The Karyotype of <Emphasis Type="Italic">Amoeba borokensis</Emphasis> from a “<Emphasis Type="Italic">proteus</Emphasis>-like” Amoeba Group (Amoebozoa: Euamoebida)

The karyotype of Amoeba borokensis was studied for the first time. At the metaphase of mitosis, this species has a haploid set of chromosomes (n = 27). This is exactly half of the diploid karyotype in Amoeba proteus (strain B). At first glance, it confirms the idea that has recently appeared that one species arises from another via multiple changes in chromosome number. However, comparative cytogenetic analysis revealed that four orthologous chromosomes from haploid sets of these two species were not distinguished by the pattern of DAPI-stained bands. It shows that the genetic distance between A. proteus and A. borokensis is higher than was believed earlier and these two species have diverged from each other. Analysis of data on the life cycles of A. proteus and A. borokensis shows that a kind of so-called “cyclic polyploidy” takes place in “proteus-like” amoebae. “Cyclic polyploidy” has recently been considered as an alternative to the sexual process for genetic recombination in agamic protists from different macrotaxons.     

Karyotyping of <Emphasis Type="Italic">Amoeba proteus</Emphasis>

In this paper, we have developed a method to prepare spreads of mitotic chromosomes of Amoeba proteus and described the process of Amoeba proteus karyotyping. This protocol allows obtaining spread chromosomes with a characteristic pattern of chromomeres on individual chromosomes. It is shown that, in the metaphase of mitosis, amoebas of strain B (one of the type strains of A. proteus in the Amoebae Cultures Collection of the Institute of Cytology, Russian Academy of Sciences) contain 27 pairs of chromosomes. It is established that the pattern of chromomeres is a chromosome-specific feature. A typical karyogram and image bank of DAPI- and YoYo1-banded individual chromosomes of A. proteus, strain B composed of five different spreads of mitotic cells are presented.     

THE CONTENT AND RELATIVE BASE RATIOS OF RIBONUCLEIC ACID IN AMOEBA

The amount and relative base ratios of ribonucleic acid (RNA) in the nucleus and cytoplasm of Amoeba proteus and A. dubia, and of homospecies cells obtained by nuclear transfer with A. proteus, have been determined by microelectrophoresis. In A. proteus the average amounts of RNA in the nucleus and the cytoplasm were 134. micromicrograms and 2520. micromicrograms; in A. dubia the averages for the nucleus and cytoplasm were 67. micromicrograms and 1427. micromicrograms. The relative base ratio of RNA of the nucleus is similar to that of the RNA of the cytoplasm within a species, but the two species differed in this respect. Homospecies nuclear transfer did not affect the relative base ratio or amount of RNA.     

Karyotypic instability of endoprophase and mitotic cells of Amoeba sp. strain Cont from the “proteus-type” group (Amoebozoa,Euamoebida, Amoebidae)

We performed karyotyping of Amoeba sp. strain Cont. Based on the results of a cytological analysis, we concluded that the chromosome number of Amoeba sp. strain Cont in mitosis was unstable. In all cases they appeared to be hypergaploid (the basic chromosome number is 30), with monosomy of all chromosomes except four shortest ones. The presence of “extrachromosomes” in the nucleus could prolong until the beginning of the anaphase. It was only then that they were ejected from the nucleus and the euploidy (haploidy) was restored. The stage of endoprophase nucleus was revealed in the cell cycle of Amoeba sp. strain Cont. This stage has not yet been found in other amoebae from the “proteus-type” group that had been previously studied (A. proteus strain B and A. borokensis). The maximum number of endoreplication rounds in the strain Cont amoebae nuclear cycle was 4 or 5. The regular extrusion of chromosomes from the nucleus into the cytoplasm occurred in each of the endoreplication rounds. Comparative cytological analysis of A. proteus strain B, A. borokensis and Amoeba sp. strain Cont karyotypes indicated that strain Cont, though rather close to the former two amoebae, is actually a distinct species.     

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 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.     

I. THE RELATION BETWEEN ATTACHMENT TO THE SUBSTRATUM AND INGESTION BY AMEBA IN STRYCHNINE SULFATE SOLUTION AND CONDITIONED MEDIA : II. BIOLOGICAL ASSAY OF CONDITIONED MEDIA

1. Strychnine sulfate 0.000069 M decreased percentage attachment to the substratum by Amoeba proteus in 0.0029 M NaCl from 77.3 to 1.3, in 0.0029 M KCl from 40.8 to 2.5, in 0.002 M CaCl₂ from 73.3 to 68.0, in 0.002 M MgCl₂ from 85.5 to 83.3. 2. Frequency of ingestion of chilomonads by Amoeba proteus is increased by adding strychnine sulfate to solutions of NaCl, KCl, or CaCl₂. Frequency of ingestion is increased in NaCl solution from 1.3 to 2.3, in KCl from 0.75 to 2.25, and in CaCl₂ from 1.1 to 1.9 chilomonads per minute. Ingestion is not significantly increased by the addition of strychnine to MgCl₂ solution. 3. Frequency of ingestion of food by Amoeba proteus is not closely correlated with attachment to the substratum in NaCl and KCl solutions to which strychnine sulfate is added. 4. Chilomonads adhere to the plasmalemma of Amoeba proteus in solutions of NaCl, KCl, or CaCl₂ containing strychnine, but in MgCl₂ plus strychnine only a few adhere to it. Strychnine appears to make the surface of the amebae and chilomonads sticky in the former but not in the latter. Frequency of ingestion is apparently correlated with adherence of chilomonads to the plasmalemma. 5. Attachment to the substratum and ingestion by Pelomyxa carolinensis is increased by dead Chilomonas, Colpidium, and Paramecium in aqueous solutions, by materials obtained from paramecia by alcoholic-ether extraction, and by solutions in which these organisms have lived. 6. Attachment to the substratum by Pelomyxa carolinensis is not closely correlated with kind or concentration of inorganic salts used in this study. 7. Materials were found in extracts of paramecia which had certain characteristics in common with choline esters. There is no reason to doubt that under certain conditions materials are present in aqueous and alcoholic extracts which are pharmacologically similar to choline and acetylcholine. 8. Aqueous suspensions of paramecia when subcutaneously injected into young mice for 21 days inhibit the gonadotropic luteinizing hormone of the pituitary. Ovaries from injected mice showed no corpora lutea, and the seminal vesicles from injected males were smaller and contained less fluid than those of the controls.     

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.     

Nuclear Lethal Effect and Nucleocytoplasmic Incompatibility Induced by Endosymbionts in Amoeba proteus1

ABSTRACT. The role of bacterial endosymbionts in the acquisition of new phenotypic characters was studied by transplanting nuclei from an uninfected strain of Amoeba proteus into the enucleated cytoplasm of a symbiont-carrying strain. After 1–10 cell cycles, the nuclei were tested for two characters: compatibility with uninfected and infected cytoplasm, and their lethal effect against amoebae of the uninfected parent strain. A significant number of transplanted nuclei displayed both of the new phenotypic traits after a few divisions in the infected cytoplasm. Thus the influence of these endosymbionts on the nucleus of A. proteus was virtually instantaneous.     

The interaction of nuclear and cytoplasmic damage after treatments with toxic chemicals

An attempt is made to show how the interaction of different degrees of nuclear and cytoplasmic damage may contribute to the ultimate whole cell damage by a chemical. It suggests that cytoplasmic, as well as nuclear damage, may be important in the action of chemical carcinogens. Using Amoeba proteus as a single cell model where nuclear and cytoplasmic damage can be separated by micrurgy, the mortality curves for the nucleus, the cytoplasm and the whole cell are examined after four different treatments: exposure to N-methyl-N-nitroso urethane, a potent carcinogen in mammalian systems; exposure to ββ₁ (dichlorodiethyl) methyl amine, an alkylating agent used in chemotherapy; exposure to methylmercury chloride, a very toxic organo-metal; and irradiation with X-rays. These illustrate how different relative nuclear/cytoplasmic sensitivities contribute to the death of the cell. The evidence for nuclear and cytoplasmic damage after treatment with the N-methyl-N-nitroso urethane is detailed, and possibilities of nuclear repair after the four different types of treatment examined. Work on Amoeba proteus makes no attempt to assess separately changes in structure or activity of any one of the cells many enzyme systems, but looks at the balance between nuclear and cytoplasmic damage as a whole.     

Optical tomography analysis of <Emphasis Type="Italic">Amoeba proteus</Emphasis> chromatin organization at various cell cycle stages

An optical tomography investigation of the nuclear cycle in large freshwater amoebae Amoeba proteus has been performed for the first time. Nuclei of cells from a synchronized culture were stained with DAPI and examined using a confocal laser scanning microscope. Detailed analysis of three-dimensional images of the intranuclear chromatin at different stages of the nuclear cycle has been performed. The materials obtained, in combination with the published data, allow for a completely new representation of the dynamics of the structural organization of the A. proteus nucleus during the cell cycle. Two-stage interphase and mitosis of a special type not matching any of the known types in the existing systems of classification of mitosis were found to occur in amoebae. Amplification of chromosomes and/or fragments thereof supposedly occurs during the cell cycle, which is consistent with the available data on nuclear DNA hyperreplication during the cell cycle of A. proteus. The number of chromosomes can vary at different stages of the cycle because of amplification, this being a putative reason for the discordant reports on the number of chromosomes in this species. The elimination of “excess” DNA mainly occurs during the transition from prophase to prometaphase. Finally, specific features of chromosome behavior during mitosis allow conclusion to be drawn that many, if not all, chromosomes are of a holocentric type.     

The Fine Structure of Four “Species” of Amoeba*

The fine structure of Amoeba discoides, Amoeba dubia, and Amoeba amazonas was studied and compared with that of Amoeba proteus. The different kinds of amebas showed general similarities but differed in the ultrastructural details of their organelles. With respect to fine structure, A. discoides was indistinguishable from A. proteus, while both A. dubia and A. amazonas had distinctive features. The nuclei of all had a prominent honeycomb-like fibrous lamina, but A. dubia differed from the others in the distribution of nucleoli within the nucleus. The mitochondria of A. amazonas were unusual in having a variable pattern of cristae, some being plate-like and others tubular. Golgi bodies in A. amazonas had a greater proportion of vesicles and a smaller number of cisternae than those of the others, while Golgi bodies in A. dubia had highly flattened cisternae without a lining of filamentous material such as is found in the other types. The plasma membrane of A. dubia also lacked the prominent filamentous cell coat common to A. proteus and other amebas. The relation between the Golgi apparatus and the cell coat and the significance of the degree of development of the cell coat for pinocytosis and other phenomena is considered. The experimental use of these cells, including the formation of hybrids by nuclear transplantation is discussed.     

Maintenance and regeneration of cytoplasmic organelles in hybrid amebae formed by nuclear transplantation

The fine structure of cytoplasmic organelles was studied in hybrid amebae that were formed by transplanting the nucleus from one kind of ameba into the cytoplasm of a different type of ameba. Four different species or strains of amebae were utilized; Amoeba proteus, A. discoides, A. dubia, and A. amazonas. The fine structure of A. discoides was indistinguishable from that of A. proteus, but A. dubia and A. amazonas had distinctive features. To determine if the fine structure of cytoplasmic organelles in hybrid cells is like that of the nuclear parent or the cytoplasmic parent, nuclei were transferred between A. proteus and A. amazonas, which differ in their cytoplasmic ultrastructure, especially in the morphology of mitochondria. The cytoplasm of these hybrids resembled most closely that of the cytoplasmic parent. The degree to which different heterologous nuclei are capable of maintaining normal cytoplasmic organization was then studied by replacing an A. proteus nucleus with a nucleus from one of the other kinds of amebae. An A. discoides nucleus maintained A. proteus cytoplasm for several weeks. A nucleus from A. dubia or A. amazonas was able to maintain A. proteus cytoplasm for a few days, but then the cytoplasm began to show ultrastructural changes characteristic of enucleated amebae. The ability of a heterologous nucleus to promote the regeneration of Golgi bodies and endoplasmic reticulum after their decline in the absence of the nucleus was tested by transplanting a heterologous nucleus into A. proteus cytoplasm that had been enucleate for 5 days. A. discoides was the most effective heterologous nucleus donor but was less successful than normal A. proteus. Insertion of a nucleus from either A. amazonas or A. dubia resulted in “reactivation” of the cytoplasm with extension of pseudopods and resumption of motility but little or no regeneration of cell organelles. Thus, the ability of a heterologous nucleus both to maintain and to regenerate normal features of cytoplasmic organelles varied with the type of heterologous nucleus and was related to the degree of morphological resemblance between the donor and recipient cells. The role of the nucleus in motility and its role in regeneration of cytoplasmic organelles were dissociated from one another in the case of some hybrid renucleates.     

Symbiont-Induced Strain-Specific Lethal Effect in Amoebae

The cytosol fraction of a symbiont-bearing strain of Amoeba proteus exerted a lethal effect when injected into symbiont-free amoebae of the original strain. The lethal factor appeared to be a protein with a molecular weight of over 200,000. While the effect of the lethal factor on the nucleus was reversible, the host cytoplasm was permanently damaged so that it could not form a viable cell when combined with a normal nucleus.     

A study of histones in amoeba nuclei by indirect immunofluorescence method.

Nuclear proteins of four species of free-living Amoebidae (Amoeba proteus, A. discoides, Chaos carolinensis and Polychaos dubia) have been studied by indirect immunofluorescence technique using specific antisera to H1, H2A, H2B, H3 and H4 histone fractions from the calf thymus. It has been shown that the nuclei of the species examined have all these five histone fractions. However, the degree of similarity between homologous fractions from amoebae and the calf thymus varies and can be expressed in terms of immunological distance. Immunological differences between amoebic and calf thymus histones are the most pronounced in H1, being least in H3 and H4. Judged by its immunochemical characteristics, the histone fraction H2A from P. dubia is closer to the corresponding fraction from the calf thymus than is H2A from the other three amoeba species.     

The Free Amino Acid Levels of Pelomyxa carolinensis,Amoeba dubia and Proteus*

SYNOPSIS. The free amino acid extracts of 3 species of the free-living amoebae, Pelomyxa carolinensis, Amoeba dubia and A. proteus, were studied. Both the free amino acid patterns and levels in each species were different. Altho it appears that the free amino acid patterns were not altered by culture routines or starvation, the free amino acid levels were altered by these factors. The possibility of using free amino acid levels as a criterion for recognizing genetic differences is discussed.     

Binding characteristics of receptors for phagocytosis on the surface of Amoeba proteus

Summary Binding of the tripeptide n-formylmethionyl-leucylphenylalanine (NFMLP) to phagocytic receptors on the surface of Amoeba proteus was examined. Peptide-binding is reversible and demonstrates saturation kinetics. The receptors for phagocytosis are internalized by a temperature-sensitive process with indications that the receptors are recycled. The amoeba is capable of down-regulating its receptors for phagocytosis in response to constant external peptide levels, and also increasing the number of surface receptors in response to food deprivation. On the basis of competition studies, there is evidence that Amoeba proteus has separate surface receptors for both pinocytosis and phagocytosis.     

THE CYTONUCLEOPROTEINS OF AMEBAE : I. Some Chemical Properties and Intracellular Distribution

Autoradiographs of whole Amoeba proteus host cells fixed after the implantation of single nuclei from A. proteus donors labeled with any one of 8 different radioactive amino acids showed that the label had become highly concentrated in the host cell nucleus as well as in the donor nucleus and that the cytoplasmic activity was relatively low. When these amebae were sectioned, the radioactivity was found to be homogeneously distributed throughout the nuclei. The effect of unlabeled amino acid "chaser," the solubility of the labeled material, and the long-term behavior of the labeled material gave evidence that the radioactivity was in protein. At equilibrium, the host cell nucleus contained approximately 30 per cent of the radioactivity distributed between the two nuclei. This unequal nuclear distribution is attributed to the presence of two classes of nuclear proteins: a non-migratory one that does not leave the nucleus during interphase, and a migratory one, called cytonucleoprotein, that shuttles between nucleus and cytoplasm in a non-random manner. It is estimated that between 12 per cent and 44 per cent of the cytonucleoproteins are present in the cytoplasm of a binucleate cell at any one moment. Nuclei of Chaos chaos host cells also concentrated label acquired from implanted radioactive A. proteus nuclei.     

Mass isolated ameba nuclei. I. Isolation procedure and determination of macromolecular composition and RNA polymerase activity

A rapid method for mass isolation of intact nuclei from Amoeba proteus has been developed. By this procedure, which includes a novel way of recovering nuclei during the filtration step, about 40% of the nuclei in the starting suspension of 4 to 5 × 10⁶ cells can be recovered within 70 min. A typical suspension of purified nuclei consists of 93% intact nuclei, 5.6% food-waste pellets, 0.4% membranes, and 1.0% extracellular debris.     

Closure of the plasma membrane around microneedle in Amoeba proteus. An ultrastructural study

Microneedle perforations of the plasma membrane of Amoeba proteus were studied on the ultrastructural level. In each individual cell one hole was produced, which subsequently was marked with an eyelash left in place. Cells were quickly fixed, and sections cut parallel to the longitudinal axis of the eyelash. It was clear that the eyelash penetrated the plasma membrane, and that its free tip was located in the interior of the cell. A gap remained between the plasma membrane and the eyelash which may correspond to the electrical leak sometimes found by microelectrode punctures. The edges of the broken plasma membrane curled back into the cytoplasm. Here, a great redundancy of the plasma membrane was observed. Within these membrane accumulations, a large quantity of dense droplets was apposed at the inner leaflet of the plasma membrane. Their involvement in the formation and expansion of the plasma membrane in Amoeba proteus and Xenopus laevis has been suggested previously [1, 18]. Present studies offer more supportive evidence to that effect. Therefore, the interpretation seems to be plausible, that these membrane accumulations are the result of membrane expansion to minimize the hole produced during injury. This is in agreement with Holtfreter's [8] and Bluemink's [1] concept that the wound closure may occur by proliferation of the plasma membrane.