Alteration of the Tetrahymena Genome During Nuclear Differentiation*†

Synopsis.
The DNA of the macro- and the micronucleus of Tetrahymena thermophila has been compared by various biochemical methods. It became evident from their thermal denaturation temperatures and buoyant densities that the 2 DNAs were very similar in overall composition. Small differences were detected when the sequence complexities of these DNAs were compared by DNA renaturation studies. The studies suggested that ˜ 10% of the micronuclear genome was lost or underrepresented in the macronucleus. Comparison of individual gene levels revealed further differences. By using the technic of gene cloning a micronuclear sequence was isolated which hybridized only with micronuclear, but not with macronuclear DNA. These results indicated the occurrence of elimination or underreplication of this sequence in the macronucleus. Gene amplification was also shown to occur. In the micronucleus only a single copy of rDNA was found integrated into the chromosome. During macro-nuclear development, amplification was observed to occur, and the amount of rDNA to increase, until there were ˜ 200 copies per haploid genome in the mature macronucleus. all of them extrachromosomal and palindromic. The 3rd case of alteration involved a simple repeated sequence, (CCCCAA)n, present in the termini of rDNA and also in many other locations of the genome. Restriction endonuclease digestion studies revealed drastic differences in the organization of the repeats between macro-and micronucleus. These differences may be interpreted as the results of chromosome fragmentation which occurs at every cluster of the repeats during macronuclear development. The relationship between this event and gene amplification and elimination is discussed.     

Regulation of Macronuclear DNA Content in Tetrahymena thermophila*†

Synopsis.
Unequal macronuclear division in Tetrahymena thermophila introduces variance into G1 macronuclei; unless eliminated such variance would result in continuous variation in DNA content. Analysis of G1 and G2 macronuclear variances reveals that the added variance is eliminated by action on the extremes of macronuclear DNA content. In this model (Model II), macronuclei with small amounts of DNA have an additional complete S phase, while those with large amounts of DNA skip S. From available data, chromatin extrusion is shown not to contribute significantly, if at all, to the elimination of variance. Computer simulations utilizing haploid subunits indicate that model II predictions apply reasonably well to experimental data in terms of coefficients of variation, mean DNA content, and frequency of additional and skipped S phases. The simulations reveal also that within certain constraints, particularly the thresholds for additional and skipped S phases, macronuclear assortment is unaffected by Model II regulation. The relationships between Model II and other aspects of the cell cycle are briefly discussed.     

Genetic Evidence Concerning the Structure of the Tetrahymena thermophila Macronucleus*†

Synopsis.
A satisfactory model of the Tetrahymena thermophila macronucleus must explain its genetic behavior in terms of its constituent molecules. Particular genetic phenomena requiring explanation are (a) phenotypic assortment , here interpreted as resulting from allelic disjunction rather than from differential gene expression; (b) unequal allelic input for some loci , interpreted as a consequence of unequal and selective replication of some alleles during early macronuclear development; (c) delayed assortment at some loci , interpreted as an effect of inequality of allelic input combined with a generalized elevation of DNA content during early clonal history; (d) linkage disruption , probably reflecting continuous somatic recombination rather than dissolution of chromosomes into small repliconic units; (e) assortment depression , brought about by the occasional association of homologous replicons (chromosomes) or else by a differential increase in some classes of replicons; (f) ploidy-related developmental differences in macronuclear primordia are interpreted on the basis of quantitative differences in DNA rather than in terms of an early perception of genic imbalance, (g) Ploidy independent macronuclear DNA content is consistent with several models of size regulation.     

Regulation of Macronuclear DNA Content in Paramecium tetraurelia*†

Synopsis.
The amitotic division of the macronucleus of Paramecium tetraurelia produces daughter macronuclei which frequently differ in DNA content. In wild-type cells these differences are small, but can be increased substantially by the action of mutant genes. The variance in macronuclear DNA content would increase continuously if there were no mechanism to regulate it. Paramecium has a very effective regulatory mechanism—all cells synthesize similar amounts of macronuclear DNA, regardless of the number of macronuclei or their prereplication DNA content. DNA synthesis is controlled at the level of macronuclear subunits, and the postreplication macronucleus consists of a mosaic of subunits that have undergone different numbers of replication events during the previous cell cycle. It is evident from experimental results that the amount of DNA synthesized can be influenced by the total size or mass of the cell. Experimental modification of the initial DNA content leads to no change in the amount of DNA synthesized, or in the subsequent protein content of the cells, but modification of cell size causes corresponding changes in the amount of DNA synthesized and in the size of the macronucleus. The implications of these observations for cell growth and the cell cycle are discussed.     

The Size of Macronuclear DNA and Its Relationship to Models for Maintaining Genic Balance*†

Synopsis.
A considerable amount of evidence is now available to indicate that the DNA in the ciliates Oxytricha and Stylonychia undergoes fragmentation when the micronucleus forms a macronucleus. Some evidence suggests that fragmentation may also occur in Tetrahymena and possibly in Paramecium . It is shown that some regulatory or nonrandom segregational mechanism must operate during cell divisions to maintain genic balance in Tetrahymena . Both the hypothesis of macronuclear subunits and also a new hypothesis based on replicative control of DNA are capable of explaining the currently know biochemical, cytological, and genetic facts.     

Selection of Nonmotile Tetrahymena with Ficoll Underlayers*,†

The mechanisms regulating the development of cilia in Tetrahymena are poorly understood but might be revealed through the study of ciliogenesis mutants. Failure to regenerate cilia after dibucaine deciliation results in continued absence of motility. Therefore, to isolate ciliogenesis mutants efficiently, methods for separating motile and nonmotile cells are essential. We examined the efficacy of Ficoll underlayers for these separations. Ciliates of T. thermophila strain mpr^-/mpr (6 mp sens IV) (6-methyl purine-sensitive; mating type IV) were mixed with Ficoll and added as underlayers to separatory funnels containing growth medium. At 27 C most of the cells remained motile and were found in the top layer; at 37 C, there was a time-dependent increase in the number of nonmotile cells and the number of cells in the Ficoll layer. After 150 min at 37 C, most of the cells became nonmotile and were found in the Ficoll layer. Other studies indicated that at 37 C, the cells remained alive and capable of regenerating cilia when deciliated. Thus, it is clear that the Ficoll underlayer effectively separates the majority of nonmotile cells from the majority of motile cells. Evidently, however, at 37 C wild-type T. thermophila exhibit temperature-sensitive phenotypic variability with regard to motility which should be minimized when selecting for mutations affecting motility and ciliogenesis.     

Partial Purification and Characterization of a Bacteriolytic Enzyme Secreted by Tetrahymena*

Synopsis. Tetrahymena pyriformis strain HSM secretes large quantities of acid hydrolases into the culture medium. An enzyme secreted by the ciliate and capable of degrading walls of streptococci was identified and purified to a considerable degree. The pH optimum of this enzyme was 3–4, and it was eluted after cytochrome c from Sephadex G-75 columns. Unlike lysozyme, the enzyme was thermolabile at pH 2.9, but relatively thermostable at pH 8.1. It degraded “C-labeled cell walls of streptococci releasing reducing groups. Cell walls prepared from different strains of streptococci differed in susceptibility to this enzyme, the most sensitive strain tested being of group A, type T12. It was shown in immunologic studies that this hydrolase released the group-specific carbohydrate from the walls. Secretions of Tetrahymena from early stationary-phase cultures had more bacterio-lytic activity than those from cells from late stationary-phase cultures. Further, cells from cultures grown in glucose-supplemented medium secreted less of the enzyme than ciliates of comparable age grown in unsupplemented proteose-peptone. The newly isolated bacteriolytic enzyme, presumably of lysosomal origin, may be helpful in characterizing streptococcal cell walls.     

Formation and Fate of Extranuclear Chromatin Bodies in Tetrahymena

The fate of extranuclear chromatin bodies (ECBs) formed by exclusion of macronuclear material at the time of karyokinesis was followed quantitatively in Tetrahymena pyriformis strain GL-I. In a logarithmic growth phase culture, 51% of the dividing cells produced one (43%) or more (8%) ECBs. Most of these gradually disappear before the next cell division, but ? 13% are retained and carried into subsequent cell cycles. The random distribution of ECBs into anterior or posterior daughter cells, their staining and morphological characteristics, and their rapid loss in cells in starvation medium, all indicate that ECBs play no more of a role in cellular activity than that of an internally produced food vacuole.     

Tip Transformation in Tetrahymena: A Morphogenetic Response to Interactions Between Mating Types*

SYNOPSIS. In Tetrahymena thermophila subline B, a morphogenic alteration of the anterior end of cells of mating types III and VII results from a cellular interaction which precedes and is a prerequisite for pairing. Cell pairing begins 1 h after starved cells of complementary mating type are mixed. The 1 h-long lag period is characterized by an actinomycin D-sensitive inductive interaction in the first 30 min, followed by a maturation period. Tip transformation begins during the maturation period and continues after pairing. Scanning electron microscopy of deciliated cells reveals ridges which form a chevron meeting in a midline seam between the oral apparatus and the anterior tip. During transformation, the seam broadens until the ridged surface is completely smooth. Melding of the ridges also occurs at the tip of the cell resulting in its blunted appearance. Cells of complementary mating types join in the region of this modified surface, which eventually becomes a specialized cell junction perforated by cytoplasmic bridges. Thus, pursuant to an inductive interaction the structure of the tip of cells is modified in anticipation of the pairing event. Rate of cell pairing might be limited by rate of tip transformation.     

Effects of Calcium Ion on Growth and Behavior of Tetrahymena pyriformis

The chelator GEDTA was used to show that Ca²⁺ is required for the growth of Tetrahymena pyriformis strain W and for normal galvanotactic responses and swimming.     

Précisions Temporelles sur les Processus de la Stomatogenèse de Tetrahymena paravorax analysés en Microscopie Photonique

Resume. Une analyse des séquences morphogénétiques du CiliéTetrahymena paravorax montre que: (A) La durée de la stomatogenèse de bipartition des formes microstomes en croissance exponentielle représente 45% du temps de génération (stade 1—20%; stade 2—3%; stade 3—3%; stade 4—5%; stade 5—5%; stade 6—9%). (B) La division cytoplasmique est inégale (les proters sont plus petits que les opisthes); la différence de taille initiale entre les 2 produits de fission est probablement compensée par une prolongation de la période de croissance chez le proter. (C) Le pourcentage maximum de réorganisation buccale microstome → macrostome pour les populations asynchrones atteint –? 70% au bout de 210 mn d'incubation dans la stomatine. (D) L'initiation de la stomatogenèse de remplacement oral est connectée avec la fin d'une période dont la durée minimale est approximativement celle du stade 0 du cycle normal d'interdivision des microstomes; cette initiation est retardée chez les microstomes exposés à la stomatine dès le début du cycle cellulaire. (E) Le primordium buccal de division peut se résorber en présence de stomatine et la stomatogenèse antérieure peut commencer avant que ne soit terminée cette résorption; la résorption n'est plus induite au-delà d'un point du stade 5 qui précède le début de la constriction du corps cellulaire. SYNOPSIS. An analysis of the morphogenetic sequences in the ciliate Tetrahymena paravorax has shown that: (A) The duration of predivision stomatogenesis in exponentially growing microstomes occupies 45% of the generation time (stage 1—20%; stage 2—3%; stage 3—3%; stage 4—5%; stage 5—5%; stage 6—9%). (B) Cytoplasmic division is unequal (the proters are smaller than opisthes); the initial size difference between the 2 fission products is presumably compensated by an increased growth period in the proter. (C) The maximum percentage of microstome-to-macrostome oral reorganization is –? 70% in asynchronous populations, 210 min after suspension in stomatin. (D) Initiation of oral replacement stomatogenesis is associated with the end of a period which has a minimum duration nearly equal to that of stage 0 characteristic of the normal inter-division cycle of the microstomes; this initiation is delayed if exposure of microstomes to stomatin is begun at the onset of the cell cycle. (E) The buccal primordium formed in division can be resorbed in presence of stomatin and anterior stomatogenesis can start before the resorption is completed: this resorption is not induced if the cells have progressed beyond a point which precedes the beginning of the cell furrowing (stage 5).     

RNA Synthesis in Division Synchronized Tetrahymena: Macronuclear and Cytoplasmic RNA*

SYNOPSIS. Synthesis of RNA in the macronucleus and appearance of RNA in the cytoplasm were studied in heat synchronized Tetrahymena pyriformis GL and compared to those found under conditions of logarithmic growth (28 C) and during heat shocks (34 C). In macronuclei of logarithmically growing cells precursors were processed to 2 rRNA species (25S and 17S). In addition, another RNA (15S), more homogeneous than the RNA (8-15S) in the cytoplasm, was observed in the macronucleus. Both 17S and 25S rRNA species were found in the cytoplasm, 17S rRNA appearing more rapidly than 25S rRNA. Synthesis of rRNA was suppressed at 34 C in cells subjected to heat synchronization; 8-15S RNA synthesis appeared to be inhibited to a lesser extent. During the time preceding the first synchronized division, the synthesis of rRNAs in the macronucleus slowly recovered. Early in the cycle, almost no newly synthesized rRNAs were extracted. By 30 min after the last heat shock (EH), most of the RNA synthesized was not identified as rRNA. By 60 min after EH, the pattern of RNA synthesis had not returned to that observed in logarithmically growing cells.     

Macro- and Micronuclei of Tetrahymena pyriformis: A Model System for Studying the Structure and Function of Eukaryotic Nuclei*†

The macro- and micronucleus of Tetrahymena pyriformis are formed from a common diploid synkaryon during conjugation. Shortly after the 2nd postzygotic division, distinct morphologic and physiologic differences develop between the 2 nuclei. Micronuclei remain small, presumably diploid, and electronmicroscopic observations indicate that micronuclear DNA is contained in a dense, fibrous, chromosome-like coil. Macronuclei contain considerably more DNA than micronuclei, and the DNA of the macronucleus is found largely in the chromatin bodies typical of ciliate nuclei. The functional differences between macro- and micronuclei in vegetative cells also are striking. The template activity of DNA in the micronucleus is highly restricted compared to that in the macronucleus. Micronuclei synthesize and contain little RNA, and do not contain either nucleoli or ribonucleoprotein granules. Macronuclei, on the other hand, synthesize and contain large amounts of RNA and have many nucleoli and ribonucleoprotein granules. Macro- and micronuclei also have distinct differences in the timing of DNA synthesis during the cell cycle and in the timing and mechanism of nuclear division. Finally, during conjugation the macronucleus becomes pycnotic and disappears while the micronucleus undergoes meiosis and fertilization, ultimately giving rise to new macro- and new micronuclei. In short, the macro- and micronuclei of Tetrahymena provide an excellent system for studying the molecular mechanisms by which the same (or related) genetic information is maintained in different structural and functional states. Methods have been devised to isolate and purify macro- and micronuclei of Tetrahymena in the hope of correlating differences in the nucleoprotein composition of these nuclei with differences in their structure and function. The DNAs of macro- and micronuclei have been found to differ markedly in their content of a methylated base, N⁶-methyl adenine, and major differences in the histones of the 2 nuclei have been observed. Macronuclei contain histones similar to those found in vertebrate nuclei, while 2 major histone fractions seem to be missing in micronuclei. In addition, histone fraction F2A1 which is found in multiple, acetylated forms in macronuclei, is present only as a single, unacetylated form in micronuclei.     

Macronuclear persistence of sequences normally eliminated during development in Tetrahymena thermophila

During conjugation in the ciliated protozoan, Tetrahymena thermophila, a somatic MAC-ronucleus develops from the germinal MICronucleus. Ten to 20 percent of the MIC genome is eliminated during this process. Three repetitive families have been identified which have different levels of repetition in the MIC and are eliminated to different degrees in the MAC. Some members of two of these families persist in the MAC. In this study, we have looked at these persistent sequences in the MAC of cell lines from a variety of sources including several inbed strains, two sets of caryonides, caryonidal subclones, and vegetatively aged cell clones. The results suggest that the sequences that remain in the MAC have a genetic predisposition to persist. However, epigenetic variations occur as the MAC develops so that only some of the persistent sequences are actually observed in a particular MAC. Polymorphisms may be generated if alternative processing of a single MIC segment occurs. These polymorphisms can later be resolved by phenotypic assortment during vegetative growth. These facultatively persistent sequences appear to differ from sequences previously described in this organism.     

Structure of Trichomonads as Revealed by Scanning Electron Microscopy*†

SYNOPSIS.
A scanning electron microscope (SEM) study of Hypotrichomonas acosta (Moskowitz), Trichomonas vaginalis Donné. Pentatrichomonas hominis (Davaine), and Tritrichomonas foetus (Riedmüller) provided new information about the structure of the periflagellar canal: emergence of the flagella from the cell body; structure of the undulating membrane; and position, shape, and size of the pelta. Of special interest were the spatial relationships of the attached part of the recurrent flagellum and the accessory filament in Hypotrichomonas and in the members of Trichomonadinae, i.e. Trichomonas and Pentatrichomonas.     

Characterizations of Phospholipids from Paramecium tetraurelia Cells and Cilia*,†

Phospholipids from Paramecium tetraurelia strains 51s and d,95 cultures and isolated cilia were characterized. The following classes of phospholipids were identified in whole cell lipids: the 1-alkyl-2–acylglyceryl and the 1,2–diacylglyceryl forms of phosphonylethanolamine, phosphorylethanolamine. and phosphorylcholine; cardiolipin: ceramide aminoethylphosphonate and 5 other sphingolipids: phosphatidylserine; phosphatidylinositol; and lyso derivatives of the major glycerophospholipids. Cilia lipids were rich in ether lipids, phosphonolipids. and sphingolipids. Net lipid biosynthesis did not occur, as determined by the weight of lipids extracted from culture medium compared with the weight of lipids extracted from culture medium and ciliates after 7 days of growth. Total lipids/cell decreased with culture age. changes in the neutral lipid fraction accounting for the major decrease. Phospholipid class distributions changed with culture age—the glyceryl phosphorylethanolamine and choline content of cells decreased, while the glyceryl ohosphonylethanolamine content remained relatively constant: hence, the ratio of phosphonolipids to total phospholipids increased. All fatty acids observed in total lipids from cells and cilia were also present in the glycerophospholipids. Total lipids from cilia contained a greater percent of polyunsaturated fatty acid than those of whole cells. Whole cells and cilia glyceryl phosphonolipids contained up to 93% eicosatetraenoic plus eicosapentaenoic fatty acids. The enrichment of phosphonolipids in cilia accounted for most of the polyunsaturated fatty acid enrichment observed in cilia total lipids. The fatty acid composition of all major whole cell glycerophospholipid classes changed dramatically with culture age, while only small changes occurred in cilia glycerophospholipid fatty acids.     

Plasmodium falciparum in Culture: Improved Continuous Flow Method*

SYNOPSIS. A new design of flow vessel provides a method for continuous culture of P. falciparum in a settled layer of human erythrocytes with a slow flow of culture medium over them. The parasitemia is kept fluctuating from ? 1%, just after addition of fresh erythrocytes. to ? 10%, 2 or 3 days later. Each vessel provides each week 3 harvests, each containing ? 0.6–1 × 10⁹ parasites.     

Transformation in Tetrahymena pyriformis: Description of an Inducible Phenotype*†

SYNOPSIS. Transformation of Tetrahymena pyriformis to a rapid-swimming (presumably dispersal) form can be induced by washing cells and suspending them in distilled H₂O, Dryl's solution or 10 mm Tris. Transformation is possible with high efficiency in mass cultures of axenically grown cells within ～ 5 h at 30 C. The radically different phenotype produced during transformation is characterized by a more elongate body form, increased numbers of somatic basal bodies and cilia, a long caudal cilium and oral membranelles positioned beneath the cell surface. DNA quantities characteristic of G1, S, and G2 cells are found in these transformed ciliates, suggesting that achievement of a particular stage in the DNA-division cycle is not a prerequisite for transformation. Preliminary observations on cells belonging to syngens 2–12 indicate that they also have a capacity to form a caudal cilium, but that the amicronucleate strain GL-C does not. Possible relevance of the transformed phenotype for taxonomy of Tetrahymena is discussed.     

Observations on Gametogenesis in Plasmodium falciparum from Continuous Culture*

Synopsis. Gametocytes of Plasmodium falciparum were produced in continuous cultures but eventually declined in numbers after 3–4 months in vitro. Their development progressed in a consistent pattern, from small rounded, through triangular, to ellipsoidal, and finally after 8 days to crescentic forms. Morphologic maturity occurred at 8–9 days, but the gametocytes would not exflagellate in vitro, even after 14–18 days of development. Thus, current culture methods cannot produce a continuous supply of functional gametes for further studies.     

Studies of Membrane Formation in Tetrahymena pyriformis. VIII. On the Origin of Membranes Surrounding Food Vacuoles*†

SYNOPSIS. A procedure was devised for the isolation of purified food vacuoles from Tetrahymena pyriformis fed particles of ferric oxide. Phospholipids extracted from vacuolar membranes were more similar in composition to the lipids of microsomes than to lipids of whole cells, cilia or post-microsomal supernatant. Fractionation of cells grown in the presence of [¹⁴C]palmitic acid or [³²P]inorganic phosphate also revealed similarities in the specific radioactivities of microsomes and vacuolar membranes. The data suggested that vacuolar membranes arise from a pool of cytoplasmic membranes.