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PubMed1991

Telomeres inhibit end to end fusion and enhance maintenance of linear DNA molecules injected into the Paramecium primaurelia macronucleus.

Direct injection into the macronucleus of Paramecium tetraurelia of DNA molecules coding for the A-antigen leads to expression of the gene and autonomous replication. When injected into Paramecium primaurelia DNA from probably any origin, procaryote or eucaryote, can replicate as linear telomerized molecules and the number of copies maintained can be very high (up to 20000 copies). We present here evidence that if the injected linear DNA molecules harbour preexisting telomeres at both extremities they are protected from degradation, the number of DNA molecules maintained being 15- to 30-fold higher than if the molecules are injected without telomeres. Some of the injected molecules replicate as multimers, but, only when the fused ends are devoid of preexisting telomeric repeats.

Animals↗

PubMed1994

Extremely short 20-33 nucleotide introns are the standard length in Paramecium tetraurelia.

Paramecium tetraurelia has the shortest known introns as its standard intron length. Sequenced introns vary between 20 and 33 nucleotides in length. The intron sequences were discovered in genomic sequences coding for a variety of different proteins, including phosphatases, kinases, and low-molecular weight GTP-binding proteins. All intron sequences begin with the conserved dinucleotide GT and end with the conserved dinucleotide AG. The sequences are more AT rich than the Paramecium coding sequences. The identified sequences were confirmed as introns by sequencing several cDNA fragments. We report here analysis of the characteristics of 50 separate introns, including size, base composition, and a consensus sequence.

Animals↗

PubMed1997

Deletion endpoint allele-specificity in the developmentally regulated elimination of an internal sequence (IES) in Paramecium.

Ciliated protozoa undergo thousands of site-specific DNA deletion events during the programmed development of micronuclear genomes to macronuclear genomes. Two deletion elements, W1 and W2, were identified in the Paramecium primaurelia wild-type 156 strain. Here, we report the characterization of both elements in wild-type strain 168 and show that they display variant deletion patterns when compared with those of strain 156. The W1 ( 168 ) element is defective for deletion. The W2 ( 168 ) element is excised utilizing two alternative boundaries on one side, both are different from the boundary utilized to excise the W2156 element. By crossing the 156 and 168 strains, we demonstrate that the definition of all deletion endpoints are each controlled by cis -acting determinant(s) rather than by strain-specific trans-acting factor(s). Sequence comparison of all deleted DNA segments indicates that the 5'-TA-3'terminal sequence is strictly required at their ends. Furthermore the identity of the first eight base pairs of these ends to a previously established consensus sequence correlates with the frequency of the corresponding deletion events. Our data implies the existence of an adaptive convergent evolution of these Paramecium deleted DNA segment end sequences.

Alleles↗

PubMed2001

Transgene-mediated post-transcriptional gene silencing is inhibited by 3' non-coding sequences in Paramecium.

Homology-dependent gene silencing is achieved in Paramecium by introduction of gene coding regions into the somatic nucleus at high copy number, resulting in reduced expression of all homologous genes. Although a powerful tool for functional analysis, the relationship of this phenomenon to gene silencing mechanisms in other organisms has remained obscure. We report here experiments using the T4a gene, a member of the trichocyst [corrected]matrix protein (TMP) multigene family encoding secretory proteins, and the ND7 gene, a single copy gene required for exocytotic membrane fusion. Silencing of either gene leads to an exocytosis-deficient phenotype easily scored on individual cells. For each gene we have tested the ability of different combinations of promoter, coding and 3' non-coding regions to provoke silencing, and analyzed transcription and steady-state RNA in the transformed cells. We provide evidence that homology-dependent gene silencing in Paramecium is post-transcriptional and that both sense and antisense RNA are transcribed from the transgenes, consistent with a role for dsRNA in triggering silencing. Constructs with and without promoters induce gene silencing. However, transgenes that contain 3' non-coding regions do not induce gene silencing, despite antisense RNA production. We present a model according to which different pathways of RNA metabolism compete for transcripts and propose that the relative efficiencies of dsRNA formation and of 3' RNA processing of sense transgene transcripts determine the outcome of transformation experiments.

3' Untranslated Regions↗

PubMed2001

Control of DNA excision efficiency in Paramecium.

Programmed excision of internal eliminated sequences (IESs) occurs at thousands of sites in ciliate genomes. How this is controlled is largely unknown. Here, we report the characterization of the non-efficiently excised 156psiG-11 IES from Paramecium primaurelia strain 156 and that of the efficiently excised 168psiG-11 IES, an allelic variant from strain 168. Then, we report a genetic and molecular analysis of IES excision efficiency in F(1) progeny derived from interstrain crosses and in F(2) homozygous progeny derived from F(1) autogamy. IES 168psiG-11 excision efficiency was approximately 100% in all cases. IES 156psiG-11 excision efficiency was 19 +/- 13% in F(1) progeny and 0.6 +/- 1.1% in F(2) progeny. No trans-excision event between IESs 156psiG-11 and 168psiG-11 was detected within the F(1) progeny. These data demonstrate that the excision efficiency of this IES is variable and controlled by a cis-acting element. This should encompass positions 8 and/or 9 of the right IES end, which display allele differences. Finally, the 30-fold stimulation of IES 156psiG-11 excision efficiency within F(1) progeny relative to F(2) progeny demonstrates that Paramecium IES excision efficiency is sensitive either to a conjugation-specific trans-acting factor provided by the zygotic genome, or to homologous chromosome cross-talk.

Alleles↗

PubMed1981

Structure and sequence of the mitochondrial 20S rRNA and tRNA tyr gene of Paramecium primaurelia.

The sequence and structure of the large (20s) mitochondrial (mt) rRNA gene and flanking regions from Paramecium primaurelia have been determined. The gene contains two regions of strong homology with other large mt rRNAs: one 44-base region near the 5' end and a 321-base region near the 3' end. Another region of strong homology to both ends of E. coli 23s RNA exists at loci consistent with these regions. The Paramecium gene appears to be 2204 bases in length and contains slightly more homology to E. coli rRNA than its mammalian or fungal counterparts. The gene, located about 1200 bp from the replicative terminal end of the linear mt DNA, is transcribed in the same polarity as replication. Previous R-looping studies detected no large introns within the gene. Here we describe sequences resembling degenerate rRNAs, one of which could represent a small intron. A tRNA tyr gene was found on the same DNA strand, 127 bp downstream from the large rRNA presumptive 3' end. The tRNA is flanked on both sides by short DNA regions of approximately 90% A + T content.

Animals↗

PubMed2006

ParameciumDB: a community resource that integrates the Paramecium tetraurelia genome sequence with genetic data.

ParameciumDB (http://paramecium.cgm.cnrs-gif.fr) is a new model organism database associated with the genome sequencing project of the unicellular eukaryote Paramecium tetraurelia. Built with the core components of the Generic Model Organism Database (GMOD) project, ParameciumDB currently contains the genome sequence and annotations, linked to available genetic data including the Gif Paramecium stock collection. It is thus possible to navigate between sequences and stocks via the genes and alleles. Phenotypes, of mutant strains and of knockdowns obtained by RNA interference, are captured using controlled vocabularies according to the Entity-Attribute-Value model. ParameciumDB currently supports browsing of phenotypes, alleles and stocks as well as querying of sequence features (genes, UniProt matches, InterPro domains, Gene Ontology terms) and of genetic data (phenotypes, stocks, RNA interference experiments). Forms allow submission of RNA interference data and some bioinformatics services are available. Future ParameciumDB development plans include coordination of human curation of the near 40 000 gene models by members of the research community.

Alleles↗

PubMed1982

Changes in intracellular pH affect calcium currents in Paramecium caudatum.

The relation between intracellular pH and membrane excitability was studied in the holotrich ciliate Paramecium caudatum. Intracellular pH (pHi) was measured with recessed-tip ion-sensitive microelectrodes (Thomas 1974) and electrical properties were examined by current stimulation and conventional two-electrode voltage clamp. Under normal conditions the resting pHi of Paramecium was 6.80 +/- 0.05. Intracellular alkalinization enhanced the early Ca current, while internal acidification depressed the Ca current. Both effects occurred in a voltage-independent manner. The late outward current was relatively unaffected by these alterations. Results obtained with replacement of extracellular Ca2+ by Ba2+ also support a direct effect of pHi on current through the Ca channel. Intracellular alkalinization to pH 7.15 converted graded, quasi-regenerative Ca responses elicited by injected current pulses into all-or-none action potentials. This change to all-or-none behaviour is presumed to be due to the increase in Ca current and a consequent change in the balance of inward and outward currents. Extracellular pH changes had little effect on pHi, resting membrane potential or the current-voltage relations. The intracellular pH was also independent of shifts in membrane potential. The results are consistent with a model in which Ca channel permeability is blocked by intracellular protonation of a single titratable site having an apparent dissociation constant of 6.2.

Acetates↗

PubMed1988

Sterol synergism in Paramecium tetraurelia.

Paramecium tetraurelia is a naturally occurring sterol auxotroph with an absolute nutritional requirement for one of a small group of structurally related phytosterols. We report here a quantitative study demonstrating that a low, otherwise sub-supportive, concentration (approximately 0.020-0.050 micrograms ml-1) of an essential phytosterol (stigmasterol) is adequate for growth of this ciliate, provided that a second, relatively non-specific sterol is available at a higher concentration (1.0 micrograms ml-1) to allow for membrane biosynthesis. This phenomenon, referred to as sterol synergism, has been observed in a broad taxonomic range of organisms, with the conclusion that small amounts of specific sterols are required to perform some previously unknown, vital metabolic or regulatory function. Paramecium promises to be an excellent model organism for the elucidation of essential sterol function.

Animals↗

PubMed2003

Host growth conditions regulate the plasticity of horizontal and vertical transmission in Holospora undulata, a bacterial parasite of the protozoan Paramecium caudatum.

A parasite might be prohibited from investing simultaneously in horizontal (infection of new hosts) and vertical (infection of the current host's offspring) transmission because of developmental, physiological, or evolutionary costs and constraints. Rather, these constraints may select for adaptive phenotypic plasticity, where the parasite uses the transmission pathway that maximizes transmission in the current ecological and epidemiological conditions. By varying environmental conditions for the host's replication, we investigated the plasticity of vertical and horizontal transmission of Holospora undulata, a micronucleus-specific bacterial parasite of the protozoan Paramecium caudatum. We observed a negative correlation between the host's growth rate and the parasite's investment in horizontal transmission. In rapidly dividing hosts, the parasite remained in the reproductive stage and was passed on vertically to the daughter nuclei during mitotic division of the Paramecium. In contrast, at low or negative growth rates of the host, the parasite's reproductive forms differentiated into infectious forms, the agents of horizontal transmission. Furthermore, in treatments that were initiated with a high proportion of individuals harboring horizontally transmitted infectious forms, rapid replication resulted in a switch back from predominantly horizontal to almost exclusively vertical transmission. These results suggest a trade-off between the efficacies of vertical and horizontal transmission, with the parasite switching to horizontal transmission only if conditions for host replication, and thus vertical transmission, deteriorate.

Adaptation, Physiological↗

PubMed1982

Characterization of a Ca2+-dependent guanylate cyclase in the excitable ciliary membrane from Paramecium.

The membraneous guanylate cyclase of cilia from Paramecium tetraurelia used MgGTP and MnGTP as substrate with Michaelis constants for GTP of 71.5 microM and 36 microM, respectively. A linear Arrhenius plot indicated that a single enzyme entity exists not sensitive to possible phase transitions of membrane lipids. Guanylate cyclase is activated by low concentrations (less than 100 microM) and inhibited by high concentrations (greater than 100 microM) of calcium, half-maximal effects were obtained with 8 microM and 500 microM Ca2+, respectively. Only strontium ions displayed partial activating and inhibiting potency, all other divalent cations tested, Ba2+, Fe2+, Co2+, Mn2+, Sn2+ and Ni2+ had no effect on guanylate cyclase activity. Ca2+ activation increased V; Km remained identical. The Ca2+ stimulated activity was not inhibited by trifluoperazine, tentatively suggesting that the stimulation may not be mediated by calmodulin. Ca2 inhibition was due to a single binding site of Ca2+ at the guanylate cyclase as evidence by a Hill coefficient h = -1 and was noncompetitive. The lanthanides La3+, Ce3+ and Tb3+ were powerful inhibitors of guanylate cyclase, with La3+ the half-maximal effect was obtained with 0.6 microM, it was kinetically a mixed-type inhibition. La3+ and CA2+ competed for the same binding site on the guanylate cyclase as determined by detailed kinetic analysis. Addition of EDTA reversed the activation and inhibition by Ca2+ and the inhibition by La3+. It is discussed that guanylate cyclase may be the initial target enzyme in the cilia for the calcium transient of the calcium-potassium action potential of Paramecium.

Animals↗

PubMed1987

RNA-dependent DNA polymerase activity in Paramecium tetraurelia: what for?

Protein extracts from the protozoan ciliate Paramecium tetraurelia revealed high levels of RNA-dependent DNA polymerase activity (reverse transcriptase). Stable and constant during the somatic phase of the cell cycle, the reverse transcriptase activity quickly diminished following the completion of the sexual phases of the cell cycle: conjugation and autogamy. The Paramecium reverse transcriptase presented a number of common features with retroviral polymerases: ability to copy synthetic templates such as poly(rCm).oligo(dG) as well as mRNA; sensitivity to various reverse transcriptase inhibitors such as HPA 23, suramin, phosphonoformate and ethidium bromide; insensitivity to the action of other DNA and RNA polymerase inhibitors and, finally, the requirement for divalent cations before the enzyme can function: either magnesium or manganese. Although the reverse transcriptase activity was not proven to be independent from one of the DNA polymerases in paramecia, its high activity predicts a role in the paramecia cell cycle. From what we are able to conceive today two possible roles could be envisaged. Participation in the anlage macronucleus formation: micronuclear sequences are first transcripted and, after rearrangements of the RNA molecules, these are retrotranscribed into the macronuclear DNA molecules or association with retrotransposons that participate in the movement of certain macronuclear sequences into the germ-line micronucleus.

Animals↗

PubMed1992

Metabolism of inositol phosphates in the protozoan Paramecium. Characterization of a novel inositol-hexakisphosphate-dephosphorylating enzyme.

Basal and stimulated levels of inositol phosphates were determined in the protozoan Paramecium labelled with myo-[3H]inositol. Under resting conditions, intracellular InsP6 (phytic acid), InsP5 and InsP4 concentrations were 140, 10 and 2 microM, respectively. InsP5 was comprised of 56% Ins(1,2,3,4,5)P5 and/or Ins(1,2,3,5,6)P5, 40% Ins(1,2,4,5,6)P5 and/or Ins(2,3,4,5,6)P5 and small amounts of Ins(1,3,4,5,6)P5 and Ins(1,2,3,4,6)P5. InsP4 was mainly Ins(1, 4, 5, 6)P4 and/or Ins(3, 4, 5, 6)P4. Other inositol phosphates were not detected at a detection limit of 50-85 nM. Using various depolarizing and hyperpolarizing stimuli, no significant changes in level of inositol phosphates were observed in vivo, indicating that in the ciliate a contribution of inositol phosphates to signal-transduction mechanisms is unlikely. In homogenates prepared from myo-[3H]inositol-labelled cells, a marked relative increase in InsP3 and InsP4 over the concentrations in vivo was observed. These inositol phosphates were identified as degradation products of endogenous InsP6. A novel separation methodology for inositol phosphates was established to allow unequivocal assignment of phosphate locations of all dephosphorylated InsP6-derived products. The dephosphorylation was catalyzed by a phytase-like enzyme with a molecular mass of 240 kDa, most likely of a hexameric structure. The enzyme had a pH optimum of 7.0 and did not require divalent cations for activity. Substrate concentrations above 300 microM were inhibitory. Dephosphorylation of InsP6 by the Paramecium enzyme differs from that of phytases from plants in that it proceeds via a sequential release of phosphate groups from positions 6, 5, 4 and 3 of the myo-inositol ring or/and positions 4, 5, 6 and 1.

Animals↗

PubMed1996

Characterization of centrin genes in Paramecium.

Centrins are highly conserved, ubiquitous cytoskeletal components which belong to the EF-hand superfamily of Ca2+-modulated proteins. We report here the molecular characterization of new members of the centrin family, Paramecium centrins. Previous studies described the organization of the infraciliary lattice (ICL), the innermost cortical cytoskeletal network of Paramecium, and showed that it was composed of a set of low-molecular-mass, Ca2+-binding polypeptides [Garreau de Loubresse, N., Klotz, C., Vigues, B., Rutin, J & Beisson, J. (1991) Biol. Cell 71, 217-225]. In this paper we show that these polypeptides are recognized by specific anti-centrin polyclonal antibodies. Their microsequences revealed four distinct N-termini. For one of them, ICL1, N-terminal and internal peptide sequences were used for PCR amplification and cloning of a DNA fragment containing a complete centrin coding sequence. The deduced amino acid sequence presents about 50% identify with those of centrins from other species. Further molecular analysis allowed us to identify two additional closely related, co-expressed ICL1 genes, providing the first example of a centrin multigenic family in a microorganism.

Amino Acid Sequence↗

PubMed1996

Cathepsin L is an intracellular and extracellular protease in Paramecium tetraurelia. Purification, cloning, sequencing and specific inhibition by its expressed propeptide.

The ciliate Paramecium tetraurelia secretes large amounts of a cysteine protease into the growth medium, presumably for extracellular food digestion. Two endoprotease isozymes (30 and 33 kDa on SDS/PAGE, respectively), both present in cell homogenates and in spent growth medium, were purified to homogeneity. Peptide sequence analysis revealed that these isozymes share identities at the amino acid level but are probably differently processed. Enzymatic characterization of the isolated proteases and sequencing of the cloned cDNA demonstrated that the enzymes belong to the cathepsin-L protease subfamily. Although the identity with mammalian and other protozoan L cathepsins was only around 30%, all important signature sequences for cathepsin L in the preproregion as well as in the catalyst of the enzyme were fully retained. The cDNA of this cysteine protease codes for a preproregion of 108 amino acids. The putative proregion of 86 amino acids which contained the characteristic conserved ERFNIN motif, was fused with a His6 tag, expressed in Escherichia coli, and purified. Both cathepsin L isozymes of Paramecium tetraurelia were inhibited by their cognate propeptide in the nanomolar concentration range. All other cysteine proteases tested (papain and mammalian cathepsin B, G and H) were unaffected by the propeptide up to 10 microM.

Amino Acid Sequence↗

PubMed1998

Thermoregulatory changes by hypoxia: lessons from the paramecium.

1. In organisms ranging from paramecia to mammals, hypoxia elicits a regulated decrease in body temperature (Tb). A decrease in Tb is an important adaptation to hypoxia primarily because it lowers metabolic rate when oxygen supply is limited, thus facilitating survival. 2. Although this beneficial response is extremely widespread among taxa, little is known of the cellular mechanisms that mediate hypoxia-induced decreases in Tb. This is due, in large part, to the extreme complexity of vertebrate thermoregulatory systems. 3. The thermoregulatory system of the unicellular paramecium is much simpler than that of vertebrates, yet it responds similarly to hypoxia. Research has explored the functional importance of hypoxia-induced decreases in Tb. In addition, a number of possible mediators and signalling pathways in hypoxia-induced reductions in Tb have been assessed. 4. In Paramecium caudatum, hypoxia appears to exert its thermoregulatory effects by inhibiting oxidative phosphorylation. Decreases in intracellular [ATP] and pH may be important intermediate signals. In addition, an endogenous opioid system appears to help mediate hypoxia-induced changes in thermoregulatory behaviour.

Animals↗

PubMed1976

Effects of actinomycin D on generation time and morphogenesis in Paramecium.

The sensitivity of Paramecium tetraurelia (=P. aurelia syngen 4) cells to pulse treatments with various doses of Actinomycin D (AMD) was estimated by comparing the generation times of treated and untreated sister cells. It was found that the delay of division in treated cells depended on the concentration of AMD, on their "age" at the time of the pulse treatment, and on their individual sensitivity. Sensitivity of Paramecium to AMD changes during the cell cycle in a predictable way. About 3 1/2 hr before the normally expected cell fission (total generation time approximately 5 1/2 hr) there is a decrease of sensitivity. Thereafter, the cell enters a new stage with a progressive increase of sensitivity. This 2nd phase ends at the "transition point" (approximately 2 hr before cell division), when sensitivity drops abruptly. The division process itself may be altered and slowed down by high concentrations of AMD, even if the drug is applied after the transition point, but this process can never be completely annulled. The impairment of the division mechanism may lead to morphologic anomalies in the offspring. Resorption of oral anlagen in P. tetraurelia probably never occurs during the cell cycle after AMD treatment. The reason for individual variability of the cells, mechanisms controlling development, and the question of an obligate sequence of gene action in each cell cycle are discussed.

Animals↗

PubMed1987

The glyceryl ethers of Paramecium phospholipids and phosphonolipids.

Two glyceryl ethers, 1-O-hexadecyl glycerol and 1-O-cis-octadec-11-enyl glycerol, chimyl and paramecyl alcohol, respectively, were quantified in total phospholipids and five glycerophospholipid classes from cells and cilia of the ciliated protozoon, Paramecium tetraurelia. The ether content of 2-aminoethyl phosphonoglycerolipid was 85-90 mole %. Concentrations of ethers were greatest in the ethanolamine phosphonolipids greater than phosphatidylcholines greater than phosphatidylserines greater than phosphatidylethanolamines greater than phosphatidylinositols. The glyceryl ether concentrations in total cellular phospholipids increased with culture age in P. tetraurelia and P. multimicronucleatum cells. The glyceryl ether concentrations in the phospholipids of P. tetraurelia cilia remained constant from mid log to stationary phase of culture growth. Paramecium tetraurelia phospholipid glyceryl ether concentrations were made greater by supplementation of cultures with chimyl alcohol.

Animals↗