Novel requirements for HAP2/GCS1-mediated gamete fusion in .

The ancestral gamete fusion protein, HAP2/GCS1, plays an essential role in fertilization in a broad range of taxa. To identify factors that may regulate HAP2/GCS1 activity, we screened mutants of the ciliate for behaviors that mimic knockout phenotypes in this species. Using this approach, we identified two new genes, and , whose products are necessary for membrane pore formation following mating type recognition and adherence.

Molecular identification of Tetrahymena species.

Mitochondrial cox1 689 bp barcodes are routinely used for identification of Tetrahymena species. Here, we examine whether two shorter nuclear sequences, the 5.8S rRNA gene region and the intergenic region between H3 and H4 histone genes, might also be useful either singly or in combination with each other or cox1.

Tetrahymena thermophila granule lattice protein 3 improves solubility of sexual stage malaria antigens expressed in Escherichia coli.

The requirement for low cost manufacturing makes bacterial cells a logical platform for the production of recombinant subunit vaccines for malaria. However, protein solubility has been a major stumbling block with prokaryotic expression systems. Notable examples include the transmission blocking vaccine candidates, Pfs25 and Pfs48/45, which are almost entirely insoluble when expressed as recombinant proteins in Escherichia coli.

Characterization and immune regulation role of an immobilization antigen from Cryptocaryon irritans on groupers.

Immobilization antigens (i-antigens) are surface membrane proteins that are widely recognized to be the ideal candidates as vaccines antigens for immunization against Cryptocaryon irritans. In this study, we cloned a putative i-antigen gene from C. irritans, which was expressed in all three stages of the C.

A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3.

Identifying monoclonal antibodies that block human voltage-gated ion channels (VGICs) is a challenging endeavor exacerbated by difficulties in producing recombinant ion channel proteins in amounts that support drug discovery programs. We have developed a general strategy to address this challenge by combining high-level expression of recombinant VGICs in Tetrahymena thermophila with immunization of phylogenetically diverse species and unique screening tools that allow deep-mining for antibodies that could potentially bind functionally important regions of the protein. Using this approach, we targeted human Kv1.

Structure-Function Studies Link Class II Viral Fusogens with the Ancestral Gamete Fusion Protein HAP2.

The conserved transmembrane protein, HAP2/GCS1, has been linked to fertility in a wide range of taxa and is hypothesized to be an ancient gamete fusogen. Using template-based structural homology modeling, we now show that the ectodomain of HAP2 orthologs from Tetrahymena thermophila and other species adopt a protein fold remarkably similar to the dengue virus E glycoprotein and related class II viral fusogens. To test the functional significance of this predicted structure, we developed a flow-cytometry-based assay that measures cytosolic exchange across the conjugation junction to rapidly probe the effects of HAP2 mutations in the Tetrahymena system.

Diversity and Universality of Endosymbiotic in the Fish Parasite .

Although the presence of endosymbiotic rickettsial bacteria, specifically Megaira, has been reported in diverse habitats and a wide range of eukaryotic hosts, it remains unclear how broadly Megaira are distributed in a single host species. In this study we seek to address whether Megaira are present in most, if not all isolates, of the parasitic ciliate . Conserved regions of bacterial 16S rRNA genes were either PCR amplified, or assembled from deep sequencing data, from 18 isolates/populations of sampled worldwide (Brazil, Taiwan, and USA).

Structure of the germline genome of and relationship to the massively rearranged somatic genome.

The germline genome of the binucleated ciliate 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 chromosome structure, locally and globally.

Molecular genetic diversity and characterization of conjugation genes in the fish parasite Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis is the etiologic agent of "white spot", a commercially important disease of freshwater fish. As a parasitic ciliate, I. multifiliis infects numerous host species across a broad geographic range.

Function of the male-gamete-specific fusion protein HAP2 in a seven-sexed ciliate.

HAP2, a male-gamete-specific protein conserved across vast evolutionary distances, has garnered considerable attention as a potential membrane fusogen required for fertilization in taxa ranging from protozoa and green algae to flowering plants and invertebrate animals [1-6]. However, its presence in Tetrahymena thermophila, a ciliated protozoan with seven sexes or mating types that bypasses the production of male gametes, raises interesting questions regarding the evolutionary origins of gamete-specific functions in sexually dimorphic species. Here we show that HAP2 is expressed in all seven mating types of T.

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.

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.

Comparative genomics of the pathogenic ciliate Ichthyophthirius multifiliis, its free-living relatives and a host species provide insights into adoption of a parasitic lifestyle and prospects for disease control.

Background: Ichthyophthirius multifiliis, commonly known as Ich, is a highly pathogenic ciliate responsible for 'white spot', a disease causing significant economic losses to the global aquaculture industry. Options for disease control are extremely limited, and Ich's obligate parasitic lifestyle makes experimental studies challenging. Unlike most well-studied protozoan parasites, Ich belongs to a phylum composed primarily of free-living members.

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.

Functional characterization of the 5'-upstream region of MTT5 metallothionein gene from Tetrahymena thermophila.

Metallothioneins are ubiquitous small, cysteine-rich, metal-binding proteins that play important roles in intracellular metal homeostasis and detoxification. Very few data are available on the promoter region and the mechanism of metallothionein transcription in Protozoa. In this study, we focused on Tetrahymena thermophila MTT5 5'-flanking region.

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

Background: Tetrahymena 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.

MTT2, a copper-inducible metallothionein gene from Tetrahymena thermophila.

Metallothioneins (MTs) are ubiquitous, cysteine-rich, metal-binding proteins whose transcriptional activation is induced by a variety of stimuli, in particular heavy metals such as cadmium, copper and zinc. Here we describe the sequence and organization of a novel copper-inducible metallothionein gene (MTT2) from Tetrahymena thermophila. Based on its deduced sequence, the gene encodes a protein 108 amino acids, containing 29 cysteine residues (30%) arranged in motifs characteristic of vertebrate and invertebrate MTs.

Metallothionein gene from Tetrahymena thermophila with a copper-inducible-repressible promoter.

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.

Genome-wide characterization of tetrahymena thermophila chromosome breakage sites. I. Cloning and identification of functional sites.

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.

Genome-wide characterization of Tetrahymena thermophila chromosome breakage sites. II. Physical and genetic mapping.

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.

TIF1 Represses rDNA replication initiation, but promotes normal S phase progression and chromosome transmission in Tetrahymena.

The non-ORC protein, TIF1, recognizes sequences in the Tetrahymena thermophila ribosomal DNA (rDNA) minichromosome that are required for origin activation. We show here that TIF1 represses rDNA origin firing, but is required for proper macronuclear S phase progression and division. TIF1 mutants exhibit an elongated macronuclear S phase and diminished rate of DNA replication.