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PubMed1988

A survey of lectin binding in Paramecium.

To better understand the general distribution of glycoproteins and the distribution of specific glycoprotein-bound sugar residues in Paramecium, a survey of the binding pattern of selected lectins was carried out in P. tetraurelia, P. caudatum, and P. multimicronucleatum. Lectins studied were concanavalin A (Con A), Griffonia simplicifolia agglutinins I and II (GS I and GS II), wheat germ agglutinin (WGA), Ulex europaeus (UEA I), peanut agglutinin (PNA), Ricinis communis toxin (RCA60) and agglutinin (RCA120), soybean agglutinin (SBA), Bauhinia purpurea agglutinin (BPA), Dolichos biflorus agglutinin (DBA), and Maclura pomifera agglutinin (MPA). Those giving the most distinctive patterns were Con A, GS II, WGA, UEA I, and PNA. No significant differences were found between the three species. Concanavalin A, a mannose/glucose-binding lectin, diffusely labeled the cell surface and cytoplasm and, unexpectedly, the nuclear envelopes. Events of nuclear division, and nuclear size and number were thus revealed. Both WGA and GS II, which are N-acetylglucosamine-binding lectins, labeled trichocyst tips, the cell surface, and the oral region, revealing stages of stomatogenesis. The lectin WGA, in addition, labeled the compartments of the phagosome-lysosome system. The lectin PNA, an N-acetyl galactosamine/galactose-binding protein, was very specific for digestive vacuoles. Finally, UEA I, a fucose-binding lectin, brightly labeled trichocysts, both their tips and body outlines. We conclude that a judicious choice of lectins can be used to localize glycoproteins and specific sugar residues as well as to study certain events of nuclear division, cellular morphogenesis, trichocyst discharge, and events in the digestive cycle of Paramecium.

Animals↗
PubMedDate not supplied

The inositol lipids of Paramecium tetraurelia and preliminary characterizations of phosphoinositide kinase activity in the ciliary membrane.

Inositol glycerolipids make up less than 10% of total phospholipids of Paramecium tetraurelia cells. Unlike inositol lipids found in mammalian and other cell types, these lipids from Paramecium lack arachidonic acid. It was demonstrated that kinase and possibly phosphatase enzymes that interconvert phosphatidylinositol (PI), phosphatidylinositol phosphate (PI-P) and phosphatidylinositol-bis-phosphate (PI-P2) exist in ciliary membranes of this ciliate. When exogenous soybean PI and [gamma-32P]ATP were provided as substrates, isolated cilia preparations exhibited PI and PI-P kinase activities as demonstrated by the incorporation of radiolabel into PI-P and PI-P2. Kinase activity was activated by millimolar [Mg2+] and inhibited by millimolar [Ca2+]. Significant inhibition of kinase activity in the presence of unlabeled excess ATP suggested that ATP is the preferred phosphate donor for this reaction. Of 4 suborganellar fractions of isolated cilia, the membrane fraction had the greatest kinase activity indicating that the enzyme(s) is membrane-associated.

1-Phosphatidylinositol 4-Kinase↗
PubMedDate not supplied

Structural and functional characterization of paramecium dynein: initial studies.

Dynein arms and isolated dynein from Paramecium tetraurelia ciliary axonemes are comparable in structure, direction of force generation, and microtubule translocation ability to other dyneins. In situ arms have dimensions and substructure similar to those of Tetrahymena. Based on spoke arrangement in intact axonemes, arms translocate axonemal microtubules in sliding such that active dynein arms are (-) end directed motors and the doublet to which the body and cape of the arms binds (N) translocates the adjacent doublet (N + 1) tipward. After salt extraction, based on ATPase activity, paramecium dynein is found as a 22S and a 14S species. The 22S dynein is a three-headed molecule that has unfolded from the in situ dimensions; the 14S dynein is single headed. Both dyneins can be photocleaved by UV light (350 nm) in the presence of Mg2+, ATP and vanadate; the photocleavage pattern of 22S dynein differs from that seen with Tetrahymena. Both isolated dyneins translocate taxol-stabilized, bovine brain microtubules in vitro. Under standard conditions, 22S dynein, like comparable dyneins from other organisms, translocates at velocities that are about three times faster than 14S dynein.

Animals↗
PubMedDate not supplied

Small GTP-binding proteins associated with secretory vesicles of Paramecium.

GTP-binding proteins act as molecular switches in a variety of membrane-associated processes, including secretion. One group of GTP-binding proteins, 20-30 kDa, is related to the product of the ras proto-oncogene. In Saccharomyces cerevisiae, ras-like GTP-binding proteins regulate vesicular traffic in secretion. The ciliate protist Paramecium tetraurelia contains secretory vesicles (trichocysts) whose protein contents are released by regulated exocytosis. Using [alpha-32P]GTP and an on-blot assay for GTP-binding, we detected at least seven GTP-binding proteins of low molecular mass (22-31 kDa) in extracts of Paramecium tetraurelia. Subcellular fractions contained characteristic subsets of these seven; cilia were enriched for the smallest (22 kDa). The pattern of GTP-binding proteins was altered in two mutants defective in the formation or discharge of trichocysts. Trichocysts isolated with their surrounding membranes intact contained two minor GTP-binding proteins (23.5 and 29 kDa) and one major GTP-binding protein (23 kDa) that were absent from demembranated trichocysts. This differential localization of GTP-binding proteins suggests functional specialization of specific GTP-binding proteins in ciliary motility and exocytosis.

Animals↗
PubMedDate not supplied

Inter- and intrasyngenic variations of the hemoglobin components in Paramecium caudatum.

The ciliated protozoan Paramecium contains hemoglobin in heterogeneous monomeric forms. In particular, Paramecium caudatum is characterized by the presence of a major component called Hb10 and a basic component named bHb. We found that in P. caudatum both of these hemoglobin components show some variation according to stock. The types and distributions of these hemoglobin components were examined on 16 stocks in five different syngens and one stock in an unidentified syngen using high performance liquid chromatography. The results indicate that in a variety of stocks the major component, Hb10, was divided into three types, A, B or A + B, and that the basic hemoglobin component was composed of a combination of two or three variants out of four possible, i.e. bHb 1, bHb 2, bHb 3 and bHb 4. Neither the Hb10 types nor the bHb variants, however, could be used to distinguish syngen in P. caudatum, since all of the Hb10 types and bHb variants were widely distributed over syngens and identical profiles appeared to some stocks in different syngens.

Animals↗
PubMedDate not supplied

Molecular characterization of the D surface protein gene subfamily in Paramecium primaurelia.

When paramecium primaurelia expresses the D serotype, a major high molecular weight mRNA species is detected in the cytoplasm. Using the cDNA derived from this mRNA as a probe, three very similar genes, D alpha, D beta and D gamma, were cloned. Of these three genes, we show that only the D alpha mRNA is present in the cytoplasm of cells expressing the D serotype and corresponds to the major mRNA species. The nucleotide sequence of the entire coding region of the D alpha gene, as well as the upstream and downstream sequences, has been determined. The 7632-nucleotide open reading frame encodes a putative protein that displays the characteristic cysteine residue periodicity of Paramecium surface antigens but does not contain central tandemly repeated sequences. Partial sequences of the two nonexpressed genes D beta and D gamma indicate a high percentage of identity (90%-95%) with the D alpha gene, suggesting that D beta and D gamma genes are either very similar surface protein genes whose transcription is repressed trough mutual exclusion, or perhaps are pseudogenes. A region of variable DNA rearrangement was identified 1 kb upstream of the D gamma gene. This macronuclear region arises from the same micronuclear locus by alternative excision of internal eliminated sequences during macronuclear development.

Amino Acid Sequence↗
PubMedDate not supplied

Molecular characterization of the D surface protein gene subfamily in Paramecium tetraurelia.

When Paramecium tetraurelia expresses the D serotype, detectable by serum tests, high molecular mRNA could be isolated, which corresponds to the molecular mass of the D surface protein. Using this D specific mRNA as a probe for screenings in different genomic libraries a subfamily of five very similar genes was found, named alpha-51D, gamma 1-51D, gamma 2-51D, delta-51D, and epsilon-51D. Each of them is about 8-kb long, they show regions of identity to each other, and there is no evidence that any are defective genes or pseudogenes. Up to now serotype D is the only known serotype showing this phenomenon. Another novel feature is that two of the D isogenes are closely linked. The sequence for the entire coding region of the alpha-51D gene has been determined, as well as the upstream and downstream noncoding regions. Its deduced amino acid sequence shows the same characteristic cysteine periodicity displayed by all other immobilization antigen (i-ag) genes from Paramecium. However, in contrast to most other such genes, tandem repeats are missing from the 7599-bp long coding region of the alpha-51D gene. When the sequences of the type 51D genes are compared to each other, the similarity is very high and extends to coding as well as to noncoding regions. Similarity within noncoding regions is usually only observed for allelic i-ag genes. We conclude that the type D genes constitute a family of isogenes that are nonallelic. They contain slightly different consensus sequences with possible functions as regulatory regions.

Amino Acid Sequence↗
PubMedDate not supplied

The 44-kDa regulatory subunit of the Paramecium cAMP-dependent protein kinase lacks a dimerization domain and may have a unique autophosphorylation site sequence.

The 44-kDa regulatory subunit (R44) of one form of cAMP-dependent protein kinase of Paramecium was purified, and two partial internal amino acid sequences from it were used to clone the corresponding cDNA. This R44 cDNA clone was 1022-bp long, including 978 bp of coding sequence and 7 bp and 37 bp of 5' and 3' untranslated sequences, respectively. A 1.1-kb mRNA was labeled on a Northern blot. The deduced R44 amino acid sequence had 31%-38% positional identity to the sequences of other cloned cAMP-dependent protein kinase regulatory subunits. R44 sequence showed equal sequence similarity to mammalian types I and II regulatory subunits. The N-terminal sequence encoding the regulatory subunit dimerization domain found in most regulatory subunits is not present in the R44 clone, confirming the lack of regulatory subunit dimer formation previously reported for the Paramecium cAMP-dependent protein kinase. The putative autophosphorylation site of R44 contains the amino acid sequence TRTS, distinct from the consensus sequence RRXS, where X is any residue, found in other autophosphorylated cAMP-dependent protein kinase regulatory subunits and many cAMP-dependent protein kinase substrates.

Amino Acid Sequence↗
PubMedDate not supplied

Introducing antisense oligodeoxynucleotides into Paramecium via electroporation.

A method utilizing electroporation to deliver antisense oligodeoxynucleotides into Paramecium tetraurelia has been developed. For these studies antisense oligonucleotides directed to different regions of the calmodulin mRNA were used. It was found that a pulse delivered at 150-250 V (375-625 V/cm field strength) for 3.9-4.2 ms using a 275 microF capacitor with resistance set at 13 Ohms was sufficient to achieve measurable incorporation of fluorescently-labeled oligodeoxynucleotides in up to 95% of the cells treated. Optimal parameters included using oligodeoxynucleotides of at least 12 bases in length with a 3' blocking group at a dose of around 10 microM. In addition, multiple oligodeoxynucleotides directed to the same target mRNA resulted in at least a 10-fold reduction in the dose of oligodeoxynucleotide required to achieve the desired effects. Taken together, these results indicate that the use of antisense oligodeoxynucleotides can be an easy and useful method for linking genes to specific functions in Paramecium tetraurelia. Finally, this report discusses how different 3' blocking groups and the use of combinations of oligodeoxynucleotides directed to different regions of the same target mRNA can help address concerns about specificity.

Animals↗
PubMedDate not supplied

Correlation between loss of a Mg2+ conductance and an adaptation defect in a mutant of Paramecium tetraurelia.

Paramecium tetraurelia responds to chronic KCl-induced depolarization by swimming backward, but the ciliate recovers within seconds and then undergoes a prolonged adaptation period during which sensitivity to external stimuli is altered radically. We examined the role of Mg2+ in this phenomenon, prompted by finding that mutations in the eccentric-A gene both suppressed a Mg(2+)-specific conductance and prevented adaptation. Adaptation of the wild type proceeded normally when extracellular Mg2+ was varied from 0-20 mM, however, suggesting that channel-mediated Mg2+ fluxes were not involved. In seeking alternative explanations for the eccentric mutant phenotype, we ascertained that there was an osmotic component to adaptation but that K(+)-induced depolarization was the primary stimulus. We also noted that wild-type and eccentric mutant cells depolarized by equivalent amounts in KCl, suggesting that the genetic lesion must lie downstream of membrane-potential change. We also examined whether the adaptation-induced behavioral changes and, indeed, the defect in eccentric might be explained in terms of Mg2+ and Na+ efflux during behavioral testing, but experimental observations failed to support this notion. Finally, we consider the possibility that eccentric gene mutation prevents adaptation by interfering with intracellular free Mg2+ homeostasis in Paramecium.

Adaptation, Physiological↗
PubMedDate not supplied

An indexed genomic library for Paramecium complementation cloning.

Recent pioneering work opened the way for cloning genes in Paramecium by functional complementation of mutants. We present here the construction and pilot utilization of a new indexed library of Paramecium macronuclear DNA. The library is made of 61,440 clones containing inserts mostly between 6 and 12 kilobases. It has already allowed the complementation cloning of four new genes, and this library has proven to be very useful for rapid hybridization cloning.

Animals↗
PubMedDate not supplied

Epigenetic mechanisms affecting macronuclear development in Paramecium and Tetrahymena.

Epigenetic inheritance includes all non-Mendelian inheritance, in fact any inheritance that does not arise from base changes. Ciliates, particularly Paramecium and Tetrahymena, undergo epigenetic changes to their macronuclei when they are formed at nuclear reorganization. Once set, however, they are reproduced in a constant fashion, except for allelic segregations, during vegetative fissions in Tetrahymena and certain life cycle changes in both Paramecium and Tetrahymena. This review is meant to be inclusive, discussing all the known cases of epigenetic changes in macronuclei. They involve virtually all traits. We find that these macronuclear changes are subject to a variety of modifications in the way that they are implemented. They constitute a major feature of ciliate genetics, probably because the separation of generative and vegetative functions to micronuclei and macronuclei makes such changes possible.

Animals↗
PubMedDate not supplied

Nuclear and cortical regulation in doublets of Paramecium: II. When and how do two cortical domains reorganize to one?

Homopolar doublets with twofold rotational symmetry were generated in Paramecium tetraurelia and in P. undecaurelia by electrofusion or by arrested conjugation. These doublets underwent a complex cortical reorganization over time, which led to their reversion to singlets. This reorganization involved a reduction in number of ciliary rows, a progressive inactivation and loss of one oral meridian, and a reduction and eventual disappearance of one cortical surface (semicell) situated between the two oral meridians. The intermediate steps of this reorganization included some processes that resemble those previously described in regulating doublets of other ciliates, and others that are peculiar to members of the "P. aurelia" species-group and some of its close relatives. The former included a disappearance of one cortical landmark (a contractile vacuole meridian) and transient appearance of another (a third cytoproct) within the narrower semicell. The latter included a reorganization of the paratene zone and the associated invariant (non-duplicating) region to occupy the entire narrower semicell and a redistribution of zones of most active basal-body proliferation within the opposite, wider semicell. The final steps of reorganization involved anterior displacement, invagination, and resorption of one of the two oral apparatuses and eventual disappearance of the associated oral meridian. An oral meridian deprived of its oral apparatus, either by spontaneous resorption or microsurgical removal, could persist for some time in "incomplete doublets" before regulating to the singlet condition. The phylogenetically widespread events encountered in the regulation of doublets to singlets suggest that Paramecium shares some of the global regulatory properties that are likely to be ancestral in ciliates. The more specific events are probably associated with the complex cytoskeletal architecture of this organism and with the frequent occurrence of autogamy that was described in the preceding study (Prajer et al. 1999).

Animals↗
PubMedDate not supplied

The vacuolar-atpase of Paramecium multimicronucleatum: gene structure of the B subunit and the dynamics of the V-ATPase-rich osmoregulatory membranes.

Previous studies have shown that the vacuolar-ATPase (V-ATPase) of the contractile vacuole complexes (CVCs) in Paramecium multimicronucleatum is necessary for fluid segregation and osmoregulation. In the current study, immunofluorescence showed that the development of a new CVC begins with the formation of a new pore around which the collecting canals form. The decorated membranes are then deposited around the newly formed collecting canals. Quick-freeze deep-etch techniques reveal that six 10-nm-wide V-ATPase V, sectors, tightly packed into a 20 x 30-nm rectangle, form two rows of these compacted sectors that helically wrap around the cytosolic side of decorated membrane tubules. During new CVC formation, packing of decorated tubules around mature CVCs was temporarily disrupted so that some of these decorated tubules became transformed into decorated vesicles. Freeze-fracturing of these decorated vesicles revealed a highly pitted E-face and a particulate P-face. The V-ATPase was purified for the first time in any ciliated protozoan and shown to contain, as in other cells, the V1 subunits A to E, and four 14-20 kDa polypeptides. The B subunit was cloned and found to be encoded by one gene containing four short introns. This subunit has 510 amino acid residues with a predicted molecular weight of 56.8 kDa, a value similar to B subunits of other organisms. Except for the N- and C-termini, it has a 75% sequence identity with other B subunits, suggesting that the B subunits in Paramecium, like other species, have been conserved and that the entire surface of this subunit may be important in interacting with other subunits.

Amino Acid Sequence↗
PubMedDate not supplied

Paramecium aurelia revisited.

The species Paramecium aurelia sensu latu, containing 15 sexually isolated subspecies (syngens), is the classic example of a sibling species complex in the ciliates. Using DNA sequence comparison, it is now possible to see whether this example parallels other studied sibling species complexes. We sequenced the internal transcribed spacer (ITS) region of the nuclear ribosomal cistron for 13 of the syngens plus two other Paramecium species and several Tetrahymena spp. Using available spirotrich sequences of the internal transcribed spacer 2 (ITS2), we established the RNA transcript folding pattern for ciliates. Ciliates exhibit the two highly conserved helices in their RNA transcript folding pattern in common with other eukaryotes, despite their unusual nuclear behavior and their presumed low copy number of micronuclear ribosomal repeats. Consequently, the set of 111-116 ITS2 nucleotide positions that are relatively conserved in evolution can be derived and used for comparative analysis. Mating behavior (i.e. gamete agglutination and fusion) is the character showing greatest correlation with the degree of ITS2 evolution in the P. aurelia complex, as also found in other eukaryotes. The degree of change in the ITS2 relatively conserved sequences found among the sibling species of P. aurelia is the same degree as found among the sibling species of the Drosophila melanogaster-mauritania-sechellia-simulans-yakuba species complex. The relatively conserved subregion of ITS2, determined from transcript secondary structure, is a tool for identifying the level of the biological species in the absence of knowledge of sexual compatibility in both micro- and macro-eukaryote species complexes.

Animals↗
PubMed2006

A multigene family encoding R-SNAREs in the ciliate Paramecium tetraurelia.

SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane interactions and are conventionally divided into Q-SNAREs and R-SNAREs according to the possession of a glutamine or arginine residue at the core of their SNARE domain. Here, we describe a set of R-SNAREs from the ciliate Paramecium tetraurelia consisting of seven families encoded by 12 genes that are expressed simultaneously. The complexity of the endomembrane system in Paramecium can explain this high number of genes. All P. tetraurelia synaptobrevins (PtSybs) possess a SNARE domain and show homology to the Longin family of R-SNAREs such as Ykt6, Sec22 and tetanus toxin-insensitive VAMP (TI-VAMP). We localized four exemplary PtSyb subfamilies with GFP constructs and antibodies on the light and electron microscopic level. PtSyb1-1, PtSyb1-2 and PtSyb3-1 were found in the endoplasmic reticulum, whereas PtSyb2 is localized exclusively in the contractile vacuole complex. PtSyb6 was found cytosolic but also resides in regularly arranged structures at the cell cortex (parasomal sacs), the cytoproct and oral apparatus, probably representing endocytotic compartments. With gene silencing, we showed that the R-SNARE of the contractile vacuole complex, PtSyb2, functions to maintain structural integrity as well as functionality of the osmoregulatory system but also affects cell division.

Amino Acid Sequence↗
PubMed1977

Localization of calcium channels in Paramecium caudatum.

1. Electrical recordings from Paramecium caudatum were made after removal of the cilia with chloral hydrate and during ciliary regrowth to study the electrical properties of that portion of the surface membrane enclosing the ciliary axoneme. 2. Removal of the somatic cilia (a 50% reduction in membrane surface area) results in an almost complete elimination of the regenerative Ca response, all-or-none Ba2+ spike, and delayed rectification. 3. A twofold increase in input resistance resulted from the 50% reduction in membrane surface area. 4. The electrical properties remained unchanged, despite prolonged exposure to the chloral hydrate, until the cilia were mechanically removed. 5. Restoration of the Ca response accompanied ciliary regrowth, so that complete excitability returns when the cilia regain their original lengths. 6. It is concluded that the voltage-sensitive Ca channels are localized to that portion of surface membrane surrounding the cilia. 7. Measurements of membrane constants before and after deciliation and estimations of the cable constants of a single cilium suggest that the cilia of Paramecium may be fully isopotential along their length and with the major cell compartment.

Action Potentials↗
PubMed1978

Calcium-dependent repolarization in Paramecium.

1. Intracellular injection, recording and current-passing methods were used to investigate the role of intracellular Ca in the modulation of electrical behaviour in the ciliate Paramecium caudatum.2. Injection of EGTA converted graded regenerative responses ascribed to Ca inward current to all-or-none action potentials. The EGTA injection also caused a discontinuity in the steady state I-V relations to outward current, but had little effect on hyperpolarizing current-voltage responses.3. The overshoot of the all-or-none spike produced by the EGTA-injected cell followed an approximate 29 mV increase for a tenfold increase in external Ca concentration and was independent of changes in external K and Na concentrations.4. The EGTA-induced all-or-none action potential tended to produce plateaus that could last up to 20 sec. During the plateau the membrane slowly repolarized to a critical potential, upon which repolarization occurred precipitously.5. Injection of 10(-6)M-free Ca(2+) as a Ca-EGTA buffer hyperpolarized the membrane and decreased the potential shifts to inward current pulses. These responses are consistent with an increase in K conductance.6. During EGTA plateaus reversed beating of the cilia indicated a rise in intracellular Ca, and thus an inability of the EGTA to complex the Ca as rapidly as it entered the cilia. Reversal of the motile apparatus thus appears to be activated at lower concentrations of intracellular Ca than are required to activate the inferred Ca-dependent K system.7. In uninjected cells removal of the cilia, which results in a loss of the voltage-activated Ca channels (Dunlap, 1977), or addition of extracellular Ba both tended to linearize the steady state I-V relations.8. Injections of Cs and TEA tended to linearize the steady state I-V relations, but did not result in either a conversion to an all-or-none spike or a discontinuity in the depolarizing steady-state I-V relations.9. It is concluded that in Paramecium a Ca-activated K conductance short-circuits the inward current of the regenerative Ca response, preventing all-or-none behaviour. The occurrence of plateau spikes following EGTA injection indicates that the Ca conductance inactivates very slowly in face of a maintained depolarization. Such slow Ca-inactivation is consistent with the slow relaxation of Ca-dependent ciliary reversal that occurs during maintained depolarization.10. The possibility is discussed that injection of EGTA may also enhance the Ca conductance.

Action Potentials↗