Axenic Paramecium caudatum. I. Mass Culture and Structure*

SYNOPSIS. To establish and grow Paramecium caudatum in mass axenic culture the culture medium of Soldo, Godoy & van Wagtendonk was modified by substituting phosphatidylethanolamine (PE) for TEM-4T and by a 10-fold increase in folic acid. Population densities of 4000 to 6000 cells/ml and a generation time of 20–26 h are regularly obtained. Optimal growth is obtained with PE-stigmasterol ratios between 40:1 to 400:1. Cells from 1-day-old axenic cultures have many lipid bodies aggregated in clumps (which disappear in 2 to 3 days) as well as foci of rough endoplasmic reticulum bordered by dictyosomes. The latter suggests a very active metabolism. Crystalline sheets found in both food vacuoles and lysosomes presumably play a role in digestion. Axenically grown cells also have abundant Golgi bodies (dictyosomes) and by late log phase become filled with lysosomes.     

Molecular Biology of the Genes for Immobilization Antigens in Paramecium1

Several genes for surface antigens of the Paramecium aurelia complex of species have been isolated. In addition lo known deletions of the 51A gene, we have obtained deletions involving the 51B gene and have developed a procedure for obtaining deletions of additional genes. Both Mendelian and non-Mendelian deletions of both the A and B genes have been found. In the non-Mendelian deletions the genes are present in the micronuclei and absent in the macronuclei. Processing of micronuclear DNA into new macronuclear DNA at conjugation and autogamy is under the control of the old macronucleus, and newly forming macronuclei become exactly like the old. Thus in the non-Mendelian mutants, macronuclei have a specific antigen gene deleted and also are impaired in their ability to direct normal DNA processing at the next conjugation or autogamy. These cases, along with others, show that this system of macronuclear control is a fundamental feature of ciliate genetics. The sequence of the 51A and 51C genes is described and compared with the 156G and 51H genes obtained by others. The 51A and 156G genes are remarkably similar while 51Cand 51H are rather different. No introns or pseudogenes have been observed. Some, possibly all, of the genes are on the ends of chromosomes. Characteristic upstream and downstream sequences adjacent to the coding portions of the genes are given. The sequences UAA and UAG are preferred over CAA and CAG for glutamine while UGA is the true stop codon.     

Enzyme Variation in Paramecium caudatum

Stocks of four “syngens” (syngens 1, 3, 12, 13) of Paramecium caudatum could not be distinguished on the basis of isozymic variations of six enzymes. Using the most common enzyme form as the standard for the syngen, we found a higher coefficient of identity between syngens (>90%) than within syngens (73%). These observations, combined with evidence for fertile matings among the groups, suggest that the groups do not warrant the status of syngens. True syngenic variation in P. caudatum is, however, clearly established on the basis of isozymic and breeding studies of wild stocks collected from various places. Some stocks from England have a close affinity with those from Japan, but stocks from Scotland and California must be placed in separate syngenic sets. Thus, P. caudatum is indeed a species complex, within which evolutionarily distinct groups (species = syngens) are identifiable. The definition of syngens on the basis of initial agglutination response is, however, unjustified.     

Temperature-Sensitive Behavior of Paramecium caudatum

SYNOPSIS. The effect of temperature on the behavior of swimming cells of Paramecium caudatum has been investigated by photographic analyses of their tracks in uniform temperature, in temperature gradient, or in temperature changing with time. When the cells were placed in the temperature gradient, the frequency of discontinuous directional changes of cells swimming toward the optimal temperature, the temperature of the culture, was much lower than that of the cells swimming in the opposite direction. This difference in the frequency of directional changes explained the observed accumulation of the cells at - the optimal temperature. When the temperature was suddenly changed toward the optimum, a transient decrease of the frequency of directional changes was observed and when the temperature was changed in the reverse direction, a transient increase of the frequency was noted. This transient response to the temperature change was the origin of the dependence of the frequency of directional changes on the swimming direction in the temperature gradient. Finally, the relation between the magnitude of the transient response and the rate of the temperature change was derived.     

Identification of Specific Antibodies Prepared Against Purified Immobilization Antigens of Paramecium aurelia

SYNOPSIS The identity of antibodies prepared against preparations of isolated immobilization antigen protein of Paramecium aurelia has been studied. Antisera were fractionated by ammonium sulfate precipitation, sucrose gradient centrifugation, and ion exchange chromatography on DEAE cellulose. The different fractions were assayed for the presence of specific antibodies by the immobilization test, and the globulins were identified by immuno-electrophoresis. It is concluded that in the sera studied in this investigation, specific antibodies are excusively of the class Ig G.     

Role of Non-Surface Antigens in Controlling Paramecium Surface Antigen Synthesis*

SYNOPSIS. Paramecium aurelia exposed to antisera prepared against cells of a different surface antigenic type are often induced to transform to a new serotype. One possible explanation is that paramecia that are so affected have antigens related to the ciliary antigens, but not accessible to immobilizing antibodies. It is these secondary antigens that are bound by the antibodies, thereby forcing the cells to alter their pattern of antigen synthesis. Examination of affected paramecia has disclosed that secondary antigens are often present but the specificity of these antigens cannot account for the activity of the antisera. It is therefore proposed that antibodies directed against substances other than the immobilization antigens may induce transformation. Two kinds of antiserum, neither of which contains immobilizing antibodies of any sort, are able to markedly alter the expression of the serotypes. One was obtained by immunizing rabbits with culture fluid in which paramecia had been growing. The 2nd was made by injecting rabbits with normal sera from other rabbits.     

Macronuclear Regeneration and Cell Division in Paramecium caudatum

SYNOPSIS. In Paramecium caudatum , occurrence of macronuclear regeneration is closely related to the time of feeding after conjugation. Macronuclear regeneration is induced with a high frequency when conjugating pairs are transferred into fresh culture medium. Feeding immediately after conjugation induces early cell division and 3 or more fissions occur without macronuclear division because of the inability of the macronuclear anlagen to divide. In the cells lacking normal macronuclear anlagen, old macronuclear fragments undergo regeneration and form vegetative macronuclei.     

Ciliary Membranes and Mating Substances in Paramecium caudatum

SYNOPSIS Cilia detached from mating reactive cells of Paramecium caudatum were fractionated for the purpose of identifying the structural component bearing mating substances. Purified axoneme fractions had no mating reactivity. The membrane fraction obtained by dialyzing against a solution of Tris-EDTA (0.1 m m EDTA, 1 m m Tris-HCI, pH 7.6) and 0.6 m KCI, and then by centrifuging over 40% (w/v) sucrose was strongly reactive. No mating reactivity was detected in the soluble fractions containing axonemal and matrix proteins. The results indicate that the mating substances in active from are localized only on the ciliary membranes.     

Disparity of native oxyhemoglobin components isolated from Paramecium caudatum and Paramecium primaurelia

• 1.1. Native oxyhemoglobin components were isolated chromatographically from Paramecium caudatum and Paramecium primaurelia, and some properties of the isolated components were investigated.
• 2.2. P. caudatum was endowed with one homogeneous hemoglobin component, while the hemoglobin in P. primaurelia was resolved into three heterogeneous components being two main and one minor.
• 3.3. Spectral properties of the isolated hemoglobin components were quite similar to each other. The isolated components, however, were distinctly different in electrophoretic mobilities.
• 4.4. Molecular weight of the isolated hemoglobin components was estimated to be about 11,000.

     

Identification of the Immobilization Antigens of Paramecium by Their Cyanogen Bromide Cleavage Patterns1

Immobilization antigens from 12 serotypes of three stocks of Paramecium tetraurelia and from one serotype of one stock of P. primaurelia were isolated and purified. Purified proteins were cleaved with cyanogen bromide, and the patterns of the fragment peptides were determined by electrophoresis on SDS-polyacrylamide gels. It was shown that each of the serotypes of stock 51 of P. tetraurelia has an antigen that produces a characteristic and unique pattern. Consequently, the antigens can be identified by their patterns. Antigens from the allelic serotypes tested had identical patterns. The method is sensitive enough for the investigation of small sample volumes, and useful as a simple biochemical technique for the identification of serotypes.     

Binding of Ca ions by Paramecium caudatum

Binding of ⁴⁵Ca by live Paramecium caudatum was determined under various external ionic conditions. It was found that calcium uptake was separable into at least two components, a rapid and a slow one. The rapid component was influenced by the presence of certain other ions in a manner which agrees with the law of mass action. It appears that an ion exchange system may be involved in a binding equilibrium established between Paramecium, Ca⁺⁺, and certain other ions. K⁺, Rb⁺, and Ba⁺⁺ in the equilibrium medium are among those ions which inhibit calcium uptake. It is proposed that liberation of Ca⁺⁺ from binding sites on Paramecium by an exchange reaction with competing ions is the first step in the mechanism of ciliary reversal in the response to external application of these ions.     

Aligning Paramecium caudatum with static magnetic fields

As they negotiate their environs, unicellular organisms adjust their swimming in response to various physical fields such as temperature, chemical gradients, and electric fields. Because of the weak magnetic properties of most biological materials, however, they do not respond to the earth's magnetic field (5 x 10(-5) Tesla) except in rare cases. Here, we show that the trajectories of Paramecium caudatum align with intense static magnetic fields >3 Tesla. Otherwise straight trajectories curve in magnetic fields and eventually orient parallel or antiparallel to the applied field direction. Neutrally buoyant immobilized paramecia also align with their long axis in the direction of the field. We model this magneto-orientation as a strictly passive, nonphysiological response to a magnetic torque exerted on the diamagnetically anisotropic components of the paramecia. We have determined the average net anisotropy of the diamagnetic susceptibility, Deltachi(p), of a whole Paramecium: Deltachi(p) = (6.7+/- 0.7) x 10(-23) m(3). We show how the measured Deltachi(p) compares to the anisotropy of the diamagnetic susceptibilities of the components in the cell. We suggest that magnetic fields can be exploited as a novel, noninvasive, quantitative means to manipulate swimming populations of unicellular organisms.