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PubMed2002

Functional genomics: the coming of age for Tetrahymena thermophila.

Over the past decade, researchers have manipulated the unique biology of Tetrahymena thermophila to generate a premier experimental organism for functional genomic analysis. A diverse array of DNA transformation methods have spearheaded in vivo strategies for discovering and dissecting universal eukaryotic processes, such as telomere addition and chromatin remodeling. Compartmentalization of this protist's genome into two functionally distinct nuclei - the silent 'germline' micronucleus and the transcriptionally active macronucleus - provides a powerful means for controlling the expression of transgenes. Heterokaryons that silently harbor homozygous recessive mutations (including lethal ones) in the germline have been exploited. The coupling of forward and reverse genetic approaches with genomics-based methods for gene discovery presents a bright future for research in this rising model eukaryote.

Animals↗
PubMedDate not supplied

Developmentally programmed excision of internal DNA sequences in Paramecium aurelia.

The development of a new somatic nucleus (macronucleus) during sexual reproduction of the ciliate Paramecium aurelia involves reproducible chromosomal rearrangements that affect the entire germline genome. Macronuclear development can be induced experimentally, which makes P. aurelia an attractive model for the study of the mechanism and the regulation of DNA rearrangements. Two major types of rearrangements have been identified: the fragmentation of the germline chromosomes, followed by the formation of the new macronuclear chromosome ends in association with imprecise DNA elimination, and the precise excision of internal eliminated sequences (IESs). All IESs identified so far are short, A/T rich and non-coding elements. They are flanked by a direct repeat of a 5'-TA-3' dinucleotide, a single copy of which remains at the macronuclear junction after excision. The number of these single-copy sequences has been estimated to be around 60,000 per haploid genome. This review focuses on the current knowledge about the genetic and epigenetic determinants of IES elimination in P. aurelia, the analysis of excision products, and the tightly regulated timing of excision throughout macronuclear development. Several models for the molecular mechanism of IES excision will be discussed in relation to those proposed for DNA elimination in other ciliates.

Animals↗
PubMed2002

Transformation of Paramecium caudatum with a novel expression vector harboring codon-optimized GFP gene.

We have developed a novel expression vector, pTub-tel3, for transformation in Paramecium caudatum. The vector was constructed by cloning P. caudatum alpha-tubulin 5' and 3' non-coding regions. To examine transformation with the pTub-tel3 construct, we chose the green fluorescent protein (GFP) as a selection marker. When a linearized pTub-tel3 vector containing a GFP open reading frame was injected into the macronucleus, the GFP transcript was expressed in many clones whereas protein expression was detected only after extensive optimization of original GFP codons. GFP-derived fluorescence was distributed throughout the nuclei and cytoplasm except for contractile and food vacuoles. Upon continuous cell division, notable heterogeneity of GFP fluorescence among descendants from the same transformant has emerged. This expression vector can be applied to the analysis of protein trafficking and localization in addition to exogenous gene expression in P. caudatum.

Animals↗
PubMed2003

Phylogenetic relationships of non-mitochondrial nucleotide transport proteins in bacteria and eukaryotes.

Current knowledge about the nucleotide metabolism of intracellular bacteria is very limited. Here we report on the identification of nucleotide transport proteins (NTT) of two obligate endoparasites, Caedibacter caryophila and Holospora obtusa, both alpha-proteobacteria, which reside in the vegetative macronucleus of Paramecium caudatum. For comparative studies, we also identified the first nucleotide transporter in chloroplasts of a red alga, i.e. Galdieria sulphuraria, and further homologs in plant chloroplasts. Heterologous expression of the NTT proteins from C. caryophila, H. obtusa, and G. sulphuraria in Escherichia coli demonstrate that the nucleotide influx mediated by these transporters is specific for ATP and ADP. The NTT proteins of C. caryophila and H. obtusa exhibit substantial sequence identity with their counterparts in chloroplasts and intracellular bacterial pathogens of humans, but not with the nucleotide transport system of mitochondria. Comprehensive phylogenetic analyses of bacterial and chloroplast NTT proteins showed that homologs in chloroplasts from plants, and green, red, stramenopile and glaucocystophyte algae are monophyletic. In contrast, the evolutionary relationships of the bacterial counterparts appear highly complex. In the presented phylogeny, NTT proteins of C. caryophila and H. obtusa are only distantly related to one another, although these two taxa are close relatives in 16S rRNA trees. The tree topology indicates that some bacterial NTT paralogs have arisen by gene duplications and others by horizontal transfer.

Adenosine Diphosphate↗
PubMed1999

The micronuclear gene encoding a putative aminoacyl-tRNA synthetase cofactor of the ciliate Euplotes octocarinatus is interrupted by two sequences that are removed during macronuclear development.

The micronuclear gene of the ciliated protozoan Euplotes octocarinatus (Eo) syngen 1 encoding the putative aminoacyl-tRNA synthetase cofactor (ARCE), as well as its macronuclear version and the corresponding cDNA, were amplified and sequenced. Analyses of the sequences revealed that the micronuclear gene contains two sequences (430 and 625bp long) that are missing in the macronuclear version of this gene. These sequences are called 'internal eliminated sequences' (IESs) and appear to occur in all ciliates. The two IESs are located in the coding region of the micronuclear gene. One IES is flanked by a pair of dinucleotide 5'-TA-3' direct repeats and the other one by a pair of hepta-nucleotide 5'-TTACTGA-3' direct repeats. Inside the two IESs, several other sequence repeats were found. The macronuclear DNA molecule carrying this gene is 1517bp long and shows characteristics typical of macronuclear chromosomes of hypotrichous ciliates. Copy number determination revealed that the molecule is amplified to only about 750 copies per macronucleus. The deduced protein is a 441-amino-acid (aa) polypeptide with a molecular mass of 50kDa. It shares a conserved endothelial monocyte-activating polypeptide II (EMAP II)-like carboxyl-terminal domain and a hydrophilic central domain containing a KEKE-motif with a group of proteins associated with aminoacyl-tRNA synthetases and tRNAs.

Amino Acid Sequence↗
PubMed2004

Adenylyl cyclases from Plasmodium, Paramecium and Tetrahymena are novel ion channel/enzyme fusion proteins.

In Paramecium, cAMP formation is stimulated by a potassium conductance, which is an intrinsic property of the adenylyl cyclase. We cloned a full-length cDNA and several gDNA fragments from Paramecium and Tetrahymena coding for adenylyl cyclases with a novel domain composition. A putative N-terminal ion channel domain contains a canonical S4 voltage-sensor and a canonical potassium pore-loop located C-terminally after the last transmembrane span on the cytoplasmic side. The adenylyl cyclase catalyst is C-terminally located. DNA microinjection of a green fluorescent protein (GFP)-tagged construct into the macronucleus of Paramecium resulted in ciliary localization of the expressed protein. An identical gene coding for an ion-channel adenylyl cyclase was cloned from the malaria parasite Plasmodium falciparum. Expression of the catalytic domain of the latter in Sf9 cells yielded an active homodimeric adenylyl cyclase. The occurrence of this highly unique subtype of adenylyl cyclase appears to be restricted to ciliates and apicomplexa.

Adenylyl Cyclases↗
PubMed1991

Developmentally controlled genomic rearrangements in ciliated protozoa.

The ciliated protozoa undergo an extensive genome reorganization during the course of forming a transcriptionally active macronucleus. The process includes numerous chromosome fragmentation and DNA breakage and rejoining events. Recent work indicates that transposable elements play a role in the process.

Animals↗
PubMed2000

An unusual complementation in non-excitable mutants in Paramecium.

A non-excitable behavioural mutant, d4-662, was previously characterized as the fourth pawn locus mutant pwD in Paramecium tetraurelia. We now provide data demonstrating that d4-662 is in fact controlled by a pwB allele that has the unusual feature of complementing other pwB alleles in heterozygous F1 progeny. Neither the cytoplasm nor the nucleoplasm of d4-662 cured the mutational defects of pwB and in the reverse combination of d4-662 and pwB, the result was the same. On the other hand, pwA, another non-excitable mutant, was cured upon cross-injection with d4-662 and mutants carrying trichocyst non-discharge marker genes were also cured. This evidence suggests that d4-662 is a new mutant belonging to pwB, and would be better designated as pwB662. Extensive crossbreeding analyses, however, showed an unusual genetic relationship between d4-662 and pwB (pwB95 or pwB96). When d4-662 was crossed with pwB mutants, many progeny expressing wild-type phenotype or mixed clones of wild-type and pawn cells were obtained in the F1. Less than 12.5% expressed the pawn phenotype. The appearance of wild-type progeny in this F1 strongly suggests that an inter-allelic interaction between pwB662 and other pwB alleles may occur during development of the macronucleus.

Animals↗
PubMed2006

Ultrastructure and phylogeny of Philasterides dicentrarchi (Ciliophora, Scuticociliatia) from farmed turbot in NW Spain.

Several species of opportunistic histophagous scuticociliates have been implicated in systemic infections of farmed fish. In turbot, scuticociliatosis is an emerging disease, and the identification of the parasite species involved is controversial. We have previously isolated Philasterides dicentrarchi from farmed turbot scuticociliatosis outbreaks in northwest Spain. In the present study, we report detailed ultrastructural studies of this parasite, and investigate phylogenetic relations with other members of the order Philasterida on the basis of sequence comparison of the small-subunit rRNA (SSUrRNA) gene. Ultrastructural study indicates the presence of dikinetids in the anterior two-thirds of the body; micronucleus closely associated with the macronucleus, though not physically connected; numerous mitochondria located below the cell cortex, parallel to the surface; numerous spherical and fusiform extrusomes located close to the plasma membrane. We consider that these characteristics are useful for diagnosis of infections by this parasite. A nested 350-bp nucleotide sequence of the SSUrRNA gene of the turbot P. dicentrachi isolate showed high identity with previously reported SSUrRNA gene sequences from 2 scuticociliates isolated from olive flounder Paralichthys olivaceus in Korea, namely P. dicentrarchi (98%) and Miamiensis avidus (99%); conversely, our P. dicentrarchi sequence showed low identity (86%) with that of Uronema marinum, a scuticociliate that has also been implicated in scuticociliatosis outbreaks in turbot in Europe and olive flounder in Asia. Phylogenetic tree construction on the basis of the SSUrRNA gene sequences, using the neighbour-joining method, confirm that the different P. dicentrarchi isolates and M. avidus are closely related and a possible synonymy between both ciliates species should be considered.

Animals↗
PubMed1989

Ubiquitinated histone H2B is preferentially located in transcriptionally active chromatin.

Using an anti-ubiquitin antibody in Western blotting experiments, we detected polyubiquitinated species of histones H2A, H2A.Z, and H2B in histone preparations of bovine thymus, chicken erythrocyte, and Tetrahymena macro- and micronuclei. Histone H2A had the greatest level of polyubiquitinated species, with tetra- to hexaubiquitinated forms of this histone being observed. The fraction of bovine thymus and chicken erythrocyte chromatin enriched in transcriptionally active gene sequences was enriched in mono- and polyubiquitinated species of histones H2A, H2B, and H2A.Z, especially in the ubiquitinated forms of histone H2B. Histones H2A and H2B were ubiquitinated in the transcriptionally active Tetrahymena macronucleus, with monoubiquitinated (u) H2B being the predominant ubiquitinated histone species. Ubiquitinated forms of histones H2A and H2B were found in transcriptionally inert micronuclei, but at lower levels than seen in macronuclear histones. Also, the level of micronuclear uH2A was greater than that of uH2B which may be from macronuclei that contaminate the preparation. These results indicate that the mono- and polyubiquitinated species of histone H2B are preferentially located in transcriptionally active chromatin regions. Ubiquitinated histone H2A is located in both expressed and repressed chromatin domains, but expressed chromatin is enriched in mono- and polyubiquitinated forms of this histone. These observations are consistent with the hypothesis that ubiquitinated histones have a role maintaining the structure of transcriptionally active chromatin.

Animals↗
PubMed2002

Organization of the macronuclear gene-sized pieces of stichotrichous ciliates into a higher order structure via telomere-matrix interactions.

Macronuclear DNA of stichotrichous ciliates occurs in small 'gene-sized' molecules with sizes of about 0.5 to 40 kb. Each of these molecules is terminated by telomeric sequences of defined length. A single macronucleus contains up to 10(8) DNA molecules; due to the high concentration of telomeric sequences in this nucleus it is an attractive model to study telomere behaviour. We recently provided evidence that macronuclear telomeres are attached to the nuclear matrix and that this interaction is mediated by the telomere binding protein (TeBP). Using various experimental approaches, we now demonstrate that telomeres as well as both subunits of the telomere binding protein are associated with the nuclear matrix. However, there is no direct binding of telomeric DNA to the matrix but telomere matrix interaction is exclusively mediated by the TeBP. In addition, we show that telomeric sequences adopt in vivo the antiparallel G-quartet structure when bound to the nuclear matrix. These data not only allow us to propose a model for macronuclear architecture but may also be relevant for further analysis of telomere-matrix interactions in higher eukaryotes.

Animals↗
PubMedDate not supplied

Z-DNA immunoreactivity in rat tissues.

Recently, it has been shown that natural and synthetic deoxynucleotide polymers can adopt a left-handed helical structure (termed Z-DNA) in appropriate conditions (see, for example, refs 1 and 2 and the references therein). In contrast to the more familiar right-handed B-DNA, Z-DNA is strongly immunogenic, and polyclonal and monoclonal antibodies against Z-DNA have been elicited. By using such antibodies, immunoreactivity for Z-DNA has been detected in the polytene chromosomes of two dipteran species, in the macronucleus of a ciliated protozoon, and in certain plant nuclei (cited in ref. 11). In view of the possible importance of Z-DNA as a genomic regulatory signal, it would be highly desirable to know whether Z-DNA also occurs in mammals. We have therefore initiated an immunohistochemical study of various rat tissues by using three antisera specific for Z-DNA, and the peroxidase-antiperoxidase technique for visualization of tissue-bound antibodies. Here we demonstrate that the nuclei of many, but not all, types of rat cells exhibit Z-DNA immunoreactivity, suggesting that Z-DNA may exist naturally in mammalian chromatin.

Animals↗
PubMed1991

Identification in situ and phylogeny of uncultured bacterial endosymbionts.

The use of Koch's technique to isolate bacteria in pure cultures has enabled thousands of bacterial species to be characterized. But for the many microorganisms that have never been cultivated, DNA amplification in vitro using the polymerase chain reaction is now making their genes accessible. Here we use this technique to study bacteria of the genus Holospora, which live in ciliates and whose phylogenetic relationship has remained unknown because they are impossible to cultivate. Species of Holospora are highly infectious and live in the nuclei of their specific host cells: H. elegans and H. undulata infect micronuclei of Paramecium caudatum, whereas H. obtusa infects the macronucleus in other strains of the same host species; Holospora species have a common developmental cycle. We have amplified, cloned and sequenced gene fragments encoding ribosomal RNA of H. obtusa. The phylogenetic position of H. obtusa in the alpha group of Proteobacteria was determined by 16S rRNA sequence analysis. The sequences were then used to design species- as well as genus-specific rRNA hybridization probes, which enabled us to detect and differentiate individual cells of the endosymbionts in situ. The large amount of rRNA in the cells indicates a high physiological activity of the endosymbionts in the host nuclei.

Animals↗
PubMed2004

The revolution of the biology of the genome.

Sequence data of entire eukaryotic genomes and their detailed comparison have provided new evidence on genome evolution. The major mechanisms involved in the increase of genome sizes are polyploidization and gene duplication. Subsequent gene silencing or mutations, preferentially in regulatory sequences of genes, modify the genome and permit the development of genes with new properties. Mechanisms such as lateral gene transfer, exon shuffling or the creation of new genes by transposition contribute to the evolution of a genome, but remain of relatively restricted relevance. Mechanisms to decrease genome sizes and, in particular, to remove specific DNA sequences, such as blocks of satellite DNAs, appear to involve the action of RNA interference (RNAi). RNAi mechanisms have been proven to be involved in chromatin packaging related with gene inactivation as well as in DNA excision during the macronucleus development in ciliates.

Animals↗
PubMed2001

Divergence between two Antarctic species of the ciliate Euplotes, E. focardii and E. nobilii, in the expression of heat-shock protein 70 genes.

Most organisms oppose many environmental stresses by rapidly enhancing synthesis of the highly conserved Hsp70 family of heat-shock proteins. Two ciliates which are endemic in Antarctic coastal seawater, Euplotes focardii and E. nobilii, and behave as psychrophile and psychrotroph micro-organisms, respectively, revealed a divergence in the capacity to respond to thermal stress with an activation of the transcription of their hsp70 genes. In both species, these genes were shown to be represented by thousands of copies in the cell's somatic functional nucleus (macronucleus). However, while a strong transcriptional activity of hsp70 genes was induced in E. nobilii cells transferred from 4 to 20 degrees C, a much smaller increase was revealed in heat-shocked cells of E. focardii. These findings suggest a closer adaptation to the stably cold Antarctic waters in the genetic response of E. focardii to thermal stress.

Animals↗
PubMed2004

Histone H3 lysine 9 methylation is required for DNA elimination in developing macronuclei in Tetrahymena.

Genome-wide DNA elimination accompanies development of the somatic macronucleus from the germ-line micronucleus during the sexual process of conjugation in the ciliated protozoan Tetrahymena thermophila. Small RNAs, referred to as "scan RNAs" (scnRNAs), that accumulate only during conjugation are highly enriched in the eliminated sequences, and mutations that prevent DNA elimination also affect the accumulation of scnRNAs, suggesting that an RNA interference (RNAi)-like mechanism is involved in DNA elimination. Histone H3 that is methylated at lysine 9 (K9) is a hallmark of heterochromatin and, in Tetrahymena, is found only in developing macronuclei (anlagen) in association with chromatin containing the sequences undergoing elimination. In this article, we demonstrate that a mutation in the TWI1 gene that eliminates the accumulation of scnRNAs also abolishes H3 methylation at K9. We created mutant strains of Tetrahymena in which the only major H3 contained a K9Q mutation. These mutants accumulated scnRNAs normally during conjugation but showed dramatically reduced efficiency of DNA elimination. These results provide strong genetic evidence linking an RNAi-like pathway, H3 K9 methylation, and DNA elimination in Tetrahymena.

Animals↗
PubMed2001

In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei.

Most eukaryotic telomeres contain a repeating motif with stretches of guanine residues that form a 3'-terminal overhang extending beyond the telomeric duplex region. The telomeric repeat of hypotrichous ciliates, d(T(4)G(4)), forms a 16-nucleotide 3'-overhang. Such sequences can adopt parallel-stranded as well as antiparallel-stranded quadruplex conformations in vitro. Although it has been proposed that guanine-quadruplex conformations may have important cellular roles including telomere function, recombination, and transcription, evidence for the existence of this DNA structure in vivo has been elusive to date. We have generated high-affinity single-chain antibody fragment (scFv) probes for the guanine-quadruplex formed by the Stylonychia telomeric repeat, by ribosome display from the Human Combinatorial Antibody Library. Of the scFvs selected, one (Sty3) had an affinity of K(d) = 125 pM for the parallel-stranded guanine-quadruplex and could discriminate with at least 1,000-fold specificity between parallel or antiparallel quadruplex conformations formed by the same sequence motif. A second scFv (Sty49) bound both the parallel and antiparallel quadruplex with similar (K(d) = 3--5 nM) affinity. Indirect immunofluorescence studies show that Sty49 reacts specifically with the macronucleus but not the micronucleus of Stylonychia lemnae. The replication band, the region where replication and telomere elongation take place, was also not stained, suggesting that the guanine-quadruplex is resolved during replication. Our results provide experimental evidence that the telomeres of Stylonychia macronuclei adopt in vivo a guanine-quadruplex structure, indicating that this structure may have an important role for telomere functioning.

Animals↗
PubMed2002

An exchanger-like protein underlies the large Mg2+ current in Paramecium.

There are very few molecules known to transport Mg(2+) in eukaryotes. The membrane of Paramecium tetraurelia passes a large Mg(2+)-selective current and exhibits a corresponding backward swimming behavior. Both are missing in a group of mutants called eccentric. By sorting an indexed WT genomic library through microinjection into the macronucleus, we have isolated a DNA fragment that complements the eccentric mutations. The Mg(2+) currents and behavior are restored fully in the transformed cells. Surprisingly, the conceptually translated protein is not homologous to any known ion channel but instead has some similarity to K(+)-dependent Na(+)Ca(2+) exchangers. Exchangers are either electrically silent or only pass very small and slow currents compared with ion-channel currents. In light of recent ion-channel crystal structures and considering the need to have narrow ion-selective filters, we speculate on how an exchanger might evolve to show channel-like activities in special circumstances. The significance of finding the molecular basis of a Mg(2+)-specific pathway is also discussed.

Amino Acid Sequence↗