Effect of Acetate on Esterase C Activity During the Growth Cycle of Paramecium*

SYNOPSIS Enhanced esterase C activity could be demonstrated by starch gel electrophoresis in various stocks of Paramecium spp. (P. primaurelia stocks 90 and 540, P. biaurelia stock 93, P. tetraurelia stock 29. P. pentaurelia stock 87, P. octaurelia stocks 31 and 300, and P. multimicronucleatum species 3, stock 8 MO) grown in Adaptation Medium. This esterase, however, was barely detectable when they were cultivated in Axenic Medium. Addition of trypticase to Adaptation Medium resulted in reduction of esterase C in the ciliates. This effect is ascribable to Na acetate present in trypticase. Since esterase C increased with the decrease in acetate concentration (as estimated by gas-liquid chromatography) during growth of Paramecium, acetate appears to be utilized by the cells. Sensitivity of esterase C to acetate occurs in all 6 species of Paramecium examined. Different stocks within a species may have different levels of sensitivity; in one case this is genetically determined. The results emphasize the importance of controlling and manipulating growth conditions for the assessment of inter- and intraspecies variations in the isozymes of Paramecium.     

Serotypes and Immobilization Antigens in Paramecium caudatum*

SYNOPSIS. Some of the serotypes in Paramecium caudatum are described in this paper. Immobilization antigens of P. caudatum have been obtained by extracting paramecia in a dilute salt solution containing 15% alcohol. Immobilization antigen F from stock 162 has been isolated and has a sedimentation coefficient of 9.3 Svedbergs, diffusion coefficient of 2.3 × 10^-7 cm/sec, and molecular weight of approximately 340,000.     

The Glyceryl Ethers of Paramecium Phospholipids and Phosphonolipids1

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 Parammecium tetraurelia. The ether content of 2-aminoethyl phosphonoglycerolipid was 85–90 mole %. Concentrations of ethers were greatest in the ethanolamine phosphonolipids > phosphatidylcholines > phosphatidylserines > phosphatidylethanolamines > phosphatidylinositols. The glyceryl ether concentrations in total cellular phospholipids increased with culture age in P. tetraurelia and P. multimteronucleatum 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.     

Esterase Variants in Four Species of the Paramecium aurelia Complex1

One hundred eighty-eight stocks of Paramecium primaurelia, P. biaurelia, P. tetraurelia, and P. octaurelia were grown axenically and tested for five esterases, visualized by starch gel electrophoresis, in a search for variant stocks. The five esterases can be distinguished on the bases of their substrate specificity, sensitivity to an inhibitor, and response to different growth conditions. This paper addresses the nature of the electrophoretic change in mobility of the variant stocks in order that species relationships can be more accurately assessed. Crosses carried out in all four species show that single genes determine the differences in mobility between variant and common subtypes. Extracts of variant stocks that gave similar patterns were run against each other, tested for their sensitivity to the inhibitor, and the pattern was compared to that found in extracts of stocks with variant and common subtypes in other species. The majority of the variants in P. primaurelia, P. tetraurelia, and P. octaurelia show an electrophoretic mobility characteristic of a common subtype, or a variant, in another species. The same proportion of variant subtypes as common subtypes have mobilities similar to esterase subtypes found in other species. Of the four species examined in this paper, P. tetraurelia and P. octaurelia appear to be most closely related on the basis of shared esterase subtypes. In P. biaurelia the mobilities of most of the variants are unique, as are the common esterase subtypes in this species. P. biaurelia stands out as having the greatest number of esterase subtypes, with very few of them homologous to subtypes found in other species. This observation supports the idea of greater diversification of stocks within P. biaurelia than for the other three species.     

Malic dehydrogenase locus of Paramecium tetraurelia

A search was undertaken for naturally occurring genetic markers for use in clonal aging studies of Paramecium tetraurelia. Clonal age is defined as the number of cell divisions since the last sexual process. Autogamy (self-fertilization) is a sexual process which can occur in aging lines, resulting in homozygosity and initiation of the next generation. Such illicit autogamies must be detected and eliminated from the aged clone. With codominant alleles, heterozygous aging lines can be established which will express a phenotype distinguishable from that of either parental type and autogamy can then be monitored by the appearance of either segregant homozygous phenotype. However, very few codominant alleles are available in this species. Electrophoretic mobilities of malic dehydrogenase (MDH) were assayed in 11 stocks of Paramecium tetraurelia by polyacrylamide gel electrophoresis. Nine stocks showed a singlebanded stock 51 type, while stock 174 and stock 29 each exhibited unique mobility. Crosses between stock 51 and the deviant stocks revealed distinct three-banded patterns indicative of heterozygosity of the F₁ generation. In the autogamous F₂ generation, 1:1 segregation of the parental types were recovered. The pattern of inheritance is consistent with codominant alleles and Mendelian inheritance. These naturally occurring biochemical markers are stable with increasing clonal age and are therefore useful genetic markers for studies of cellular aging.This work was supported by NSF Grant PCM 7704315.     

Distribution of the Paramecium aurelia Species Complex (Protozoa,Ciliophora) in the Carpathian Chain of Poland*

Four species of the Paramecium aurelia complex were found in the Carpathian chain: P. primaurelia, P. biaurelia, P. tetraurelia and P. novaurelia. However, variation in the frequency of settlement of these species in the region was observed; P. biaurelia and P. novaurelia appeared with the greatest frequency, P. Primaurelia occurred less frequently. while P. tetraurelia was rather rare being found only in the Tatras.     

Localization of the chemoreceptive properties of the surface membrane ofParamecium tetraurelia

Summary The plasma membrane ofParamecium tetraurelia comprises two morphologically distinct components; a membrane that encloses the cell body and a ciliary membrane. In order to investigate the relative contributions of the two membranes to attractant-induced membrane potential changes, cells were deciliated with ethanol and their subsequent responses to attractants examined.Deciliation did not significantly affect the magnitude of the hyperpolarizations evoked by acetic or lactic acids, and had no effect on the concentration dependence of responses to folic acid. We conclude that the components necessary for detection and response to attractants are not exclusive to the ciliary membrane ofP. tetraurelia. Deciliation ofParamecium concomitantly permits localized chemical stimuli to be applied directly to the cell surface in the absence of strong fluid currents that are generated by the activity of the locomotory organelles. By systematically applying K₂ folate to a number of sites on the cell surface, it has been possible to demonstrate an anterior-posterior gradient of chemosensitivity on the cell body ofP. tetraurelia.     

Seasonal and spatial variability of species occurrence of the <Emphasis Type="Italic">Paramecium aurelia</Emphasis> complex in a single natural pond (Protista,Ciliophora)

The geographic distribution and temporal occurrence of ciliates are still little known. In the present article, the occurrence of the Paramecium aurelia species complex in a natural pond situated in Kraków (Opatkowice) was investigated in different seasons in two following years. A sequence of species occurrence of the P. aurelia complex was observed. Always, paramecia were found only in some sampling points among six points sampled each time and not necessarily in the same ones. Paramecia appearing in one habitat (water body) might occupy different niches characterized by various environmental features suitable for paramecia. The following species were found in the pond: P. biaurelia, P. triaurelia, P. tetraurelia, P. pentaurelia, and P. dodecaurelia. The occurrence of some rare species (P. tetraurelia, P. pentaurelia, and P. dodecaurelia) may be connected with migrating birds which can transport paramecia with drops of water from other water bodies. If a species was observed in successive seasons or years, the possible genetic variation was investigated by analysis of sequences of LSU rDNA and mitochondrial cytb gene fragments. Among the studied species (P. biaurelia, P. triaurelia, P. pentaurelia, and P. dodecaurelia) only P. dodecaurelia showed haplotype variation in different seasons and sampling points, probably caused by the colonization of the pond by different populations of paramecia.     

L-Glutamate-induced membrane hyperpolarization and behavioural responses inParamecium tetraurelia

Summary Paramecium tetraurelia is attracted to L-glutamic acid concentrations of 10^–9 M to 10^–4 M in a behavioural assay. Electrophysiological studies show thatP. tetraurelia responds to L-glutamate application with hyperpolarization. This response is transient, even in the continued presence of the stimulus. The concentration dependence of the membrane potential response is similar to that of the behavioural responses, although the threshold concentration of L-glutamate required for hyperpolarization is three orders of magnitude lower than for attraction. The membrane potential response to L-glutamate persists following artificial deciliation ofP. tetraurelia.While application of L-glutamate toP. tetraurelia invariably elicits a hyperpolarization, withdrawal of the stimulus frequently results in a second transient membrane response, in the form of either a hyperpolarization or a depolarization. It is suggested that these off-responses may have a significant role in maintaining a behavioural response to L-glutamate.Abbreviation I_che index of chemokinesis     

Host specificity of Salmonella weltevreden typing phages

The host range of the six S. weltevreden typing phages was studied on 1469 strains belonging to 37 different Salmonella serotypes. In addition to S. weltevreden, only S. nchanga, S. give, S. lexington and S. anatum, all belonging to O group E₁, showed varying degrees of susceptibility to the action of some of the typing phages.Typing phage VI lysed only one strain other than S. weltevreden. All serotypes tested other than S. weltevreden were resistant to phages III and IV even at 1000 times the routine test dilution. Thus, typing phages III and IV were specific for S. weltevreden. The sensitivity patterns of S. weltevreden typing phages were not found to bear much correlation with either somatic of flagellar antigens of Salmonellae.     

Alternative Phenotypic States in Genomically Identical Cells: Interstock Genetics of a Trichocyst Phenotype in PARAMECIUM TETRAURELIA

The trichocysts of most wild stocks of Paramecium tetraurelia discharge en masse in response to picric acid. In most nonresponding wild stocks, the defective phenotype is simply determined by a single recessive gene difference from the standard wild type, stock 51. However, two wild stocks, 146 and 148, which are completely homozygous at all loci, express either a nondischarge, ND, or discharge, DI, phenotype. In stock 146, both ND and DI sublines generally reproduce true to type, but observed changes are highly biased. Changes from ND to DI occur more than ten times as often as changes from DI to ND. After conjugation between ND and DI cells, genomically identical exconjugant lines from the ND parent may be either ND or DI, while those from the DI parent invariably remain DI.—Interstock crosses between stocks 146 and 51 indicate that stock 146 possesses a recessive gene, nd146, which, when homozygous in stock 51 background, produces a distinct nondischarge phenotype, KO. Crosses between stock 146 and KO phenotype nd146 homozygotes in stock 51 background demonstrate that stock 146 possesses a dominant gene, M-nd146, which modifies the defect of nd146 homozygotes, resulting in either the ND or DI phenotype. The two loci, M-nd146 and nd146, are linked and estimated to be 5.3 centiMorgans apart. Stock 148 has the same alleles as stock 146 at these loci.—Presumably M-nd146 is involved in the dual phenotypic states in stock 146, but M-nd146 nd146 homozygotes backcrossed into stock 51 are invariably discharging. The possibility that the original ND state is independent of these genes is discussed and is regarded as unlikely. The phenotypic and genetic relationship discovered in these stocks should remind population biologists that phenotypic and genotypic variability do not always have a simple relationship. The nature and frequency of epistasis in the highly inbreeding P. tetraurelia are reviewed.     

Developmentally Controlled Rearrangement of Surface Protein Genes in Paramecium tetraurelia1

ABSTRACT Early research on Paramecium genetics highlighted the role of the cytoplasm on inheritance. Today this tradition continues as recent investigations of macronuclear development in Paramecium have revealed unusual cytoplasmic effects that are not easily explained within current paradigms. It is generally assumed that most programmed DNA rearrangements in ciliates are regulated by cis acting signals encoded within the germline (micronuclear) DNA, but there are increasing examples in which the old macronucleus acts through the cytoplasm (in trans) to affect the loss and rearrangement of DNA in the developing macronucleus. The remarkable specificity of this effect has forced a reevaluation of the standard view of macronuclear determination in Paramecium. This review summarizes our knowledge of the effect of the old macronucleus on the developmentally controlled rearrangements of the P. tetraurelia, stock 51A and B variable surface protein genes.     

Structure of Mitochondrial Dna From Paramecium Tetraurelia*

Mitochondrial DNA (mtDNA) from endosymbiote-free stocks of Paramecium tetraurelia was isolated by 2 procedures. the buoyant density of the mtDNA in neutral CsCI was 1.702 gm/cm³. a value consistent with the melting temperature of the mtDNA. Only linear molecules were observed by electron microscopy. These molecules were homogeneous in size with a monomer molecular weight of 25.6 × 10⁶ daltons. the size of the mtDNA determined after digestion with the restriction endonucleases EcoRI or Hind III agreed with the value obtained by electron microscopy. These studies also revealed that the digestion pattern of mtDNA from stock 172 differed from that of the other 3 stocks (51, 127. 203) examined. Some mtDNA molecules exhibited snapback reassociation following denaturation.     

Comparison of one-dimensional IEF patterns for serologically detectable HLA-A and B allotypes

A new mouse monoclonal antibody (MoAb) 4E, which detects an epitope shared by HLA-B locus antigens, together with the MoAb W6/32, detecting a common HLA, B, C, determinant, and the MoAb 4B, detecting HLA-A2 and A28, were used to isolate HLA-A and -B antigens in sequential immunoprecipitation. The HLA antigens obtained from metabolically labeled cell extracts of B-lymphoblastoid cell lines or from phytohemagglutinin (PHA) activated peripheral blood lymphocytes were compared by one-dimensional isoelectric focusing (1D-IEF). The IEF banding patterns obtained with native HLA antigens segregated in a family with HLA. Neuraminidase treatment of isolated antigens reduced the number of bands to one or two, simplifying the analysis of characteristic patterns. Thus, we have cataloged IEF banding patterns for the majority of the serologically recognized HLA-A and -B allotypes obtained from 57 unrelated American Caucasians. While no HLA-A locus or HLA-B locus specific banding patterns were observed, the HLA-A antigens had, in general, slightly higher pl values than the HLA-B antigens. HLA-C antigens could not be detected in this assay system. The polymorphism detected by IEF banding patterns was as extensive as the serologically detected polymorphism identified by classical HLA serology. Moreover, variants for some HLA allotypes could be detected by this method. In addition to previously recognized A2 variants, new variants were identified for HLA-A1, A26, and Bw44. Each A1 and Bw44 variant was associated with particular haplotypes. The HLA-A2 antigens occurred on 43 HLA haplotypes in the unrelated Caucasian population. Only one of each A2 variants was identified in this population.     

Stable and unstable transformation by microinjection of macronucleoplasm in Paramecium

Transformation by microinjection of macronucleoplasm in Paramecium caudatum was investigated. Macronucleoplasm with three genetic markers (behavior, trichocyst, and mating type) was injected into the macronucleus. To facilitate microinjection, in most cases, paramecia were immobilized in a gelatin (7.5%) solution. The injected cells began to express a dominant gene (cnrA⁺ or cnrB⁺) of the donor 9–24 hr after injection. Expression did not require cell division suggesting injected macronucleoplasm was capable of expressing a phenotype. The amount of injected macronucleoplasm appears to correlate with the frequency of successful expression but not to correlate with the time required for expression. After a number of fissions, the injected cells produced clones which had cells expressing the phenotype of the donor. This suggests that injected macronucleoplasm was replicated and expressed in the recipient cell lines. The transformed clones were classified into two groups. In one group, transformation was stable. All cell lines derived from the injected cells expressed a phenotype similar to the heterozygote of donor and recipient cells. In the other group, transformation was unstable. During the first five to seven fissions after injection, at each division, cells produced one daughter cell which later reverted to the recipient phenotype. After this unstable period, cells no longer produced the recipient phenotype but produced the donor phenotype exclusively. Donor and recipient phenotypes were, thus, segregated in different cell lines. Observation of genetic markers and analysis by computer simulation shed light on the mode of transmission of injected macronucleoplasm. In stable transformation, injected macronucleoplasm appears to be distributed equally to daughter cells. In unstable transformation, injected macronucleoplasm is distributed only to one of the daughter cells at every division until about the fifth to seventh fission after injection and then begins to assort equally to daughter cells. The cell cycle stage at injection may influence the mode of transformation. Interspecific microinjection of macronucleoplasm from P. multimicronucleatum and P. tetraurelia to P. caudatum. resulted in the expression of foreign genes in P. caudatum. In one case, injection of macronucleoplasm of P. tetraurelia produced a stable transformant indicating replication of foreign macronucleoplasm in P. caudatum. This work reveals the mode of transformation by injected macronucleoplasm and shows the possibility of transformation among Paramecium species, which is significant in the study of the conservation of gene products and the mechanism of gene expression in different species. © 1992 Wiley-Liss, Inc.     

Biotin chemoresponse in Paramecium

Paramecium tetraurelia locate their␣foodsource by detecting bacterial metabolites and altering swimming behavior to congregate near bacterial populations on which they feed. Several attractants, such as folate, glutamate, cAMP and acetate have been identified and various aspects of chemoreception, signal transduction and effector mechanisms have been described. Here we characterize the Paramecium chemoresponse to biotin. An essential enzymatic cofactor in all cells, biotin is secreted by a large number of bacterial species during growth phase. P. tetraurelia are strongly attracted to biotin with a half-maximal behavioral response at 0.3 mmol · 1⁻¹ in T-maze assays. Physiological recordings from whole cells show that cells hyperpolarize in a concentration-dependent manner in biotin. Whole-cell binding assays utilizing ³H-biotin identify a saturable and specific binding site with an apparent dissociation constant of 0.4 mmol · l⁻¹. The biotin analogs desthiobiotin and biotin methyl ester are also strong attractants. Diaminobiotin fails to attract P. tetraurelia at 1 mmol · l⁻¹, but does interfere with the biotin chemoresponse and displaces ³H-biotin from whole cells. We hypothesize that the keto group and/or fidelity of the ureido ring of biotin are necessary for biotin chemoresponse. Accepted: 23 April 1998     

Role of arginine kinase in Paramecium tetraurelia (Ciliophora,Peniculida): Subcellular localization of AK3 and phosphoarginine shuttle system in cilia

The ciliate Paramecium tetraurelia has four arginine kinase genes (AK1, AK2, AK3, and AK4). Of these genes, only AK3 has a signal sequence for farnesylation, a post-translational modification that enables anchoring of the modified enzyme to the ciliary membrane. To confirm this modification, AK3 was synthesized using a cell-free protein synthesis system and the peptide masses were analyzed using peptide mass fingerprinting (PMF). The PMF analysis indicated that the C-terminal peptide of AK3 is farnesylated. Thus, AK3 can be farnesylated under physiologically appropriate conditions. To determine the subcellular localization of P. tetraurelia AK3, Western blot analysis was performed using an AK3 polyclonal antibody for the proteins extracted from intact cells and ciliary fractions. When extraction was performed using Triton X-100, AK3 was detected the ciliary fraction. This result suggested that the ciliary fraction contains AK3. In addition, we investigated the role of P. tetraurelia AKs in ciliary movement using the feeding RNA interference method. The swimming velocity of AK1- and AK3-silenced cells was significantly reduced to half the value of that control cells. In summary, P. tetraurelia AK3 is likely to be located in the ciliary membrane and influences swimming velocity, presumably through the phosphoarginine shuttle system present in cilia.     

Hydrophobic and Hydrophilic Radio-Iodination, Crosslinking, and Differential Extraction of Cell Surface Proteins in Paramecium tetraurelia Cells

We combined widely different biochemical methods to analyze proteins of the cell surface of P. tetraurelia since so far one can isolate only a subfraction of cell membrane vesicles enriched in the GPI-anchored surface antigens (``immoblization' or ``i-AGs'). We also found that i-AGs may undergo partial degradation by endogenous proteases. Genuine intrinsic membrane proteins were recognized particularly with lipophilic 5-[¹²⁵I]-iodonaphthalene-1-azide (INA) labeling which reportedly ``sees' integral proteins and cytoplasmic cell membrane-associated proteins. With INA (+DTT), bands of ≤55 kDa were similar as with hydrophilic iodogen (+DTT), but instead of large size bands including i-AGs, a group of 122, 104 and 94 kDa appeared. Several bands of the non i-AG type are compatible with integral (possibly oligomeric) or associated proteins of the cell membrane of established molecular identity, as we discuss. In summary, we can discriminate between i-AGs and some functionally important minor cell membrane components. Our methodical approach might be relevant also for an analysis of some related protozoan parasites. Received: 5 April 1999/Revised: 19 July 1999