STUDIES ON THE ORIGIN OF RIBOSOMES IN AMOEBA PROTEUS

The origin of cytoplasmic RNA and ribosomes was studied in Amoeba proteus by transplantation of a radioactive nucleus into an unlabeled cell followed by examination of the cytoplasm of the recipient for the presence of label. When a RNA-labeled nucleus was used, label appeared in the ribosomes, ribosomal RNA, and soluble RNA. Since the kinetics of appearance of labeled RNA indicates that the nucleus was not injured during the transfer, and since the transferred nuclear pool of labeled acid-soluble RNA precursors is inadequate to account for the amount of cytoplasmic RNA label, it is concluded that cytoplasmic ribosomal RNA is derived from acid-insoluble nuclear RNA and is probably transported as an intact molecule. Likewise, cytoplasmic soluble RNA probably originated in the nucleus, although labeling by terminal exchange in the cytoplasm is also possible. The results were completely different when a protein-labeled nucleus was grafted into an unlabeled host. In this case, label was found only in soluble proteins in the host cell cytoplasm, and there were no (or very few) radioactive ribosomes. This suggests that the nuclear pool of ribosomal protein and ribosomal protein precursors is relatively small and perhaps nonexistent (and, furthermore, shows that there was no cytoplasmic ribosomal contamination of the transferred nucleus).     

INFECTIVE ORGANISMS IN THE CYTOPLASM OF AMOEBA PROTEUS

Evidence from electron and phase microscopy is given which shows that infective organisms are present in the cytoplasm of Amoeba proteus. Vesicles containing living organisms have been observed after repeated washing and starvation of the amebae for a period of 2 weeks. Exposure to γ-radiation in conjunction with starvation, repeated washing, isolation of single amebae, refeeding with contaminant-free Tetrahymena, and clone selection has produced clones with reduced cytoplasmic infection. These findings are discussed in regard to the autoradiographic studies of other investigators on Amoeba proteus. The controversies over whether DNA and RNA are synthesized in the cytoplasm may be resolved by the finding of cytoplasmic infection.     

ELECTRON MICROSCOPY OF THE NUCLEAR MEMBRANE OF AMOEBA PROTEUS

An electron microscope study of the nuclear membrane of Amoeba proteus by thin sectioning techniques has revealed an ultrastructure in the outer layer of the membrane that is homologous to the pores and annuli observed in the nuclear membranes of many other cell types studied by these techniques. An inner honeycombed layer apparently unique to Amoeba proteus is also described.     

CYTOPLASMIC FILAMENTS OF AMOEBA PROTEUS : I. The Role of Filaments in Consistency Changes and Movement

The role of filaments in consistency changes and movement in a motile cytoplasmic extract of Amoeba proteus was investigated by correlating light and electron microscopic observations with viscosity measurements. The extract is prepared by the method of Thompson and Wolpert (1963). At 0°C, this extract is nonmotile and similar in structure to ameba cytoplasm, consisting of groundplasm, vesicles, mitochondria, and a few 160 A filaments. The extract undergoes striking ATP-stimulated streaming when warmed to 22°C. Two phases of movement are distinguished. During the first phase, the apparent viscosity usually increases and numerous 50–70 A filaments appear in samples of the extract prepared for electron microscopy, suggesting that the increase in viscosity in caused, at least in part, by the formation of these thin filaments. During this initial phase of ATP-stimulated movement, these thin filaments are not detectable by phase-contrast or polarization microscopy, but later, in the second phase of movement, 70 A filaments aggregate to form birefringent microscopic fibrils. A preparation of pure groundplasm with no 160 A filaments or membranous organelles exhibits little or no ATP-stimulated movement, but 50–70 A filaments form and aggregate into birefringent fibrils. This observation and the structural relationship of the 70 A and the 160 A filaments in the motile extract suggest that both types of filaments may be required for movement. These two types of filaments, 50–70 A and 160 A, are also present in the cytoplasm of intact amebas. Fixed cells could not be used to study the distribution of these filaments during natural ameboid movement because of difficulties in preserving the normal structure of the ameba during preparation for electron microscopy.     

CYTOPLASMIC EFFECTS ON THE DIFFERENTIATION OF ANTHERS AND OVULES OF COTTON

Reciprocal crosses of Upland cotton with a hybrid derived from (Gossypium anomalum × G. thurberi) × G. hirsutum produced progenies differing significantly in anther development and in the production of supernumerary ovules outside the ovary. Plants with G. anomalum cytoplasm produced fewer anthers than the reciprocal hybrids with G. hirsutum cytoplasm, had a higher percentage of sterile anthers, and were far more likely to form external ovules on an abnormally thickened tip of the staminal tube. The number of locules and ovules within the ovary was not significantly affected by cytoplasm.     

CYTOPLASMIC DNA SYNTHESIS IN AMOEBA PROTEUS : III. Further Studies on the Nature of the DNA-Containing Elements

The application of electron microscope autoradiography to Amoeba proteus cells labeled with tritiated thymidine has permitted the identification of morphologically distinct particles in the cytoplasm as the sites of incorporated DNA precursor. The particles correspond to those previously described from light microscope studies, with respect to both H³Tdr incorporation and distribution in centrifugally stratified amoebae. Ingested bacteria differ from the particles, in morphology as well as in the absence of associated label. Attempts to introduce a normal particle labeling pattern by incubating amoebae with labeled sediment derived from used amoeba medium failed. The resultant conclusion, that the particles are maintained in the amoeba by self-duplication, is supported by the presence of particles in configurations suggestive of division.     

CYTOPLASMIC DNA SYNTHESIS IN AMOEBA PROTEUS : I. On the Particulate Nature of the DNA-Containing Elements

The incorporation of tritiated thymidine in Amoeba proteus was reinvestigated in order to see if it could be associated with microscopically detectable structures. Staining experiments with basic dyes, including the fluorochrome acridine orange, revealed the presence of large numbers of 0.3 to 0.5 µ particles in the cytoplasm of all cells studied. The effect of nuclease digestion on the dye affinity of the particles suggests that they contain DNA as well as RNA. Centrifugation of living cells at 10,000 g leads to the sedimentation of the particles in the centrifugal third of the ameba near the nucleus. Analysis of centrifuged cells which had been incubated with H₃-thymidine showed a very high degree of correlation between the location of the nucleic acid-containing granules and that of acid-insoluble, deoxyribonuclease-sensitive labeled molecules and leads to the conclusion that cytoplasmic DNA synthesis in Amoeba proteus occurs in association with these particles.     

THE EFFECTS OF KIN-STRUCTURED COLONIZATION ON NUCLEAR AND CYTOPLASMIC GENETIC DIVERSITY

We investigate kin-structured migration and its effects on patterns of genetic variation in cytoplasmic and nuclear genomes. We show that colonization events involving close relatives, such as those that might characterize range expansion and the invasion of new habitats, can change the patterns of nuclear and cytoplasmic genetic variation expected at equilibrium. The difference in the patterns of variation between the nuclear and mitochondrial genomes suggests that it may be possible to estimate the time of the effective kin-structured colonization event. Observations in several taxa are discussed in light of these findings and in relation to their known geological history.     

CYTOPLASMIC DNA SYNTHESIS IN AMOEBA PROTEUS : II. On the Behavior and Possible Nature of the DNA-Containing Elements

Nucleic acid-containing particles in the cytoplasm of Amoeba proteus (cf. reference 1) were counted after acridine orange staining. The number of particles per ameba was found to be correlated with cell age and size. Fresh daughters had a mean particle number of 5400, whereas predivision amebae contained around 11,000 particles. Amebae from two other strains contained similar particles. The particles were found to be clustered in fasted cells and redispersed after feeding. A marked increase in the particle population was noted in anucleate fragments. These results, together with those previously presented, suggest that the particles multiply intracellularly. Their nature and their relationship to previous work on nucleic acid labeling in Amoeba are discussed.     

THE EFFECT OF ENUCLEATION ON THE DPN LEVEL OF AMEBA

1. Amebae contain DPN at levels of from 1 to 4 x 10^–13 moles per cell. 2. Following enucleation, nucleate and enucleate halves continue to have equal DPN contents over the six day period studied. Similarly, starving whole amebae maintain their DPN level over this period. 3. No reduced DPN could be detected in these aerobic animals. This remained true for whole amebae and for nucleate and enucleate halves over 5 days of starvation. 4. A method is described for the preparation and rapid separation of nucleate and enucleate ameba halves, based on a response of amebae to light.     

RELATIONSHIP BETWEEN PINOCYTOSIS AND ADHESION IN AMOEBA PROTEUS

Induction of pinocytosis in Amoeba proteus is independent of adhesion. It is manifested by non-adhering floating specimens, as well as by amoebae moderately adhering and locomoting on the glass, or tightly attached to the polylysine-coated substratum. The formation of pinocytotic rosettes results in de-adhesion, at the beginning of pinocytosis on the glass, but at its end on the polylysine. It suggests an opposition between adhesion and cell shape transformation. Pinocytosis and adhesion are both inhibited, by an unknown mechanism, in the presence of gelatin either in the substratum or in solution.