Chengjie Fu

Transcriptome Analysis of the Model Protozoan, Tetrahymena thermophila, Using Deep RNA Sequencing

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

Tetrahymena Functional Genomics Database (TetraFGD): an integrated resource for Tetrahymena functional genomics

The ciliated protozoan Tetrahymena thermophila is a useful unicellular model organism for studies of eukaryotic cellular and molecular biology. Researches on T. thermophila have contributed to a series of remarkable basic biological principles. After the macronuclear genome was sequenced, substantial progress has been made in functional genomics research on T. thermophila, including genome-wide microarray analysis of the T. thermophila life cycle, a T. thermophila gene network analysis based on the microarray data and transcriptome analysis by deep RNA sequencing. To meet the growing demands for the Tetrahymena research community, we integrated these data to provide a public access database: Tetrahymena functional genomics database (TetraFGD). TetraFGD contains three major resources, including the RNA-Seq transcriptome, microarray and gene networks. The RNA-Seq data define gene structures and transcriptome, with special emphasis on exon–intron boundaries; the microarray data describe gene expression of 20 time points during three major stages of the T. thermophila life cycle; the gene network data identify potential gene–gene interactions of 15 049 genes. The TetraFGD provides user-friendly search functions that assist researchers in accessing gene models, transcripts, gene expression data and gene–gene relationships. In conclusion, the TetraFGD is an important functional genomic resource for researchers who focus on the Tetrahymena or other ciliates. Database URL: http://tfgd.ihb.ac.cn/

Genome-wide identification and characterization of cytochrome P450 monooxygenase genes in the ciliate Tetrahymena thermophila

BackgroundCytochrome P450 monooxygenases play key roles in the metabolism of a wide variety of substrates and they are closely associated with endocellular physiological processes or detoxification metabolism under environmental exposure. To date, however, none has been systematically characterized in the phylum Ciliophora. T. thermophila possess many advantages as a eukaryotic model organism and it exhibits rapid and sensitive responses to xenobiotics, making it an ideal model system to study the evolutionary and functional diversity of the P450 monooxygenase gene family.ResultsA total of 44 putative functional cytochrome P450 genes were identified and could be classified into 13 families and 21 sub-families according to standard nomenclature. The characteristics of both the conserved intron-exon organization and scaffold localization of tandem repeats within each P450 family clade suggested that the enlargement of T. thermophila P450 families probably resulted from recent separate small duplication events. Gene expression patterns of all T. thermophila P450s during three important cell physiological stages (vegetative growth, starvation and conjugation) were analyzed based on EST and microarray data, and three main categories of expression patterns were postulated. Evolutionary analysis including codon usage preference, site-specific selection and gene-expression evolution patterns were investigated and the results indicated remarkable divergences among the T. thermophila P450 genes.ConclusionThe characterization, expression and evolutionary analysis of T. thermophila P450 monooxygenase genes in the current study provides useful information for understanding the characteristics and diversities of the P450 genes in the Ciliophora, and provides the baseline for functional analyses of individual P450 isoforms in this model ciliate species.

The Giant Zooxanthellae‐Bearing Ciliate Maristentor dinoferus (Heterotrichea) is Closely Related to Folliculinidae

Abstract. The small subunit rDNA sequence of Maristentor dinoferus (Lobban, Schefter, Simpson, Pochon, Pawlowski, and Foissner, 2002) was determined and compared with sequences from other Heterotrichea and Karyorelictea. Maristentor resembles Stentor in basic morphology and had been provisionally assigned to Stentoridae. However, our phylogenetic analyses show that Maristentor is more closely related to Folliculinidae. Our results support the creation of a separate family for Maristentor, Maristentoridae n. fam., and also confirm the phylogenetic grouping of Folliculindae, Stentoridae, Blepharismidae, and Maristentoridae, which we informally call ‘stentorids’. Maristentor, rather than Stentor itself, appears to be most significant in understanding the origins of folliculinids from their aloricate ancestors. Our analyses suggest continued uncertainty in the exact placement of the root of heterotrichs with this phylogenetic marker.

Gene network landscape of the ciliate Tetrahymena thermophila.

Background Genome-wide expression data of gene microarrays can be used to infer gene networks. At a cellular level, a gene network provides a picture of the modules in which genes are densely connected, and of the hub genes, which are highly connected with other genes. A gene network is useful to identify the genes involved in the same pathway, in a protein complex or that are co-regulated. In this study, we used different methods to find gene networks in the ciliate Tetrahymena thermophila, and describe some important properties of this network, such as modules and hubs. Methodology/Principal Findings Using 67 single channel microarrays, we constructed the Tetrahymena gene network (TGN) using three methods: the Pearson correlation coefficient (PCC), the Spearman correlation coefficient (SCC) and the context likelihood of relatedness (CLR) algorithm. The accuracy and coverage of the three networks were evaluated using four conserved protein complexes in yeast. The CLR network with a Z-score threshold 3.49 was determined to be the most robust. The TGN was partitioned, and 55 modules were found. In addition, analysis of the arbitrarily determined 1200 hubs showed that these hubs could be sorted into six groups according to their expression profiles. We also investigated human disease orthologs in Tetrahymena that are missing in yeast and provide evidence indicating that some of these are involved in the same process in Tetrahymena as in human. Conclusions/Significance This study constructed a Tetrahymena gene network, provided new insights to the properties of this biological network, and presents an important resource to study Tetrahymena genes at the pathway level.

Cloning, characterization, and gene expression analysis of a novel cadmium metallothionein gene in Tetrahymena pigmentosa

A novel cadmium-inducible metallothionein (MT) gene (Tpig-MT1) was cloned and sequenced from the ciliate Tetrahymena pigmentosa. The number of deduced amino acids is 118. The polypeptide possesses CCC and CC clusters characteristic of typical Tetrahymena Cd-inducible MTs. The structure of Tpig-MT1 is different from the reported Cd-MT in T. pyriformis, T. thermophila and T. pigmentosa. Tpig-MT1 contains two intragenic tandem repeats with 72.9% identity described as Tpig-MT1 (repeat A1) and Tpig-MT1 (repeat A2). The transcriptional response of Tpig-MT1 gene to different heavy metals (Cd, Cu, Zn, Hg, Pb) and oxidative stress (H(2)O(2)) was measured using real-time quantitative PCR. The results showed that the gene was quickly induced (1 h) by the five heavy metals and the order of expression level was Hg>Pb>Cd>Cu>Zn. The induction effect of H(2)O(2) was 5-fold after about 15 min, but soon decreased to a non-significant level (30 min). The genetic diversity of Tetrahymena MT genes is discussed in relation to the unique structure of the Tpig-MT1 gene and other reported Cd-MT isoforms.

Genome-wide identification and evolution of ATP-binding cassette transporters in the ciliate Tetrahymena thermophila : A case of functional divergence in a multigene family

BackgroundIn eukaryotes, ABC transporters that utilize the energy of ATP hydrolysis to expel cellular substrates into the environment are responsible for most of the efflux from cells. Many members of the superfamily of ABC transporters have been linked with resistance to multiple drugs or toxins. Owing to their medical and toxicological importance, members of the ABC superfamily have been studied in several model organisms and warrant examination in newly sequenced genomes.ResultsA total of 165 ABC transporter genes, constituting a highly expanded superfamily relative to its size in other eukaryotes, were identified in the macronuclear genome of the ciliate Tetrahymena thermophila. Based on ortholog comparisons, phylogenetic topologies and intron characterizations, each highly expanded ABC transporter family of T. thermophila was classified into several distinct groups, and hypotheses about their evolutionary relationships are presented. A comprehensive microarray analysis revealed divergent expression patterns among the members of the ABC transporter superfamily during different states of physiology and development. Many of the relatively recently formed duplicate pairs within individual ABC transporter families exhibit significantly different expression patterns. Further analysis showed that multiple mechanisms have led to functional divergence that is responsible for the preservation of duplicated genes.ConclusionGene duplications have resulted in an extensive expansion of the superfamily of ABC transporters in the Tetrahymena genome, making it the largest example of its kind reported in any organism to date. Multiple independent duplications and subsequent divergence contributed to the formation of different families of ABC transporter genes. Many of the members within a gene family exhibit different expression patterns. The combination of gene duplication followed by both sequence divergence and acquisition of new patterns of expression likely plays a role in the adaptation of Tetrahymen a to its environment.

A P450 gene associated with robust resistance to DDT in ciliated protozoan, Tetrahymena thermophila by efficient degradation

Analysis of metabolic mechanisms of dichlorodiphenyltrichloroethane (DDT) accumulation and degradation in microorganisms, which could be used to reduce its hazard to higher organisms at the higher in the food chain, have not been investigated. Robust resistance to DDT (grows well in 256 mg/L DDT) and a surprising ability to degrade DDT (more than 70% DDT within 4h) were found in the ciliated protozoan Tetrahymena thermophila. A P450 gene (CYP5013C2) was found to respond specifically to DDT treatment. In the presence of 256 mg/L DDT, cells with overexpressing CYP5013C2 (p450-OE) grew faster and degraded DDT more efficiently than wild-type (WT) cells, while cells with CYP5013C2 partially knocked down (p450-KD) grew slower and exhibited reduced ability to degrade DDT compared to WT cells. Both dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD) were detected in cells after exposure to DDT, and the concentration of DDD in the p450-OE strain gradually decreased from 0.5 to 4h. Thus, we argue that this P450 gene (CYP5013C2), by efficiently degrading DDT to DDD, is associated with robust resistance to DDT in Tetrahymena, and that a strain overexpressing this gene has the potential to serve as bioreactor that degrades environmental DDT.

Nonsense-mediated mRNA decay in Tetrahymena is EJC independent and requires a protozoa-specific nuclease

Abstract Nonsense-mediated mRNA decay (NMD) is essential for removing premature termination codon-containing transcripts from cells. Studying the NMD pathway in model organisms can help to elucidate the NMD mechanism in humans and improve our understanding of how this biologically important process has evolved. Ciliates are among the earliest branching eukaryotes; their NMD mechanism is poorly understood and may be primordial. We demonstrate that highly conserved Upf proteins (Upf1a, Upf2 and Upf3) are involved in the NMD pathway of the ciliate, Tetrahymena thermophila. We further show that a novel protozoa-specific nuclease, Smg6L, is responsible for destroying many NMD-targeted transcripts. Transcriptome-wide identification and characterization of NMD-targeted transcripts in vegetative Tetrahymena cells showed that many have exon–exon junctions downstream of the termination codon. However, Tetrahymena may lack a functional exon junction complex (EJC), and the Tetrahymena ortholog of an EJC core component, Mago nashi (Mag1), is dispensable for NMD. Therefore, NMD is EJC independent in this early branching eukaryote.

Cd-Metallothioneins in Three Additional Tetrahymena Species: Intragenic Repeat Patterns and Induction by Metal Ions

Ciliate metallothioneins (MTs) possess many unique features compared to the “classic” MTs in other organisms, but they have only been studied in a small number of species. In this study, we investigated cDNAs encoding subfamily 7a metallothioneins (CdMTs) in three Tetrahymena species (T. hegewischi, T. malaccensis, and T. mobilis). Four CdMT genes (ThegMT1, ThegMT2, TmalMT1, and TmobMT1) were cloned and characterized. They share high sequence similarity to previously identified subfamily 7a MT members. Tetrahymena CdMTs exhibit a remarkably regular intragenic repeat homology. The CdMT sequences were divided into two main types of modules, which had been previously described, and which we name “A” and “B”. ThegMT2 was identified as the first MT isoform solely composed of module “B”. A phylogenetic analysis of individual modules of every characterized Tetrahymena CdMT rigorously documents the conclusion that modules are important units of CdMT evolution, which have undergone frequent and rapid gain/loss and shuffling. The transcriptional activity of the four newly identified genes was measured under different heavy metal exposure (Cd, Cu, Zn, Pb) using real‐time quantitative PCR. The results showed that these genes were differentially induced after short (1 h) or long (24 h) metal exposure. The evolutionary diversity of Tetrahymena CdMTs is further discussed with regard to their induction by metal ions.