Elimination of the germ nuclei of Paramecium tetraurelia by laser microbeam irradiation

The germ nuclei (micronuclei) of Paramecium tetraurelia can be eliminated successfully by irradiating the micronucleus with an argon-ion laser microbeam after sensitization with the dye acridine orange. No immediate cytological damage of the irradiated micronuclei is visible, but they are lost before they enter the next division. This method produces cell lines lacking micronuclei (i.e., amicronucleates). These amicronucleates provide favorable materials for the study of micronuclear functions as well as intra-and inter-specific nucleocytoplasmic interactions. Some preliminary observations show that the micronucleus is not required for macronuclear fragmentation and macronuclear regeneration during sexual reproduction, but suggest that the micronucleus might participate in some asexual cellular function in addition to their gametic role.     

Mating type transformation by transfer of macronuclear chromatin in Paramecium tetraurelia

Macronuclear chromatin from vegetative cells of one mating type (O, or E) in Paramecium tetraurelia was transferred by micropipetting into the macronucleus of vegetative cells of the opposite mating type (E, or O). A few percent (<5%) of the recipient cells gave rise to, by asexual propagation, progenies amongst which some were found to have transformed their mating type in accordance with the donor chromatin. This demonstrates the transformation of mating type during asexual propagation of the cells. In the case of E chromatin transfer to O recipients, many asexual progenies of the recipients transformed from O to E mating type nevertheless remained O after one sexual cycle. Such results indicate two distinctive macronuclear activities in mating type determination: one determining mating type of vegetative cells and the other influencing the differentiation of the developing post-zygotic macronucleus for mating type. The results are interpreted by the hypothesis that the quantity of E macronuclear chromatin required for differentiation of the developing post-zygotic macronucleus from mating type is larger than required for mating type determination in vegetative cells.     

Purine Composition of the Crystalline Cytoplasmic Inclusions of Paramecium tetraurelia

Crystalline cytoplasmic inclusions were isolated by differential centrifugation from mass cultures of Paramecium tetraurelia feeding on Klebsiella pneumonia. Physical and chemical measurements of intact and solubilized crystals determined that they consist primarily of guanine and hypoxanthine with traces of xanthine. Crystals from the mutant sombre consist primarily of xanthine, suggesting there is a disorder of purine metabolism in this mutant.     

The effective sites of some mutations affecting exocytosis in Paramecium tetraurelia

Summary Using a microinjection technique, the functional competence of the trichocysts or of the nontrichocyst cytoplasms of wild-type and mutant stocks of Paramecium tetraurelia was tested. The results indicate that the exocytic (trichocyst discharge) phenotype of P. tetraurelia depends upon the functional competence of the trichocysts themselves and also upon the function of apparently trichocyst-specific cytoplasmic components. Thus, the mutants tam8, ndA and ndB are shown to contain defective trichocysts, but have apparently functional cytoplasms which can properly utilize normal trichocysts if these are supplied. Conversely, the mutant nd9 contains apparently normal trichocysts but is deficient in some cytoplasmic component required for normal trichocyst discharge. Injections of genetically complementary cytoplasm apparently supply nd9 with the missing component and can thus repair the nd9 trichocyst exocytic phenotype.     

Induction of autogamy by transfer of macronuclear karyoplasm in Paramecium tetraurelia

Macronuclear karyoplasm was transplanted from pre-autogamous donor cells (clonal age, 22 fissions) into the macronucleus of young recipient cells (2 fissions after autogamy occurred) by means of microinjection. A reciprocal experiment was carried out by injecting karyoplasm from young clonal age donors into pre-autogamous recipients. In the case of karyoplasm transfer from pre-autogamous donors to young recipients, autogamy occurred early in 67% of injected cells, whereas reciprocal injections had no influence on the onset of autogamy, and all of the injected cells underwent autogamy. Such results indicate a distinct role of pre-autogamous cells of macronucleus in the induction of autogamy.     

Timing of oral morphogenesis and its relation to commitment to division in Paramecium tetraurelia

The interval between commitment to division and fission in synchronous cell samples is a constant fraction of the cell cycle (0.2) in cell cycles up to 6.5 h in duration. In longer cell cycles this interval has a fixed duration of about 80 min. The point of commitment to division is associated with the six-rowed anlage stage of oral primordium development (stage V). At this stage cells carrying the cc1 mutation are not blocked by transfer to restrictive conditions but rather proceed to division. Stage V is also the stabilization point for oral anlagen. When shifted to restrictive conditions prior to this stage, development is arrested and resorption of anlagen is initiated. The cc1 mutation also blocks contractile vacuole duplication and migration under restrictive conditions. The cc1 gene function is required continuously prior to the transition point. The timing of morphogenetic stages in asynchronous cells is roughly similar to that in synchronous cells. There are, however, significant differences in timing as estimated by the two experimental procedures.     

Mitochondrial chromatin in Paramecium aurelia

Summary Chromatin-like structures have been observed in material extracted from the mitochondria of Paramecium aurelia and evidence is presented which establishes that these structures do not originate from nuclear contamination of mitochondrial preparations but are exclusively of mitochondrial origin. Extraction of these chromatin-like structures with dilute acid followed by gel electrophoresis of the extract shows the presence of five major basic proteins which are of similar electrophoretic mobility to histones. Digestion of mitochondrial extracts with endogenous nuclease, followed by electrophoresis of the extracted DNA, demonstrated that the mitochondrial genome is protected from nuclease digestion by regular repeating units very similar to those observed in the nuclease digestion of chromatin.This research was supported by a postgraduate studentship from University of Edinburgh to E. Olszewska and by the Science Research Council.     

Transplantation of synkaryon in Paramecium caudatum. Analysis of its competence as germ nucleus

To establish whether the synkaryon of Paramecium can perform the function as a germ nucleus without a special process of differentiation into germ nucleus, synkaryons were transplanted into amicronucleate vegetative cells. The transplanted synkaryons underwent not only mitosis but also meiosis and the subsequent nuclear changes in the same way as the normal germ nucleus. The result suggests that no ‘determination’ of germ nucleus is necessary for the synkaryon to function as a germ nucleus. In Paramecium, the germ line is continuous through generations.     

Symbiotic Chlorella enhances the thermal tolerance in Paramecium bursaria

Symbiotic Chlorella enhanced the tolerance to high temperature in Paramecium bursaria. We found that 50% of Chlorella-free P. bursaria died within 85 s of exposure to 41°C in a standard saline solution, while the presence of Chlorella almost doubled the survival time of P. bursaria (160 s, P<0.001). The degree of tolerance in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor for photosynthesis) treated Chlorella-containing P. bursaria and Chlorella-containing organisms kept in the dark for 24 h was as low as in Chlorella-free organisms. The degree of tolerance to high temperature in Chlorella-free P. bursaria in solutions containing maltose, glucose, fructose or O₂, was as high as that of normal Chlorella-containing organisms. The degree of thermal tolerance in Chlorella-containing P. bursaria was not affected in the presence of these carbohydrates or oxygen.     

The timing of initiation of DNA synthesis in Paramecium tetraurelia is established during the preceding cell cycle as cells become committed to cell division

The timing of initiation of DNA synthesis (IDS) in Paramecium is established before cell division at a point located at about 0.75 in the preceding cell cycle. This point occurs about 90 min prior to fission and coincides with the point at which cells become committed to cell division. The location of the point at which the timing of IDS is set was deduced from a series of nutrient-shift experiments. Changes in nutrient level lead to changes in the duration of the subsequent G1 interval when they occur more than 90 min prior to fission. Perturbation of the cell cycle so that the timing of commitment to cell division is altered, results in a parallel shift in the point at which the timing of IDS is established.     

Transplantation of germ nucleus in Paramecium caudatum I : Nuclei in the pre-meiotic S phase can enter into mitotic cycle

Cells of Paramecium caudatum have two functionally different nuclei, somatic macronucleus and germinal micronucleus. We succeeded in transplanting the micronucleus of the pre-meiotic S phase into vegetative cells in the stationary phase by microinjection. Transplanted micronucleus in pre-meiotic S phase stopped proceeding into meiosis but entered the mitotic cycle when the recipient cells were grown. The result shows that commitment to meiosis in the micronucleus occurs some time after pre-meiotic DNA synthesis.     

A gene function required for cell cycle progression during the G1 portion of the cell cycle and for maintenance of macronuclear DNA synthesis in Paramecium tetraurelia

The ccl mutation in Paramecium tetraurelia reversibly and rapidly blocks cell cycle progression and DNA synthesis at the restrictive temperature. Progression through the cell cycle is blocked during both the G1 and S portions of the cell cycle, while at the restrictive temperature there is neither residual cell cycle progression nor induction of excess delay of subsequent cell cycle events. DNA synthesis activity is reduced to 50% of the normal level in about 5 min and is completely blocked at 30 min after a shift to restrictive temperature. On return to permissive conditions, DNA synthesis is reactivated with similar kinetics.     

Autogamy is induced in Paramecium bursaria by methyl cellulose

When isolated single cells of Paramecium bursaria were treated with 1.25% methyl cellulose, most of the single cells showed nuclear changes which were substantially similar to those seen in conjugation, suggesting that the cells undergo autogamy. Additional cytological observations indicated that the cells underwent normal nuclear processes during autogamy. The ability to induce autogamy will facilitate genetic studies in P. bursaria.     

Inhibition of lysosomal fusion with symbiont-containing vacuoles in Paramecium bursaria

Paramecium bursaria harbors several hundred intracellular Chlorella symbionts which remain undigested at the same time that the host cell phagocytizes and digests other organisms. Using electron microscopy and thorotrast labelling, we have shown that secondary lysosomes fuse with food vacuoles, but do not fuse with vacuoles containing symbiotic algae. From these and other data we suggest that the symbiotic algae alter the membrane of the vacuole which surrounds them, thus inhibiting fusion with secondary lysosomes.     

Analysis of germinal aging in Paramecium caudatum by micronuclear transplantation

The role of the micronucleus in the age-dependent increase in mortality after conjugation in Paramecium has been investigated using micronuclear transplantation. The clone of Paramecium caudatum used for this study had a lifespan of about 750 fissions. In this clone, the fission rate began to decrease about 450 fissions after conjugation. Mortality after selfing conjugation also began to appear at about 450 fissions and gradually increased with clonal age. Cells at about 650 fissions showed 10–70% survival after selfing conjugation but when their micronuclei were transplanted into amicronucleate cells of about 450 fissions, the progeny survival increased to 70–90%. When micronuclei from cells 700–750 fissions old were transplanted into amicronucleate cells of 100–150 fissions, however, increase in progeny survival was very rare. The results indicate that micronuclei in cells up to the age of 650 fissions can function normally if the cytoplasmic environment is young.     

Expression of A mating type genes of Coprinus cinereus in a heterologous basidiomycete host

The A mating factor of Coprinus cinereus determines compatibility in mating by regulating part of a developmental sequence that leads to dikaryon formation. The A genes that trigger development encode two different classes of homeodomain proteins, and for a successful mating, a protein of one class, HD 1, must interact with a protein of the other class, HD 2. In this report we show that C. cinereus A genes that encode HD 2 proteins, a2-1 and b2-1, can elicit A-regulated development in the heterologous host C. bilanatus. Transformation rates were very low, suggesting that the genes were poorly transcribed. The fact that the HD 2 genes are functionally expressed implies successful heteromultimeric association of putative DNA-binding proteins coded by the two Coprinus species. This interaction was sufficient to satisfy the need for different A factors in the formation of a fertile C. bilanatus dikaryon, but fertile dikaryons were more readily produced in matings with the a2-1 gene transformants. The C. cinereus A genes, b1-1 and d1-1, which encode HD1 proteins, were either not expressed or their proteins were non-functional in C. bilanatus. These experiments raise some interesting questions regarding HD1–HD2 protein interactions.