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PubMed1999

How gravity acts on Paramecium: new insights from free-fall experiments.

The swimming behaviour of Paramecium is affected by media of various specific gravities. At 1g, the negative gravitaxis of Paramecium virtually disappears in solutions the specific gravity of which is about the same as that of the organism (1.04). In solutions with a higher specific gravity (1.08), Paramecium becomes positively gravitactic. We recorded the swimming tracks of Paramecium in these media on videotape before, during and after free-falls. The records show that the density-dependent differences in the swimming behaviour disappeared immediately following the onset of the free-fall. The recorded tracks and distributions of cells in the experimental chambers were compared with computer-simulated traces and distributions based on gravitactic and gravikinetic models proposed for Paramecium. Our preliminary analysis favors a novel gravitactic mechanism involving modification of the ciliary movement The drop shaft at the Japan Microgravity Center, Hokkaido (JAMIC) was used for the free-fall experiments.

Animals↗

PubMed1984

AMP-dependence of the cyanide-insensitive pathway in the respiratory chain of Paramecium tetraurelia.

The AMP-dependent stimulation of the cyanide-insensitive respiration of Paramecium mitochondria was investigated. The nucleotides exhibiting a stimulatory effect on the cyanide-insensitive oxidation of pyruvate (+ malate) in a medium supplemented with EDTA or carboxyatractyloside were, in decreasing order of efficiency, AMP, GMP, IMP, UMP and TMP. On the other hand, ADP, ATP and cyclic AMP were ineffective. In the presence of carboxyatractyloside, addition of AMP to Paramecium mitochondria incubated with pyruvate (+malate) led to an increase in membrane potential. In the absence of light, the photoactivable derivative of AMP, 3'-[4-[N-(4-azido-2-nitrophenyl)amino]butyryl]-AMP (NAP4-AMP) added to Paramecium mitochondria opposed the stimulatory effect of AMP on the cyanide-insensitive respiration; the Ki for NAP4-AMP was much lower than the Km for AMP, 0.2 microM compared with 120 microM. The ADP-stimulated respiration was not affected. Photoirradiation of Paramecium mitochondria in the presence of NAP4-AMP resulted in irreversible inhibition of the AMP-stimulated cyanide-insensitive respiration. No effect on the ADP-stimulated respiration was observed. A heatlabile cyanide-insensitive ubiquinol oxidase was extracted from Paramecium mitochondria with the detergent NN-dimethyl-N-(3-laurylamidopropyl)amine oxide. The quinol oxidase activity was slightly stimulated by AMP.

Adenosine Diphosphate↗

PubMed1997

Identification of isoforms of the exocytosis-sensitive phosphoprotein PP63/parafusin in Paramecium tetraurelia and demonstration of phosphoglucomutase activity.

PP63 (parafusin) is a 63 kDa phosphoprotein which is very rapidly (within 80 ms) dephosphorylated (to P63) during triggered trichocyst exocytosis; this occurs selectively in exocytosis-competent Paramecium tetraurelia strains. In the present work, two cDNAs coding for PP63/parafusin have been isolated, one of which is a new isoform. These isoforms are 99.6% identical and are derived from two different genes. Similarity searches revealed 43-51% identity of the deduced amino acid sequences with known phosphoglucomutases from yeast and mammals. The sequences of two proteolytic peptides obtained from PP63/parafusin isolated from Paramecium are identical to parts of the amino acid sequence deduced from the major cDNA. The major cDNA was mutated from the macronuclear ciliate genetic code into the universal genetic code and expressed in Escherichia coli. The recombinant protein shows the same biochemical and immunological characteristics as the (P)P63/parafusin originally isolated from Paramecium. It has the same specific phosphoglucomutase activity as phosphoglucomutase from chicken muscle. We also show that recombinant P63-1 parafusin 1 is a substrate of an endogenous casein kinase from Paramecium, as is the originally isolated P63/parafusin. Polyclonal antibodies against recombinant P63-1/parafusin 1 were raised which recognized phosphoglucomutases from different sources. Thus we show that PP63/parafusin and phosphoglucomutase in Paramecium are identical.

Amino Acid Sequence↗

PubMed1992

3'-modified antisense oligodeoxyribonucleotides complementary to calmodulin mRNA alter behavioral responses in Paramecium.

The calcium-binding protein calmodulin has been shown to modulate the Ca(2+)-dependent ion channels of Paramecium tetraurelia. Mutations in the calmodulin gene of Paramecium result in an altered pattern of behavioral responses. Antisense oligodeoxyribonucleotides (ODNs), complementary to calmodulin mRNA in Paramecium, were synthesized from a modified solid support that introduced a 3'-hydroxyhexyl phosphate. These 3'-modified ODNs were tested for their ability to alter the behavioral response of Paramecium. The microinjection of antisense ODNs temporarily reduced the backward swimming behavior of the cells in test solutions containing Na+. The injection of sense and random 3'-modified ODNs, or unmodified antisense ODNs, had no effect. The antisense ODN-induced effect was reversed by the injection of calmodulin protein. The pattern of response of the injected cells in various behavioral test solutions indicated that the calmodulin antisense ODNs reduce the Ca(2+)-dependent Na+ current. Antisense ODNs, complementary either to the 5' start site or to an internal sequence of the calmodulin mRNA, were similarly effective in altering behavior. These results show that antisense ODNs may be utilized in ciliated protozoa as a tool for reducing the expression of specific gene products. In addition, Paramecium represents a powerful model system with which to study and develop antisense ODN technology.

Animals↗

PubMed1999

Guanylyl cyclases with the topology of mammalian adenylyl cyclases and an N-terminal P-type ATPase-like domain in Paramecium, Tetrahymena and Plasmodium.

We cloned a guanylyl cyclase of 280 kDa from the ciliate Paramecium which has an N-terminus similar to that of a P-type ATPase and a C-terminus with a topology identical to mammalian adenylyl cyclases. Respective signature sequence motifs are conserved in both domains. The cytosolic catalytic C1a and C2a segments of the cyclase are inverted. Genes coding for topologically identical proteins with substantial sequence similarities have been cloned from Tetrahymena and were detected in sequences from Plasmodium deposited by the Malaria Genome Project. After 99 point mutations to convert the Paramecium TAA/TAG-Gln triplets to CAA/CAG, together with partial gene synthesis, the gene from Paramecium was heterologously expressed. In Sf9 cells, the holoenzyme is proteolytically processed into the two domains. Immunocytochemistry demonstrates expression of the protein in Paramecium and localizes it to cell surface membranes. The data provide a novel structural link between class III adenylyl and guanylyl cyclases and imply that the protozoan guanylyl cyclases evolved from an ancestral adenylyl cyclase independently of the mammalian guanylyl cyclase isoforms. Further, signal transmission in Ciliophora (Paramecium, Tetrahymena) and in the most important endoparasitic phylum Apicomplexa (Plasmodium) is, quite unexpectedly, closely related.

Amino Acid Sequence↗

PubMed1990

Protozoan myoglobin from Paramecium caudatum. Its autoxidation reaction and hemichrome formation.

Native oxymyoglobin (MbO2) was isolated directly from the cells of Paramecium caudatum with complete separation from metmyoglobin (metMb) on a DEAE-cellulose column. It was examined for its spectral and stability properties. When compared with sperm whale MbO2 used as a reference, Paramecium MbO2 was found to be much more susceptible to autoxidation over a wide range of pH (4-11) in 0.1 M buffer at 25 degrees C. Kinetic analysis has revealed that a proton-catalyzed displacement of O2- from MbO2 by an entering water molecule can play a dominant role in the autoxidation reaction of Paramecium MbO2 to metMb, as in the case of sperm whale MbO2 involving the distal histidine as its catalytic residue. At pH values higher than 9.5, however, Paramecium MbO2 was found to be oxidized to yield a hemichrome. The spontaneous formation of hemichromes is at variance with the other known myoglobins and is therefore discussed in relation to the unusual amino acid sequence of Paramecium myoglobin having a large number of deletion.

Animals↗

PubMed1987

Growth of Paramecium tetraurelia in bacterized, monoxenic cultures.

Wild type and mutant Paramecium tetraurelia were grown in monoxenic cultures by first growing Enterobacter aerogenes on a defined medium and then adding the Paramecium to the stationary phase bacterial culture. The bacterial growth was proportional to the concentration of the carbon source (citrate), and the Paramecium growth was dependent upon both the bacterial density and the starting density of Paramecium. The behavior, electrophysiological properties, ciliary lipid composition, and growth characteristics were similar to the commonly used bacterized medium (Cerophyl) except that 5-10 times greater Paramecium yields were reliably obtained.

Animals↗

PubMedDate not supplied

Chemorepellents in Paramecium and Tetrahymena.

Although Paramecium has been widely used as a model sensory cell to study the cellular responses to thermal, mechanical and chemoattractant stimuli, little is known about their responses to chemorepellents. We have used a convenient capillary tube repellent bioassay to describe 4 different compounds that are chemorepellents for Paramecium and compared their response with those of Tetrahymena. The classical Paramecium t-maze chemokinesis test was also used to verify that this is a reliable chemorepellent assay. The first two compounds, GTP and the oxidant NBT, are known to be depolarizing chemorepellents in Paramecium but this is the first report of them as repellents in Tetrahymena. The second two compounds, the secretagogue alcian blue and the dye cibacron blue, have not previously been described as chemorepellents in either of these ciliates. Two other compounds, the secretagogue AED and the oxidant cytochrome c, were found to be repellents to Paramecium but not to Tetrahymena. The repellent nature of each of these compounds is not related to toxicity because cells are completely viable in all of them. More importantly, all of these repellents are effective at micromolar to nanomolar concentrations, providing an opportunity to use them as excitatory ligands in future works concerning their membrane receptors and possible receptor operated ion channels.

Alcian Blue↗

PubMed2003

PAK paradox: Paramecium appears to have more K(+)-channel genes than humans.

K(+)-selective ion channels (K(+) channels) have been found in bacteria, archaea, eucarya, and viruses. In Paramecium and other ciliates, K(+) currents play an essential role in cilia-based motility. We have retrieved and sequenced seven closely related Paramecium K(+)-channel gene (PAK) sequences by using previously reported fragments. An additional eight unique K(+)-channel sequences were retrieved from an indexed library recently used in a pilot genome sequencing project. Alignments of these protein translations indicate that while these 15 genes have diverged at different times, they all maintain many characteristics associated with just one subclass of metazoan K(+) channels (CNG/ERG type). Our results indicate that most of the genes are expressed, because all predicted frameshifts and several gaps in the homolog alignments contain Paramecium intron sequences deleted from reverse transcription-PCR products. Some of the variations in the 15 genomic nucleotide sequences involve an absence of introns, even between very closely related sequences, suggesting a potential occurrence of reverse transcription in the past. Extrapolation from the available genome sequence indicates that Paramecium harbors as many as several hundred of this one type of K(+)-channel gene. This quantity is far more numerous than those of K(+)-channel genes of all types known in any metazoan (e.g., approximately 80 in humans, approximately 30 in flies, and approximately 15 in Arabidopsis). In an effort to understand this plurality, we discuss several possible reasons for their maintenance, including variations in expression levels in response to changes in the freshwater environment, like that seen with other major plasma membrane proteins in Paramecium.

Alternative Splicing↗

PubMed1996

A single telomerase RNA is sufficient for the synthesis of variable telomeric DNA repeats in ciliates of the genus Paramecium.

Paramecium telomeric DNA consists largely of a random distribution of TTGGGG and TTTGGG repeats. Given the precise nature of other ciliate telomerases, it has been postulated that there are two distinct types of the Paramecium enzyme, each synthesizing perfect telomeric repeats: one with a template RNA that specifies the addition of TTTGGG and the second dictating the synthesis of TTGGGG repeats. We have cloned and sequenced telomerase RNA genes from Paramecium tetraurelia, P. primaurelia, P. multimicronucleatum, and P. caudatum. Surprisingly, a single gene encodes telomerase RNA in all four species, although an apparently nontranscribed pseudogene is also present in the genome of P. primaurelia. The overall lengths of the telomerase RNAs range between 202 and 209 nucleotides, and they can be folded into a conserved secondary structure similar to that derived for other ciliate RNAs. All Paramecium telomerase RNAs examined include a template specific for the synthesis of TTGGGG telomeric repeats, which has not been posttranscriptionally edited to account for the conventional synthesis of TTTGGG repeats. On the basis of these results, possible mechanisms for the synthesis of variable telomeric repeats by Paramecium telomerase are discussed.

Animals↗

PubMed1985

Pressure-induced changes in Ca2+-channel excitability in Paramecium.

The behaviour of swimming Paramecium is markedly affected by hydrostatic pressure (50-200 atm, 1 atm = 101 325 Pa). To investigate whether pressure might alter behaviour by acting directly on specific ion channels that mediate the behavioural responses, we examined the effects of K+, Na+ and Ba2+ ions on swimming speed and the reversal response during pressurization and decompression. If pressure acted on the channels that transport these ions, then the pressure-induced responses of swimming Paramecium should be exaggerated or diminished, according to which ions were present in the experimental buffer. Pressurization to 100 atm in standard buffer inhibited the brief reversal of swimming direction that occurred at atmospheric pressure when a paramecium encountered the wall of the pressure chamber. To determine whether pressure impaired mechanoreceptor function or directly blocked the Ca2+-channels that control ciliary reversal, we added Ba2+ or Na+ to standard buffer to induce multiple spontaneous reversals. Pressurization blocked these reversals, suggesting that channel opening is directly inhibited by pressure. Decompression in standard buffer elicited momentary ciliary reversal and backward swimming. Buffers with a high ratio of K+ to Ca2+ suppressed this response, and the decompression-induced reversal was exaggerated in the presence of Ba2+ or Na+, consistent with the effects that these ions are known to have on Paramecium's reversal response. These data imply that, upon decompression, the Ca2+-channels that mediate ciliary reversal open transiently. In addition to blocking the reversal response, pressurization slowed forward swimming. By examining the response to pressurization of Paramecium immobilized by Ni2+, we found that hydrostatic pressure apparently slows swimming by reorientating the direction of ciliary beat.

Animals↗

PubMed2003

Responses to hypergravity in proliferation of Paramecium tetraurelia.

It has been reported that Paramecium proliferates faster when cultured under microgravity in orbit, and slower when cultured under hypergravity. This shows that the proliferation rate of Paramecium affected by gravity. The effect of gravity on Paramecium proliferation has been argued to be direct in a paper with an axenic culture under hypergravity. To clear up uncertainties with regard to the effect of gravity, Paramecium tetraurelia was cultured axenically under hypergravity (20 x g) and the time course of the proliferation was investigated quantitatively by a new non-invasive method, laser-beam optical slice, for measuring the cell density. This method includes optical slicing a part of the culture and computer-aided counting of cells in the sliced volume. The effects of hypergravity were assessed by comparing the kinetic parameters of proliferation that were obtained through a numerical analysis based on the logistic growth equation. Cells grown under 20 x g conditions had a significantly lower proliferation rate, and had a lower population density at the stationary phase. The lowered proliferation rate continued as long as cells were exposed to hypergravity (> one month). Hypergravity reduced the cell size of Paramecium. The long and short axes of the cell became shorter at 20 x g than those of control cells, which indicates a decrease in volume of the cell grown under hypergravity and is consistent with the reported increase in cell volume under microgravity. The reduced proliferation rate implies changes in biological time defined by fission age. In fact the length of autogamy immaturity decreased by measure of clock time, whereas it remained unchanged by measure of fission age.

Animals↗

PubMed1995

A multigene family of novel K+ channels from Paramecium tetraurelia.

Paramecium tetraurelia has an excitable membrane with electrical properties resembling those of nerve and muscle cells from metazoans. However, the relationship between the molecules underlying electrical excitability in Paramecium and those found in metazoans has been unexplored. Using a PCR-based screen, we identified a multigene family of putative potassium channels in the ciliate protist Paramecium tetraurelia. Two distinct genomic clones were isolated, each encoding an apparently complete channel, Pak1 and Pak2. Fragments of three additional genes were also isolated. The deduced amino acid sequences appear to define a family of novel K+ channel genes which are only distantly related to metazoan potassium channels; the K+-selective pore region is exceptionally well conserved, but outside this region, PaK1 and PaK2 bear little resemblance to the metazoan voltage-gated K+ channels, Shaker Shal, Shab or Shaw. The uniqueness of this large set of K+ channel genes may be accounted for by the distinct evolutionary pressures at play in the single celled Paramecium versus the multicellular Metazoans. Since a large number of K+ channel mutants exist in Paramecium, the sequences of the K+ channel gene family reported here may represent a significant step towards the molecular characterization of these important behavioral mutants.

Amino Acid Sequence↗

PubMedDate not supplied

Intraspecific genetic variation in Paramecium revealed by mitochondrial cytochrome C oxidase I sequences.

Studies of intraspecific genetic diversity of ciliates, such as population genetics and biogeography, are particularly hampered by the lack of suitable DNA markers. For example, sequences of the non-coding ribosomal internal transcribed spacer (ITS) regions are often too conserved for intraspecific analyses. We have therefore identified primers for the mitochondrial cytochrome c oxidase I (COI) gene and applied them for intraspecific investigations in Paramecium caudatum and Paramecium multimicronucleatum. Furthermore, we obtained sequences of the ITS regions from the same strains and carried out comparative sequence analyses of both data sets. The mitochondrial sequences revealed substantially higher variation in both Paramecium species, with intraspecific divergences up to 7% in P. caudatum and 9.5% in P. multimicronucleatum. Moreover, an initial survey of the population structure discovered different mitochondrial haplotypes of P. caudatum in one pond, thereby demonstrating the potential of this genetic marker for population genetic analyses. Our primers successfully amplified the COI gene of other Paramecium. This is the first report of intraspecific variation in free-living protozoans based on mitochondrial sequence data. Our results show that the high variation in mitochondrial DNA makes it a suitable marker for intraspecific and population genetic studies.

Animals↗

PubMed2000

Control of ciliary orientation through cAMP-dependent phosphorylation of axonemal proteins in paramecium caudatum.

Ciliary reorientations in response to cAMP do not take place after a brief digestion with trypsin in ciliated cortical sheets from Triton-glycerol-extracted Paramecium. In this study, we examined the effects of tryptic digestion on the cAMP-dependent phosphorylation of axonemal proteins to clarify the relationship between phosphorylation and ciliary reorientation. As reported for Paramecium tetraurelia, cAMP stimulated phosphorylations of the 29 kDa and 65 kDa axonemal polypeptides also in Paramecium caudatum. After a brief digestion of axonemes by trypsin, none of the cAMP-dependent phosphorylations occurred. On the other hand, the 29 kDa polypeptide still remained to be labeled after a brief digestion of axonemes that had previously been labeled with (32)P in the presence of cAMP, which indicates that this brief digestion breaks down endogenous cAMP-dependent protein kinases but not phosphorylated proteins. This must be the reason that trypsin-treated cilia on the sheets cannot reorient towards the posterior part of the cell. Our results indicate that cAMP regulates not only the beat frequency but also the ciliary orientation via phosphorylation of dynein subunits in Paramecium.

Animals↗

PubMed1997

Occurrence of a para-nitrophenyl phosphate-phosphatase with calcineurin-like characteristics in Paramecium tetraurelia.

Using para-nitrophenyl phosphate (pNPP) as a substrate for enzymatic activity, we sought to identify CaN in Paramecium. We isolated three different pNPP-phosphatases from the soluble fraction of Paramecium cells by anion-exchange and affinity column chromatographies. One, pNPP-phosphatase Peak I, is very similar to mammalian CaN. Divalent cation dependency, inhibition by calmodulin (CaM) antagonists (trifluoperazine, calmidazolium), and insensitivity to various phosphatase inhibitors (heparin, okadaic acid, sodium vanadate, etc.) show similarity to mammalian CaN rather than to any other Paramecium pNPP-hydrolyzing enzymes tested. Polyclonal antibodies against bovine brain CaN recognizing subunits A (61 or 58 kDa) and B (17 kDa) of brain CaN cross-reacted with a 63-kDa protein in fractions containing Peak IpNPP-phosphatase activity and coeluted calmodulin. Overlay assays using biotinylated brain calmodulin indicated Ca2+-dependent CaM-binding by the 63-kDa protein. A Ca2+-binding protein with the same electrophoretic mobility as CaN B (17 kDa) was also present, though in other fractions from DEAE-cellulose chromatography. This finding strongly suggests that, in the absence of Ca2+, both subunits, A and B, were separated either before or during chromatographic processing. Our data support the existence of both subunits of a CaN-like phosphatase in Paramecium cells.

4-Nitrophenylphosphatase↗

PubMed1980

Macronuclear DNA organization and transcription in Paramecium primaurelia.

Macronuclear DNA was isolated from Paramecium primaurelia, stock 168. Although the macronucleus is polyploid to the extent of 840C, in other respect the DNA appears to be simply organized, having neither satellite sequences nor substantial amounts of intermediately repetitive sequence. The sequence complexity of macronuclear DNA is quite low for a eukaryote cell, being approximately 19 times more complex than the genome of Escherichia coli. In addition, the GC content is low (25%) and the isolated DNA molecules have lengths mostly in the range 0.2-5 micrometer. In these various respects, the macronuclear DNA of Paramecium is similar to that of other ciliates. A clone of Paramecium cultured under controlled conditions contains polyadenylated RNA sequences which are homologous to 5-8% of the macronuclear DNA. Sequence complexity analysis indicates that the polyadenylated RNA contains two abundance classes of molecules, one present at low frequency and transcribed from approximately 10(4) genes, the other at 100 times greater concentration and transcribed from about 100 genes. The relevance of these results to the control of gene expression in Paramecium is discussed.

Animals↗

PubMed1982

Altered genetic code in Paramecium mitochondria: possible evolutionary trends.

The sequence and presumptive structure of a tRNA trp gene from Paramecium tetraaurelia are given. The gene is located 1,500 bp downstream from the 13S rRNA gene, in about the middle of the genome. Paramecium tRNA trp has a completely normal TpsiC loop and stem, however its anticodon (UCA) constitutes an alteration in the "universal" genetic code, similar to those seen in fungal and mammalian mitochondria. Most features of Paramecium tRNA trp resemble other mitochondrial counterparts; however, its sequence is more homologous to the "unaltered" tRNA trp (anticodon CCA) from E. coli. Paramecium mitochondria may resemble a primitive stage of organelle evolution.

Base Sequence↗