigs and 3220 singletons. Extrapolation of the data based on rates of EST discovery suggests that more than half the expected protein-coding genes of I. multifiliis are represented in this data. BLASTX comparisons against GenBank nr, UniProtKB (SwissProt and TrEMBL), as well as Tetrahymena thermophila, Plasmodium falciparum, and Paramecium tetraurelia protein databases produced 3694 significant (E-value ≤1e(-10)) hits, of which 1178 were annotated using gene ontology (GO) analysis. A high proportion of UniScripts (63%) showed similarity to other ciliate proteins. When combined with expression profiling data, GO ontology analysis of Biological Process, Cellular Component, and Molecular Function revealed interesting differences in gene families expressed in the two stages. Indeed, the most abundant transcripts were highly stage-specific and coincided with the metabolic activities associated with each stage. This work provides an effective genomics resource to further our understanding of Ichthyophthirius biology, and lays the groundwork for the identification of potential drug targets and vaccines candidates for the control of this devastating fish pathogen.

Tetrahymena in the Classroom

Tetrahymena has been a useful model in basic research in part due to the fact it is easy to grow in culture and exhibits a range of complex processes, all within a single cell. For these same reasons Tetrahymena has shown enormous potential as a teaching tool for fundamental principles of biology at multiple science education levels that can be integrated into K-12 classrooms and undergraduate and graduate college laboratory courses. These Tetrahymena-based teaching modules are inquiry-based experiences that are also effective at teaching scientific concepts, retaining students in science, and exciting students about the scientific process. Two learning communities have been developed that utilize Tetrahymena-based teaching modules. Advancing Secondary Science Education with Tetrahymena (ASSET) and the Ciliate Genomics Consortium (CGC) have developed modules for K-12 students and college-level curriculums, respectively. These modules range from addressing topics in ecology, taxonomy, and environmental toxicity to more advanced concepts in biochemistry, proteomics, bioinformatics, cell biology, and molecular biology. An overview of the current modules and their learning outcomes are discussed, as are assessment, dissemination, and sustainability strategies for K-12 and college-level curriculum.