[Show abstract][Hide abstract] ABSTRACT: Nanoscale titanium dioxide (TiO2) is increasingly used in consumer goods and entering waste streams, thereby exposing and potentially affecting environmental microbes. Protozoans could either directly uptake TiO2 from water and sediments or acquire TiO2 during bactivory of TiO2-encrusted bacteria. Here, the TiO2 exposure route to the ciliated protozoan Tetrahymena thermophila was varied, and growth, plus uptake and accumulation of TiO2 in T.thermophila, were measured. While TiO2 did not affect T. thermophila swimming or cellular morphology, direct TiO2 exposure in rich growth medium resulted in a lower population yield. When TiO2 exposure was by bactivory of Pseudomonas aeruginosa, T. thermophila population yield plus growth rate were reduced compared to bactivory of non-TiO2-encrusted bacteria. Regardless of feeding mode, T. thermophila cells internalized TiO2 into their food vacuoles. Biomagnification of TiO2 was not observed, attributed to the observation that TiO2 appeared unable to cross the food vacuole membrane and enter the cytoplasm. Nevertheless, our findings imply that TiO2 could be transferred into higher trophic levels within food webs and that the food web could be affected by decreased growth rate/yield of organisms near the base of the web.
Applied and Environmental Microbiology 07/2013; · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The unicellular eukaryote Tetrahymena thermophila has seven mating types. Cells can mate only when they recognize cells of a different mating type as non-self. As a ciliate, Tetrahymena separates its germline and soma into two nuclei. During growth the somatic nucleus is responsible for all gene transcription while the germline nucleus remains silent. During mating, a new somatic nucleus is differentiated from a germline nucleus and mating type is decided by a stochastic process. We report here that the somatic mating type locus contains a pair of genes arranged head-to-head. Each gene encodes a mating type-specific segment and a transmembrane domain that is shared by all mating types. Somatic gene knockouts showed both genes are required for efficient non-self recognition and successful mating, as assessed by pair formation and progeny production. The germline mating type locus consists of a tandem array of incomplete gene pairs representing each potential mating type. During mating, a complete new gene pair is assembled at the somatic mating type locus; the incomplete genes of one gene pair are completed by joining to gene segments at each end of germline array. All other germline gene pairs are deleted in the process. These programmed DNA rearrangements make this a fascinating system of mating type determination.
[Show abstract][Hide abstract] ABSTRACT: The differentiation of germline and somatic genomes in Tetrahymena thermophila results in two independent systems of genetic transmission. One is the conserved, sexual Mendelian genetics system of the germline genome. The other is a random genetic assortment mechanism, which operates in the somatic genome during asexual propagation. This chapter describes both systems, their interplay, and how they are exploited to construct useful biological reagents and powerful tools, which can be used to answer a variety of experimental questions.
Methods in cell biology 01/2012; 109:301-25. · 1.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ciliated protozoan Tetrahymena thermophila is a well-studied single-celled eukaryote model organism for cellular and molecular biology. However, the lack of extensive T. thermophila cDNA libraries or a large expressed sequence tag (EST) database limited the quality of the original genome annotation.
This RNA-seq study describes the first deep sequencing analysis of the T. thermophila transcriptome during the three major stages of the life cycle: growth, starvation and conjugation. Uniquely mapped reads covered more than 96% of the 24,725 predicted gene models in the somatic genome. More than 1,000 new transcribed regions were identified. The great dynamic range of RNA-seq allowed detection of a nearly six order-of-magnitude range of measurable gene expression orchestrated by this cell. RNA-seq also allowed the first prediction of transcript untranslated regions (UTRs) and an updated (larger) size estimate of the T. thermophila transcriptome: 57 Mb, or about 55% of the somatic genome. Our study identified nearly 1,500 alternative splicing (AS) events distributed over 5.2% of T. thermophila genes. This percentage represents a two order-of-magnitude increase over previous EST-based estimates in Tetrahymena. Evidence of stage-specific regulation of alternative splicing was also obtained. Finally, our study allowed us to completely confirm about 26.8% of the genes originally predicted by the gene finder, to correct coding sequence boundaries and intron-exon junctions for about a third, and to reassign microarray probes and correct earlier microarray data.
RNA-seq data significantly improve the genome annotation and provide a fully comprehensive view of the global transcriptome of T. thermophila. To our knowledge, 5.2% of T. thermophila genes with AS is the highest percentage of genes showing AS reported in a unicellular eukaryote. Tetrahymena thus becomes an excellent unicellular model eukaryote in which to investigate mechanisms of alternative splicing.
PLoS ONE 01/2012; 7(2):e30630. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genetically programmed DNA rearrangements can regulate mRNA expression at an individual locus or, for some organisms, on a genome-wide scale. Ciliates rely on a remarkable process of whole-genome remodeling by DNA elimination to differentiate an expressed macronucleus (MAC) from a copy of the germline micronucleus (MIC) in each cycle of sexual reproduction. Here we describe results from the first high-throughput sequencing effort to investigate ciliate genome restructuring, comparing Sanger long-read sequences from a Tetrahymena thermophila MIC genome library to the MAC genome assembly. With almost 25% coverage of the unique-sequence MAC genome by MIC genome sequence reads, we created a resource for positional analysis of MIC-specific DNA removal that pinpoints MAC genome sites of DNA elimination at nucleotide resolution. The widespread distribution of internal eliminated sequences (IES) in promoter regions and introns suggests that MAC genome restructuring is essential not only for what it removes (for example, active transposons) but also for what it creates (for example, splicing-competent introns). Consistent with the heterogeneous boundaries and epigenetically modulated efficiency of individual IES deletions studied to date, we find that IES sites are dramatically under-represented in the ∼25% of the MAC genome encoding exons. As an exception to this general rule, we discovered a previously unknown class of small (<500 bp) IES with precise elimination boundaries that can contribute the 3' exon of an mRNA expressed during genome restructuring, providing a new mechanism for expanding mRNA complexity in a developmentally regulated manner.
[Show abstract][Hide abstract] ABSTRACT: Tetrahymena thermophila is a ciliate--a unicellular eukaryote. Remarkably, every cell maintains differentiated germline and somatic genomes: one silent, the other expressed. Moreover, the two genomes undergo diverse processes, some as extreme as life and death, simultaneously in the same cytoplasm. Conserved eukaryotic mechanisms have been modified in ciliates to selectively deal with the two genomes. We describe research in several areas of Tetrahymena biology, including meiosis, amitosis, genetic assortment, selective nuclear pore transport, somatic RNAi-guided heterochromatin formation, DNA excision and programmed nuclear death by autophagy, which has enriched and broadened knowledge of those mechanisms.
Research in Microbiology 05/2011; 162(6):578-86. · 2.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies have shown that engineered nanomaterials can be transferred from prey to predator, but the ecological impacts of this are mostly unknown. In particular, it is not known if these materials can be biomagnified-a process in which higher concentrations of materials accumulate in organisms higher up in the food chain. Here, we show that bare CdSe quantum dots that have accumulated in Pseudomonas aeruginosa bacteria can be transferred to and biomagnified in the Tetrahymena thermophila protozoa that prey on the bacteria. Cadmium concentrations in the protozoa predator were approximately five times higher than their bacterial prey. Quantum-dot-treated bacteria were differentially toxic to the protozoa, in that they inhibited their own digestion in the protozoan food vacuoles. Because the protozoa did not lyse, largely intact quantum dots remain available to higher trophic levels. The observed biomagnification from bacterial prey is significant because bacteria are at the base of environmental food webs. Our findings illustrate the potential for biomagnification as an ecological impact of nanomaterials.
[Show abstract][Hide abstract] ABSTRACT: The model eukaryote, Tetrahymena thermophila, is the first ciliated protozoan whose genome has been sequenced, enabling genome-wide analysis of gene expression.
A genome-wide microarray platform containing the predicted coding sequences (putative genes) for T. thermophila is described, validated and used to study gene expression during the three major stages of the organism's life cycle: growth, starvation and conjugation.
Of the approximately 27,000 predicted open reading frames, transcripts homologous to only approximately 5900 are not detectable in any of these life cycle stages, indicating that this single-celled organism does indeed contain a large number of functional genes. Transcripts from over 5000 predicted genes are expressed at levels >5x corrected background and 95 genes are expressed at >250x corrected background in all stages. Transcripts homologous to 91 predicted genes are specifically expressed and 155 more are highly up-regulated in growing cells, while 90 are specifically expressed and 616 are up-regulated during starvation. Strikingly, transcripts homologous to 1068 predicted genes are specifically expressed and 1753 are significantly up-regulated during conjugation. The patterns of gene expression during conjugation correlate well with the developmental stages of meiosis, nuclear differentiation and DNA elimination. The relationship between gene expression and chromosome fragmentation is analyzed. Genes encoding proteins known to interact or to function in complexes show similar expression patterns, indicating that co-ordinate expression with putative genes of known function can identify genes with related functions. New candidate genes associated with the RNAi-like process of DNA elimination and with meiosis are identified and the late stages of conjugation are shown to be characterized by specific expression of an unexpectedly large and diverse number of genes not involved in nuclear functions.
PLoS ONE 02/2009; 4(2):e4429. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metallothioneins are ubiquitous small, cysteine-rich, multifunctional proteins which can bind heavy metals.
We report the results of phylogenetic and gene expression analyses that include two new Tetrahymena thermophila metallothionein genes (MTT3 and MTT5). Sequence alignments of all known Tetrahymena metallothioneins have allowed us to rationalize the structure of these proteins. We now formally subdivide the known metallothioneins from the ciliate genus Tetrahymena into two well defined subfamilies, 7a and 7b, based on phylogenetic analysis, on the pattern of clustering of Cys residues, and on the pattern of inducibility by the heavy metals Cd and Cu. Sequence alignment also reveals a remarkably regular, conserved and hierarchical modular structure of all five subfamily 7a MTs, which include MTT3 and MTT5. The former has three modules, while the latter has only two. Induction levels of the three T. thermophila genes were determined using quantitative real time RT-PCR. Various stressors (including heavy metals) brought about dramatically different fold-inductions for each gene; MTT5 showed the highest fold-induction. Conserved DNA motifs with potential regulatory significance were identified, in an unbiased way, upstream of the start codons of subfamily 7a MTs. EST evidence for alternative splicing in the 3' UTR of the MTT5 mRNA with potential regulatory activity is reported.
The small number and remarkably regular structure of Tetrahymena MTs, coupled with the experimental tractability of this model organism for studies of in vivo function, make it an attractive system for the experimental dissection of the roles, structure/function relationships, regulation of gene expression, and adaptive evolution of these proteins, as well as for the development of biotechnological applications for the environmental monitoring of toxic substances.
[Show abstract][Hide abstract] ABSTRACT: Tetrahymena thermophila is the best studied of the ciliates, a diversified and successful lineage of eukaryotic protists. Mirroring the way in which many metazoans partition their germ line and soma into distinct cell types, ciliates separate germ line and soma into two distinct nuclei in a single cell. The diploid, transcriptionally silent micronucleus undergoes meiosis and fertilization during sexual reproduction and determines the genotype of the progeny; in contrast, the expressed macronucleus contains many copies of hundreds of small chromosomes, determines the cell's phenotype, and is inherited only through vegetative reproduction. Here we demonstrate the power of HAPPY physical mapping to aid the complete assembly of T. thermophila macronuclear chromosomes from shotgun sequence scaffolds. The finished genome, one of only two ciliate genomes shotgun sequenced, will shed valuable additional light upon the biology of this extraordinary, diverse, and, from a genomics standpoint, as yet largely unexplored evolutionary branch of eukaryotes.
[Show abstract][Hide abstract] ABSTRACT: The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.
[Show abstract][Hide abstract] ABSTRACT: The insecticide dichlorodiphenyltrichloroethane (DDT) is persistent in the environment, and continues to cause health problems. Tetrahymena has potential as a model organism for assaying low levels of DDT and for analysing the mechanisms of its toxicity. We constructed the suppression subtractive hybridization library of T. thermophila exposed to DDT, and screened out 90 Expressed Sequence Tags whose expressions were significantly up- or downregulated with DDT treatment. From this, a series of important genes related to the DDT metabolism and detoxification were discovered, such as P450 gene, glutathione S-transferase gene and sterol carrier protein 2 gene. Furthermore, their expressions under different concentrations of DDT treatment were detected by real-time fluorescent quantitative PCR. The results show that Tetrahymena is a relevant and useful model organism for detecting DDT in the environment and for discovering biomarkers that can be used to develop specific bio-reporters at the molecular and genomic levels.
[Show abstract][Hide abstract] ABSTRACT: As a typical ciliate, Tetrahymena thermophila is a unicellular eukaryote that exhibits nuclear dimorphism: each cell contains a diploid, germ line micronucleus (MICN) and a polyploid, somatic macronucleus (MACN). During conjugation, when a new MACN differentiates from a mitotic descendant of the diploid fertilization nucleus, the five MICN chromosomes are site-specifically fragmented into 250 to 300 MACN chromosomes. The classic chromosome breakage sequence (CBS) is a 15-bp element (TAAACCAACCTCTTT) reported to be necessary and sufficient for chromosome breakage. To determine whether a CBS is present at every site of chromosome fragmentation and to assess the range of sequence variation tolerated, 31 CBSs were isolated without preconception as to the sequence of the chromosome breakage element. Additional CBS-related sequences were identified in the whole-genome sequence by their similarities to the classic CBS. Forty CBS elements behaved as authentic chromosome breakage sites. The CBS nucleotide sequence is more diverse than previously thought: nearly half of the CBS elements identified by unbiased methods have a variant of the classic CBS. Only an internal 10-bp core is completely conserved, but the entire 15-bp chromosome breakage sequence shows significant sequence conservation. Our results suggest that any one member of the CBS family provides a necessary and sufficient cis element for chromosome breakage. No chromosome breakage element totally unrelated to the classic CBS element was found; such elements, if they exist at all, must be rare.
[Show abstract][Hide abstract] ABSTRACT: The chromosomes of the macronuclear (expressed) genome of Tetrahymena thermophila are generated by developmental fragmentation of the five micronuclear (germline) chromosomes. This fragmentation is site specific and directed by a conserved 15-bp chromosome breakage sequence (Cbs element). This article reports the construction of a library enriched for chromosome breakage junctions and the development of a successful scheme for the genome-wide isolation and characterization of functional Cbs junctions. Twenty-three new Cbs junctions were characterized and each was assigned to a specific micronuclear chromosome or chromosome arm. Two distinct previously unreported variant chromosome breakage sequences were found, each in two or more functional Cbs elements. Analysis of natural Cbs junctions confirmed that microheterogeneity in the macronuclear telomere addition site is associated with chromosome fragmentation. The physical and genetic characterization of these functional chromosome breakage junctions is reported in the accompanying article in this issue. The whole-genome shotgun sequencing and auto-assembly phase of the Tetrahymena Genome Initiative has recently been completed at The Institute for Genome Research (TIGR). By providing unique sequence from the natural ends of macronuclear chromosomes, Cbs junctions characterized in the work reported here will serve as useful sequence tags for relating macro- and micronuclear genetic, physical, and sequence maps.
[Show abstract][Hide abstract] ABSTRACT: The chromosomes of the macronuclear (expressed) genome of Tetrahymena thermophila are generated by developmental fragmentation of the five micronuclear (germline) chromosomes. This fragmentation is site specific, directed by a conserved chromosome breakage sequence (Cbs element). An accompanying article in this issue reports the development of a successful scheme for the genome-wide cloning and identification of functional chromosome breakage sites. This article reports the physical and genetic characterization of 30 functional chromosome breakage junctions. Unique sequence tags and physical sizes were obtained for the pair of macronuclear chromosomes generated by fragmentation at each Cbs. Cbs-associated polymorphisms were used to genetically map 11 junctions to micronuclear linkage groups and macronuclear coassortment groups. Two pairs of junctions showed statistically significant similarity of the sequences flanking the Cbs, suggestive of relatively recent duplications of entire Cbs junctions during Tetrahymena genome evolution. Two macronuclear chromosomes that lose at least one end in an age-related manner were also identified. The whole-genome shotgun sequencing of the Tetrahymena macronucleus has recently been completed at The Institute for Genome Research (TIGR). By providing unique sequence from natural ends of macronuclear chromosomes, Cbs junctions will provide useful sequence tags for relating macro- and micronuclear genetic, physical, and whole-genome sequence maps.
[Show abstract][Hide abstract] ABSTRACT: Tetrahymena thermophila is a valuable model organism for the study of metallothioneins, which play important roles in the metabolism of essential metal ions and in heavy-metal detoxification. Starting from a T. thermophila expressed sequence tag (EST) originally annotated as a Zn–Cu–Cd metallothionein (GenBank Acc #BM394105), we determined the genomic sequence corresponding to this EST and its flaking region. The sequence contains two adjacent genes encoding metallothioneins of the copper-induced type: the MTT2 gene (submitted to GenBank by Boldrin et al. as Cu-inducible while this work was in progress) and a second, novel gene (MTT4), located ∼1.4 kb downstream from and with same orientation as MTT2. Our entire sequenced segment is contained in the T. thermophila whole-genome sequence recently completed at TIGR. Both MTT4 and MTT2 are intronless and encode two nearly identical proteins (two differences out of 108 amino acids). The 5′ and 3′-flanking regions of both the metallothionein genes show significant sequence similarity as well, suggesting recent gene duplication. All glutamines use the CAA codon, reminiscent of codon usage observed in highly expressed Tetrahymena genes. MTT4 and MTT2 map to a 1.5-Mb macronuclear chromosome from the right arm of micronuclear chromosome 2.Supported by NIH grant RR02931. Sequence data were obtained from The Institute for Genomic Research Tetrahymena website, http://www.tigr.org/tdb/e2k1/ttg/
Journal of Eukaryotic Microbiology 03/2005; 52(2). · 2.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Experimental data on mating type determination in T. thermophila, collected by Nanney, Allen, and their collaborators over a period of 25 years, are reinterpreted in the light of our current understanding of macronuclear genetics. A strong case is developed supporting the idea that mating type determination involves the developmental alteration of somatic DNA that occurs regularly in developing macronuclei in conjugating pairs. A. testable DNA deletion/splicing model is developed that although based on a few simple, plausible assumptions, explains the observations remarkably well. The model is in (at least) superficial analogy to the mechanism that must be involved to explain the somatic differentiation and alteration of DNA sequences that ultimately constitute an expressed vertebrate immunoglobulin gene. Because of the genetic, biochemical, and micromanipulative versatility of Tetrahymena, it may well turn out to be a uniquely suitable microbial eukaryotic experimental system for the study of developmental alterations of somatic DNA.
[Show abstract][Hide abstract] ABSTRACT: In Ciliates, the diploid, mitotic, genetically silent micronucleus is the cell's germline, while the macronucleus is the exclusive site of gene expression. Pre-genomic studies in Tetrahymena thermophila have shown that•The macronucleus contains 250–300 chromosomes, derived from the five germline chromosomes;•G2 copies of each chromosome are distributed amitotically (randomly) during macronuclear division.In heterozygotes, this results in the segregation of vegetative descendants expressing only one of the two alleles (assortment). Phenotypic assortment rate measurements for a handful of loci suggested a uniform macronuclear chromosome G1 copy number (∼45 per macronucleus; rDNA minichromosome excepted). The NIGMS/NSF-supported, whole-genome-shotgun sequencing of the T. thermophila macronuclear genome was recently completed at TIGR. (Sequence and scaffold databases, and related resources, are publicly available for blasting or downloading without restriction at http://www.tigr.org/tdb/e2k1/ttg/). Analysis of the depth of sequence coverage shows that (rDNA excepted) at least 95%—and possibly all—of the macronuclear genome is in chromosomes with uniform copy number. This implies the existence of regulatory mechanisms that independently correct the number of copies of nearly 300 different chromosomes after random distribution at every cell cycle.Supported by NIH grant RR02931. Preliminary sequence data were obtained from The Institute for Genomic Research website.
Journal of Eukaryotic Microbiology 01/2005; 52(2). · 2.16 Impact Factor