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PubMed2000

Phylogenetic relationships of the genus Paramecium inferred from small subunit rRNA gene sequences.

The genus Paramecium includes species that are well known and very common in freshwater environments. Species of Paramecium are morphologically divided into two distinct groups: the "bursaria" subgroup (foot-shaped) and the "aurelia" subgroup (cigar-shaped). Their placement within the class Oligohymenophorea has been supported by the analysis of the small subunit rRNA gene sequence of P. tetraurelia. To confirm the stability of this placement and to resolve relationships within the genus, small subunit rRNA gene sequences of P. bursaria, P. calkinsi, P. duboscqui, P. jenningsi, P. nephridiatum, P. primaurelia, and P. polycaryum were determined and aligned. Trees constructed using distance-matrix, maximum-likelihood, and maximum-parsimony methods all depicted the genus as a monophyletic group, clustering with the other oligohymenophorean taxa. Within the Paramecium clade, P. bursaria branches basal to the other species, although the remaining species of the morphologically defined "bursaria" subgroup do not group with P. bursaria, nor do they form a monophyletic subgroup. However, the species of the "aurelia" subgroup are closely related and strongly supported as a monophyletic group.

Animals↗
PubMed1993

Antimalarial drugs inhibit calcium-dependent backward swimming and calcium currents in Paramecium calkinsi.

The antimalarial drugs, quinacrine, chloroquine, quinine, primaquine, and mefloquine, share structural similarities with W-7, a compound that inhibits calcium-dependent backward swimming and calcium currents in Paramecium. Therefore, we tested whether antimalarial drugs also inhibit backward swimming and calcium currents in P. calkinsi. When the Paramecium is depolarized in high potassium medium, voltage-dependent calcium channels in the ciliary membrane open causing the cell to swim backward for 30 to 70 s. Application of calcium channel inhibitors, such as W-7, reduce the duration of backward swimming. In 0.05 mM calcium, quinacrine, mefloquine, quinine, chloroquine, primaquine and W-7 all reduced the duration of backward swimming. These effects were seen in sodium-containing and sodium-free high potassium solutions as well as sodium-free depolarizing solutions containing potassium channel blockers. In these low calcium solutions, backward swimming was inhibited by 50% at concentrations ranging from 100 nM to 30 microM. At higher calcium concentrations (1 mM or 15 mM), the effects of the antimalarials and W-7 were reduced. The effects of quinacrine and W-7 were tested directly on calcium currents using the two microelectrode voltage clamp technique. In 15 mM calcium, 100 microM quinacrine and 100 microM W-7 reduced the peak calcium current by 51% and 42%, respectively. Thus, antimalarial drugs reduce calcium currents in Paramecium calkinsi.

Animals↗
PubMed1980

Evolutionary divergence of mitochondrial DNA from Paramecium aurelia.

Mitochondrial (mt) DNA from four sibling species within the Paramecium aurelia complex, including stocks of different geographic origin and mutants, were analyzed using four 6-bp recognition site and one 4-bp recognition site endonucleases and the sequence divergence was estimated using Upholt's (1977) statistical procedure. All four species were readily distinguishable regardless of the restriction endonuclease employed. With intraspecies comparisons, no differences were observed which could be accounted for on the basis of geographic origin. Except for species 4, each stock (and mutant) gave a species-specific fragment pattern. For species 4, while the patterns were distinct from the other species, two species-specific type of patterns were found, designated A and B. The sequence divergence between these was estimated to be between 1 and 2 percent. With interspecies comparisons, the sequence divergence ranged from 3.9 to 10.3% with the greatest divergence being between species 1 and 4, and the least between species 1 and 5. The similarity between species 1 and 5 is in accord with other criteria for interspecies comparisons. The degree of sequence divergence measured here in Paramecium mt DNA is well within the range reported for rodents and primates. All four species mt DNA were cleaved to many DNA fragments by DPN II, an enzyme which recognizes non-methylated sites, and not by DPNI, the methyl-site specific counterpart of DPN II, suggesting that mt DNA from Paramecium aurelia is not appreciably methylated, if at all.

Base Sequence↗
PubMed1975

Studies on Macronuclear DNA from Paramecium aurelia.

Macronuclear DNA was isolated from purified macronuclei of Paramecium aurelia and the size distribution was determined with regard to growth phase and method of extraction. DNA molecules as long as 105 microns and as short as 0.2 microns were observed. It was concluded that the method of extraction affected the observed length of DNA extracted and that macronuclear DNA isolated from cells in balanced growth was less susceptible to nuclease degradation than was DNA isolated from cells in stationary phase. Renaturation studies were performed on macronuclear DNA and a kinetic complexity of 22-times E. coli DNA was determined. This value was similar to those values reported for Tetrahymena and Stylonychia macronuclear DNA. Correcting for GC base content yielded a kinetic complexity for Paramecium macronuclear DNA of 11-times E. coli DNA which corresponded to 3 X 10(10) daltons. There would be about 1400 copies of a unit genome of this complexity within each newly replicated macronucleus. Density gradient analysis indicated that the genes coding for ribosomal RNA had a greater density in CsCl than the bulk DNA. Molecular hybridization studies indicated that the genes coding for 25 S RNA represented 0.14 percent of the total macronuclear DNA. Correcting for GC base content, this corresponded to 30-35 25 S RNA genes per unit genome. These results on Paramecium are discussed in relationship to other ciliate macronuclear DNA.

Animals↗
PubMed1986

Accumulation of DNA damages in aging Paramecium tetraurelia.

Paramecium tetraurelia cells of ages 4, 15, and 27 days were labeled with [14C]-thymidine. In addition, cells were grown clonally for 27 days (108 generations) and labeled with [14C]-thymidine in the presence of 0.5 or 7.5 micrograms/ml of mitomycin-C (MMC) or no MMC. These cells were gently deposited on a filter membrane, which impedes the passage of DNA strands. The cells were then lysed with detergents and the cellular components washed through the filters, leaving double-stranded DNA intact on the surface. Proteinase K was used to remove histone or DNA-bound proteins. The DNA was then eluted under alkaline conditions, which denatures double-stranded DNA and converts apurinic/apyrimidinic sites into single-strand breaks. The results obtained with the cells of ages 4, 15, and 27 days (16, 60, and 108 generations, respectively) indicate that as Paramecium tetraurelia ages during asexual reproduction, apurinic/apyrimidinic lesions, strand breaks or single-strand gaps accumulate. This accumulation may be the basic mechanism of aging in such cells. In the MMC-treated cells of 27 days (108 generations), the MMC reduced elution of DNA fragments more at the higher than at the lower pH's used; random MMC cross-links should occur more often in longer strands than in shorter strands. The reductions in elution preferentially at higher pH, at which longer single strands would be eluted, confirmed the pH-versus-length relationship for Paramecium DNA eluted under our conditions.

Animals↗
PubMed1986

Lectin binding sites in Paramecium tetraurelia cells. II. Labeling analysis predominantly of non-secretory components.

All the lectin-FITC conjugates tested (ConA, RCA II, WGA) bind to the surface of Paramecium cells. Yet only WGA yields a distinct fluorescent pattern; it contours the basis of cilia and in some cells it brilliantly stains a few neighbouring rows of the regular surface fields in the anterioventral region (a region known to contain extensive fields of linear aggregates of freeze-fracture particles and to be engaged in conjugation). Incubation in vivo with WGA-FITC resulted in the selective labeling of the cytopharyngeal region as well as of the cytoproct. On Lowicryl K4M sections, WGA-gold probes concomitantly labeled disk-shaped vesicles that are assumed in the literature to serve as shuttle vesicles between these two cell regions and, thus, to connect forming and defecating digesting vacuoles (stages DV I and DV IV). On K4M sections WGA-Au stains also most other components of the lysosomal system. Also on K4M sections RCA II-Au labeled the walls of bacteria contained in DV I and II type digesting vacuoles (but not lysosomes identified bona fide by their size and shape and by their frequent vicinity to or continuity with digesting vacuoles). The WGA data largely support previous conclusions on the possible functional connection of all these elements (DV I-IV, smaller lysosomes, disk-shaped vesicles etc.) of the lysosomal system in Paramecium, as proposed by Allen and his group on the basis of other lines of evidence. As shown in the accompanying paper, ConA-FITC stained ghosts (formed after massive trichocyst exocytosis) also abut into DV-like structures. The different results obtained with the three lectins tested reflect the complex sorting machinery contained in the elaborate lysosomal system of a Paramecium cell. In the cytosol, finally, there occurs a particularly intense staining with ConA-gold, applied to Lowicryl sections, that probably represents glycogen-like particles. The same procedure reveals some weak staining of secretory contents and of nuclear structures.

Animals↗
PubMed1991

Studies of the cyclic adenosine monophosphate chemoreceptor of Paramecium.

A doublet of proteins (approximately 48,000 Mr) from the Paramecium cell body membrane fits several criteria for the external cAMP chemoreceptor. These criteria include: (i) selective elution from a cAMP affinity column, matching a specificity that could be predicted from the behavioral response and whole-cell binding; (ii) binding to wheat germ agglutinin indicating the presence of carbohydrate moieties indicating surface exposure; and (iii) selective inhibition of the intact cells' chemoresponse to cAMP by antibodies against the doublet. Additional evidence for the existence of a receptor, in general, comes from selective elimination of the cAMP chemoresponse by photoaffinity labeling of while cells with 8-N3-cAMP. The doublet proteins are not identical to the regulatory subunit of a cAMP-dependent protein kinase from Paramecium, the Dictyostelium cAMP chemoreceptor, or the 42-45 kDa range proteins related to the large surface glycoprotein in Paramecium. The doublet proteins are not readily separable and, as in Dictyostelium, may represent two different covalent modification states of the same protein. Amino acid analysis indicates that the proteins are similar, but does not distinguish between the possibilities of proteolysis and covalent modification. Once cloned, this doublet may prove to be only the fifth external, eukaryotic chemoreceptor to be identified.

Amino Acids↗
PubMed1990

Calmodulin defects cause the loss of Ca2(+)-dependent K+ currents in two pantophobiac mutants of Paramecium tetraurelia.

Two behavioral mutants of Paramecium tetraurelia, pantophobiacs A1 and A2, have single amino acid defects in the structure of calmodulin. The mutants exhibit several major ion current defects under voltage clamp: (i) the Ca2(+)-dependent K+ current activated upon depolarization of Paramecium is greatly reduced or missing in both mutants, (ii) both mutants lack a Ca2(+)-dependent K+ current activated upon hyperpolarization, and (iii) the Ca2(+)-dependent Na+ current is significantly smaller in pantophobiac A1 compared with the wild type, whereas this current is slightly increased in pantophobiac A2. Other, minor defects include a reduction in peak amplitude of the depolarization-activated Ca2+ current in pantophobiac A2, increased rates of voltage-dependent inactivation of this Ca2+ current in both pantophobiac A1 and pantophobiac A2, and an increase in the time required for the hyperpolarization-activated Ca2+ current to recover from inactivation in the pantophobiacs. The diversity of the pantophobiac mutations' effects on ion current function may indicate specific associations of calmodulin with a variety of Ca2(+)-related ion channel species in Paramecium.

Amino Acid Sequence↗
PubMed1989

Calcium-dependent potassium channel in Paramecium studied under patch clamp.

We have studied a class of Ca2+i-dependent K channels in inside-out excised membrane patches from Paramecium under patch clamp. single channels had a conductance of 72 +/- 9.0 pS in a solution containing 100 mM K+. The channels were selective for K+ over Rb+ with the permeability ratio of 1: 0.56, and over Na+, Cs+ or NH+4 with a ratio 1: less than 0.1. The channel activity was dependent on Ca2+i, which was applied to the cytoplasmic side; the Ca2+i concentration for the half maximal activation was 2 microM. The Hill coefficient for the Ca2+i dependence of the channel activity was 2.58, indicating that more than two Ca2+i bindings are necessary for full activation. Unlike most Ca2+i-dependent K channels in other organisms, the channels in Paramecium were slightly more active upon hyperpolarization than upon depolarization. The voltage dependence was fitted to a Boltzmann curve with 41.2 mV per e-fold change in channel activity. While a high Ca2+i concentration activated the channels, it also irreversibly reduced the channel activity over time. The decay of channel activity occurred faster at higher Ca2+i concentrations. Quaternary ammonium ions suppressed ion passage through the channel; more highly alkylated quaternary ammonium ions were more efficient in blocking. Ba2+i and Ca2+i were relatively ineffective in blockage. it was concluded that these Ca2+i-dependent K channels in Paramecium are different from the previously described Ca2+i-dependent K channels, and are perhaps of a novel class.

Animals↗
PubMed1989

Proteolytic activation of a hyperpolarization- and calcium-dependent potassium channel in Paramecium.

The effects of proteolysis on a hyperpolarization- and Ca2+-dependent K channel from the surface membrane of Paramecium tetraurelia were examined in the inside-out excised patch mode. Treatment with trypsin, pronase or thermolysin removed the Ca2+-dependence of the channel activation, yielding an increase in channel activity greater than 2.5-fold at all Ca2+ concentrations between 10(-4) and 10(-8) M. Thermolysin additionally removed the voltage dependence of channel opening and gave the most activation among the three proteases tested. Proteolysis did not affect the single-channel conductance. In an analogy to the mechanism of activation of many Ca2+-dependent enzymes it is suggested that this Paramecium channel has a cytoplasmic inhibitory domain which can be removed by proteolysis, and that the physiological activation by Ca2+ is due to a temporary removal of this inhibition. Moreover, these findings indicate structural differences between depolarization-, Ca2+-dependent K channels (BK channels) and the hyperpolarization-, Ca2+-dependent K channels in Paramecium.

Animals↗
PubMed1978

A potassium conductance activated by hyperpolarization in paramecium.

Voltage clamp studies show that the wild-type membrane of Paramecium tetraurelia contains a conductance component which is sensitive to hyperpolarization. This component manifests itself as "anomalous", or "inward going", rectification of membrane voltage in response to applied constant current pulses and as a "hyperpolarizing spike" when no K is added to the external solution (Y. Satow, C. Kung, 1977. J. Comp. Physiol. 119:99). Like the conductances which underlie anomalous rectification in other cells, the hyperpolarization-sensitive conductance in Paramecium is specific for K, and the magnitude of the voltage-dependent conductance change depends not only on voltage but also on external potassium concentration. The internal potassium ion concentration of Paramecium is calculated to be between 17 and 18mM.

Animals↗
PubMed2000

Polyamine triggering of exocytosis in Paramecium involves an extracellular Ca(2+)/(polyvalent cation)-sensing receptor, subplasmalemmal Ca-store mobilization and store-operated Ca(2+)-influx via unspecific cation channels.

The polyamine secretagogue, aminoethyldextran (AED), causes a cortical [Ca(2+)] transient in Paramecium cells, as analyzed by fluorochrome imaging. Our most essential findings are: (i) Cortical Ca(2+) signals also occur when AED is applied in presence of the fast Ca(2+) chelator, BAPTA. (ii) Extracellular La(3+) application causes within seconds a rapid, reversible fluorescence signal whose reversibility can be attributed to a physiological [Ca(2+)](i) transient (while injected La(3+) causes a sustained fluorescence signal). (iii) Simply increasing [Ca(2+)](o) causes a similar rapid, short-lived [Ca(2+)](i) transient. All these phenomena, (i-iii), are compatible with activation of an extracellular "Ca(2+)/(polyvalent cation)-sensing receptor" known from some higher eukaryotic systems, where this sensor (responding to Ca(2+), La(3+) and some multiply charged cations) is linked to cortical calcium stores which, thus, are activated. In Paramecium, such subplasmalemmal stores ("alveolar sacs") are physically linked to the cell membrane and they can also be activated by the Ca(2+) releasing agent, 4-chloro-m-cresol, just like in Sarcoplasmic Reticulum. Since this drug causes a cortical Ca(2+) signal also in absence of Ca(2+)(o) we largely exclude a "Ca(2+)-induced Ca(2+) release" (CICR) mechanism. Our finding of increased cortical Ca(2+) signals after store depletion and re-addition of extracellular Ca(2+) can be explained by a "store-operated Ca(2+) influx" (SOC), i.e., a Ca(2+) influx superimposing store activation. AED stimulation in presence of Mn(2+)(o) causes fluorescence quenching in Fura-2 loaded cells, indicating involvement of unspecific cation channels. Such channels, known to occur in Paramecium, share some general characteristics of SOC-type Ca(2+) influx channels. In conclusion, we assume the following sequence of events during AED stimulated exocytosis: (i) activation of an extracellular Ca(2+)/polyamine-sensing receptor, (ii) release of Ca(2+) from subplasmalemmal stores, (iii) and Ca(2+) influx via unspecific cation channels. All three steps are required to produce a steep cortical [Ca(2+)] signal increase to a level required for full exocytosis activation. In addition, we show formation of [Ca(2+)] microdomains (</=0.5 microm, </=33 msec) upon stimulation.

Animals↗
PubMed1996

Cold-sensitive Ca2+ influx in Paramecium.

The concentration of intracellular calcium, [Ca2+]i, in Paramecium was imaged during cold-sensitive response by monitoring fluorescence of two calcium-sensitive dyes, Fluo-3 and Fura-Red. Cooling of a deciliated Paramecium caused a transient increase in [Ca2+]i at the anterior region of the cell. Increase in [Ca2+]i was not observed at any region in Ca(2+)-free solution. Under the electrophysiological recording, a transient depolarization of the cell was observed in response to cooling. On the voltage-clamped cell, cooling induced a transient inward current under conditions where K+ currents were suppressed. These membrane depolarizations and inward currents in response to cooling were lost upon removing extracellular Ca2+. The cold-induced inward current was lost upon replacing extracellular Ca2+ with equimolar concentration of Co2+, Mg2+ or Mn2+, but it was not affected significantly by replacing with equimolar concentration of Ba2+ or Sr2+. These results indicate that Paramecium cells have Ca2+ channels that are permeable to Ca2+, Ba2+ and Sr2+ in the anterior soma membrane and the channels are opened by cooling.

Animals↗
PubMed2006

Multiple tubulin forms in ciliated protozoan Tetrahymena and Paramecium species.

Tetrahymena and Paramecium species are widely used representatives of the phylum Ciliata. Ciliates are particularly suitable model organisms for studying the functional heterogeneity of tubulins, since they provide a wide range of different microtubular structures in a single cell. Sequencing projects of the genomes of members of these two genera are in progress. Nearly all members of the tubulin superfamily (alpha-, beta-, gamma-, delta-, epsilon-, eta-, theta-, iota-, and kappa-tubulins) have been identified in Paramecium tetraurelia. In Tetrahymena spp., the functional consequences of different posttranslational tubulin modifications (acetylation, tyrosination and detyrosination, phosphorylation, glutamylation, and glycylation) have been studied by different approaches. These model organisms provide the opportunity to determine the function of tubulins found in ciliates, as well as in humans, but absent in some other model organisms. They also give us an opportunity to explore the mechanisms underlying microtubule diversity. Here we review current knowledge concerning the diversity of microtubular structures, tubulin genes, and posttranslational modifications in Tetrahymena and Paramecium species.

Animals↗
PubMed1976

Defective ion regulation in a class of membrane-excitation mutants in Paramecium.

The "paranoiac" mutants of Paramecium aurelia show prolonged backward swimming in solutions containing Na+, unlike wild-type paramecia, which jerk back and forth in Na+ solutions. The paranoiac mutants in Na+ solutions also show large losses of cellular K+ and large influxes of Na+. Three different paranoiac mutants all show similar defects in ion regulation but to different degrees. Wild-type Paramecium, in contrast, shows no Na+ -dependent loss of cellular K+ and a much smaller Na+ influx. In K+ -containing solutions, there is no difference between wild-type and paranoiac paramecia with respect to their cellular K+ content. The Na+ influx, the K+ loss, and the duration of backward swimming are all proportional to the extracellular Na+ concentration. Electrophysiologically, the backward swimming of the paranoiac mutants corresponds to a prolonged depolarization of the membrane potential, while the backward jerks of wild-type Paramecium correspond to a series of transient depolarizations. We propose that the large Na+ influxes and the large K+ effluxes in paranoiacs occur during the periods of backward swimming, while the membrane is depolarized.

Animals↗
PubMed1979

Biochemical studies of the excitable membrane of Paramecium tetraurelia. II. Phospholipids of ciliary and other membranes.

The phospholipids of cilia and deciliated bodies of Paramecium tetraurelia were isolated and characterized. 1-alkyl-2-acyl-sn-glycero-3-(2'-aminoethyl) phosphonate (GAEPL), phosphatidylethanolamine, and 1-alkyl-2-acyl-sn-glycero-3-phosphorylcholine (GPC) were the major lipids of Paramecium, and the minor lipids included phosphatidylinositol, cardiolipin, ceramide-(2-aminoethyl) phosphonate (CAEP), ceramide phosphorylethanolamine (COPE) and four sphingolipids whose identity was not established. The deciliated bodies contained 4% cardiolipin, 15% GAEPL, 41% phosphatidylethanolamine, 30% GPC and 3% each of CAEP and phosphatidylinositol; the cilia contained no cardiolipin, 24% GAEPL, 37% phosphatidylethanolamine, 15% GPC, 15% CAEP, 3% phosphatidylinositol, 2% COPE and small amounts (approx. 1%) of the four uncharacterized sphingolipids. No alteration in phospholipid composition was found among cells harvested in the various stages of growth. The phospholipids of six Paramecium mutants of three distinct phenotypes (pawn, paranoiac and fast) were also examined. Only one significant difference was found on comparison of the whole cell, deciliated body and cilia fraction of the mutants with the analogous fractions from wild type cells: the fast mutant, fA 97, had two extra, minor phospholipids (approx. 2%) in the deciliated body fraction that were tentatively identified as 1,2-diacyl-sn-glycero-3-(2'-aminoethyl) phosphonate (AEPL) and 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamine (GPE).

Animals↗
PubMed1984

Ionic regulation of cyclic AMP levels in Paramecium tetraurelia in vivo.

cAMP levels in Paramecium increased dose dependently after a step increase of [Ca] or [Sr] in the incubation, provided K was present. Two mM Ca or Sr tripled cAMP concentrations within 3 s and induced an increase in forward swimming speed. The increase in cAMP formation was strictly dependent on the Donnan ratio [K]: square root [Ca]. Na, Li, or tetraethylammonium could not replace K. The data provide evidence for regulation of cAMP in Paramecium by the membrane surface charge as determined specifically by the regulation of cAMP in Paramecium by the membrane surface charge as determined specifically by the K: Ca ratio.

Animals↗
PubMed1995

Structure and evolution of Paramecium hemoglobin genes.

Hemoglobin (Hb) genes have been cloned from three different species of ciliated protists, P. multimicronucleatum, P. triaurelia and P. jenningsi. Southern blotting of the genomic DNAs using the P. caudatum Hb cDNA showed both intraspecies variation in different stocks of P. caudatum and interspecies variation within the genus Paramecium. The isolated Hb genes were composed of 118, 117 and 117 codons, and interrupted by a short intron with 27, 29 and 29 bp at the same position, in P. multimicronucleatum, P. triaurelia and P. jenningsi, respectively. This suggests that the one-intron and two-exon structure has been conserved in the Hb genes in this genus. The amino acid sequences of the Paramecium Hbs were more than 87% identical to one another and homologous to those from the other ciliated protists Tetrahymena thermophila and T. pyriformis, the green alga Chlamydomonas eugametos, and the cyanobacterium Nostoc commune Hbs, all of which consist of about 120 amino acid residues (120-aa group). In particular, the amino acid sequences of the P. triaurelia and P. jenningsi Hbs were the same, although there were 20 nucleotide differences between the coding regions in the two genes. A maximum likelihood inference as to the phylogenetic relationships among these genes suggests that the Paramecium Hbs genes have evolved more rapidly than the other genes in the 120-aa group, and that P. triaurelia and P. genningsi are sibling species and the P. aurelia complex became a small cell after it separated from P. jenningsi.

Amino Acid Sequence↗