Relation Between Cell Union and Nuclear Reorganization in Triplet Conjugants of Paramecium caudatum and Paramecium multimicronucleatum

SYNOPSIS Triplet conjugants of Paramecium caudatum which appeared naturally in mating mixtures and those of Paramecium multimicronucleatum which were produced by conjugation-inducing chemicals were isolated. Triplet conjugants lasting for more than 3 h were stained to examine macronuclear events. In P. caudatum , only 2 triplets among 182 (1%) contained macronuclear fragmentation in all 3 members. The most frequently occurring triplets (79%) were those producing 1 cell without and 2 cells with macronuclear fragments. There were also triplets (17%) producing 1 cell with, and 2 without macronuclear fragments, and some (3%) with 3 cells that contained no fragments. The length of persistence of the triplet was not responsible for the occurrence of macronuclear fragmentation in the 3rd cell of the triplet. In P. multimicronucleatum , the same 4 classes of triplets occurred, but the most frequently occurring class was that consisting of 3 cells (91%) with macronuclear fragments. Induction of nearly 100% of triplets with 3 such cells was possible by isolating the triplets' from a culture which was treated chemically at about 24 h after the last feeding. Treatment with chemicals in starved cultures resulted in triplets with incompletely fragmented or nonfragmented macronuclei. Further, in P. multimicronucleatum , chemicallyinduced triplets involving only holdfast pairs to which the 3rd cells were uniting often produced 3 cells with fragmented macronuclei.     

Analysis of mating type determination by transplantation of O macronuclear karyoplasm in Paramecium tetraurelia

The odd (O) or even (E) mating type in Paramecium tetraurelia is determined during the first cell cycle after new macronuclear development. The present paper demonstrates that mating type E is irreversibly determined at the end of the first cell cycle. Direct evidence comes from transplanting O macronuclear karyoplasm containing O-determining factor into E autogamous cells during a new postzygotic macronuclear development. Transplantation of O macronuclear karyoplasm into E autogamous cells at 7–8 hr after the origin of the macronucleus from a product of the synkaryon produces nearly 100% O mating type among the exautogamous cell lines but almost none 10–11 hr after the origin of the macronucleus (around the end of the first cell cycle). The macronuclear anlagen at the stage at which mating type E seems to be fixed contains about 20 times as much DNA as the vegetative G1 micronucleus. The O-determining factor shifting E cells toward O mating type by transplanting O macronuclear karyoplasm is also produced by the newly developed macronucleus in an effective concentration at 10–11 hr after the sensitive period and produced at full levels by the third cell cycle. The level of O factor in the macronucleus then gradually declines with subsequent repeated rounds of DNA synthesis and is finally lost by the eighth cell cycle.     

DNA methylation in vegetative and conjugating cells of a protozoan ciliate: Blepharisma japonicum

We report here the presence of N⁶-methyladenine (MeAde) in the macronuclear DNA (maDNA) of Blepharisma japonicum vegetative cells. We have further investigated the relationship between DNA methylation and cell union in cells activated for conjugation. Such activation was induced by treating cells of mating type I with complementary gamone 2. We found a reduction of about 24% of MeAde content in gamone-treated cells ready for cell union. First indications of the presence and reduction of MeAde content came from electrophoresis of maDNA digested by appropriate restriction endonucleases. Chromatographic determination of the amount of methylated base by HPLC substantiated these observations. In vegetative cells, 1.576 ± 0.02% of total adenine was found to be methylated as opposed to 1.193 ± 0.04% in activated cells. The HPLC analysis of maDNA also revealed a peak with a retention time corresponding to that of 5-hydroxymethyluracil, already found in some species of dinoflagellates. In that gamone treatment is correlated with a differential gene expression (indicated by a differential RNA and protein synthesis), our results suggest that there is a relationship between macronuclear genome activation and demethylation of maDNA. This is the first report of a correlation between gene activation and adenine demethylation in a eukaryotic organism.     

Tetrahymena Telomerase RNA levels increase during macronuclear development

Telomeres, the G-rich sequences found at the ends of eukaryotic chromosomes, ensure chromosome stability and prevent sequence loss from chromosome ends during DNA replication. During macronuclear development in Tetrahymena, the chromosomes fragment into pieces ranging from 20 kb to 1,500 kb. Tetrahymena telomerase, a ribonucleoprotein, adds telomeric (TTGGGG)_n repeats onto telomeres and onto the newly generated macronuclear DNA ends. We have investigated whether telomerase RNA levels increase during macronuclear development, since such an increase might be expected during chromosomal fragmentation. The steady-state level of the telomerase RNA component was used to estimate the abundance of telomerase present in mating and nonmating Tetrahymena. Northern blot analysis revealed that in vegetatively growing Tetrahymena, there were 18,000–40,000 copies of telomerase RNA per cell. In mating cultures, the levels of RNA increased 2-to 5-fold at 9–15 h, and 1.5- to 3.5-fold in starved nonmating cultures. This increase in telomerase RNA paralleled telomerase activity, which also increased slightly in mating and starved nonmating cells. © 1992 Wiley-Liss, Inc.     

Disturbance of the determination of germinal and somatic nuclei by heat shock in Paramecium caudatum

During conjugation of Paramecium caudatum, nuclear determination occurs soon after the third postzygotic division: one of the four anterior nuclei becomes the micronucleus and the remaining three degenerate, while four posterior nuclei differentiate into macronuclear anlagen. Macronuclear differentiation is supposed to be dependent on a cytoplasmic differentiation factor. In this study, postzygotic cells were subjected to heat shock for 30 min and nuclear changes were observed by staining with carbol fuchsin solution. When heat shock was initiated during the period from metaphase to telophase of the third postzygotic division, cells showed an excess of macronuclear anlagen and were typically amicronucleate. Abnormal nuclear localization around the end of the third (last) postzygotic division may explain the origin of these kinds of cells. A similar phenomenon appeared after treatment with actinomycin D or emetine. Since heat shock did not inhibit macronuclear differentiation but destroyed the formation of micronuclei, some factor(s) probably plays an essential role in nuclear determination, especially in the protection of the micronuclei.     

Timing and Regulation of Nuclear and Cortical Events in the Cell Cycle of Tetrahymena pyriformis*

SYNOPSIS. Using continuous flow cultures based on the chemostat principle, we varied the cell generation times of the ciliate Tetrahymena pyriformis strain GL, from 4.9 to 22.2 hr and studied various parameters of the cell cycle at 28 C. These included: the duration of the periods required for oral morphogenesis, macronuclear division, cell division, G₁ S, and G₂. The size of individual cells was also measured. Independent of the growth rate, the period of oral morphogenesis occurred during the last 90 min of the cell cycle. In all cases macronuclear and cell divisions took place during the last part of these 90 min, and the final macronuclear separation occurred just before final cell separation. The S-period increased slightly, while the G₁ and G₂ both increased in roughly the same relative proportion to the increasing generation times. Slowly growing cells (generation time 20.5 hr) were shorter but broader and somewhat larger in volume than quickly growing cells (generation time 4.9 hr).     

Kinetics of Incorporation of DNA Precursors from Ingested Bacteria into Macronuclear DNA of Paramecium aurelia12

SYNOPSIS. The kinetics of transfer of tritium-labeled material from the DNA of ingested bacteria into macronuclear DNA of Paramecium was examined by autoradiography. Bacteria labeled with tritiated thymidine were almost immediately incorporated into food vacuoles, thus becoming available for digestion and a potential source of labeled DNA precursors. Soluble label derived from food vacuoles appeared in low concentrations in the cytoplasm soon after cells were transferred to medium with labeled bacteria; incorporation of labeled precursors into macronuclear DNA began within 5 min. Labeled food vacuoles remained as potential sources of tritiated DNA precursors for a long and variable period after removal of labeled cells to non-labeled medium. The activity of the soluble cytoplasmic DNA precursors decreased parallel to the loss of labeled food vacuoles and no soluble DNA precursors were carried over from one macronuclear DNA synthetic period to the next. Labeling experiments were designed, using this information, which allowed determination of the pattern of macronuclear DNA synthesis and nuclear mass increase during the cell cycle. Macronuclear DNA synthesis began 25–30% of the way thru the cell cycle, continued at a constant rate during the middle half, and decreased in rate during the last quarter. Macronuclear mass increased in an approximately linear fashion, beginning with the onset of DNA synthesis and doubling by the time of karyokinesis.     

Selective inhibition of DNA synthesis in macronuclear fragments in Paramecium aurelia exconjugants and its reversal during macronuclear regeneration

In Paramecium exconjugants very rapid DNA synthesis takes place in the developing macronuclear anlagen, while DNA synthesis is suppressed in macronuclear fragments. The rate of DNA synthesis in fragments (as a percentage of the rate in anlagen or macronuclei in the same cells) decreases by about 40% during each successive cell cycle over at least the first five cell cycles after conjugation, even though macronuclear anlagen are fully mature by the end of the second cell cycle. — Suppression of DNA synthesis in macronuclear fragments is reversible. If macronuclear anlagen are removed at fission, a very high rate of DNA synthesis resumes in macronuclear fragments after a two-hour lag. The total rate of synthesis in the ensemble of macronuclear fragments in cells without anlagen is greater than that in anlagen in control cells. Thus, suppression of DNA synthesis in macronuclear fragments is not the result of any stable differentiation or irreversible change in the fragments but is the result of, and dependent on, the presence of macronuclear anlagen. — The results of injection of cytoplasm from vegetative cells into normal exeonjugants suggest that normal macronuclei produce an inhibitor which selectively suppresses DNA synthesis in macronuclear fragments. In control cells the relative rate of DNA synthesis in fragments ranged from 40 to 70% of that in anlagen in the same cells, while in injected cells the relative rate of incorporation of DNA precursors was suppressed to as little as 7%. The mean level of incorporation into fragments in injected cells was significantly lower than that in controls, suggesting that the injected cytoplasm contained an inhibitor.Contribution 822, Zoology Department, Indiana University. Supported in part by contract COO-235-66 of the USAEC and by grant No. Gm 15410-05 of the USPHS to T. M. Sonneborn.This paper is a portion of a dissertation submitted in partial fulfillment of the equirements for the degree of Doctor of Philosophy.     

Internuclear control of DNA synthesis in exconjugant cells of Paramecium caudatum

An exconjugant cell of Paramecium caudatum has two kinds of macronuclei, fragmented prezygotic macronuclei and postzygotic new macronuclei (anlagen). Although the DNA synthesis in the fragmented prezygotic macronucleus continues until the third cell cycle after conjugation, selective suppression of the DNA synthesis in the prezygotic macronucleus takes place at the fourth cell cycle. The inhibition of DNA synthesis in prezygotic fragmented macronuclei is due to the presence of a postzygotic macronucleus (anlage) in the same cytoplasm because the inhibition does not occur when the postzygotic macronucleus (anlage) is removed by micromanipulation during the third or fourth cell cycle. Well-developed postzygotic macronuclei (anlagen) with full ability to divide have the ability to depress the DNA synthesis of prezygotic macronuclear fragments. The suppression of DNA synthesis in prezygotic macronuclear fragments seems to be irreversible. Competition for the limited amount of DNA precursors also plays an important role in the onset of the selective suppression of the DNA synthesis.     

Nuclear Behavior During Reconjugation in Euplotes patella (Ciliophora,Hypotrichida)1

Nuclear behavior during reconjugation and the ultimate fate of the ex-reconjugants were followed after induction of reconjugation in Euplotes patella. An exconjugant could reconjugate with a vegetative cell or with another exconjugant. Exconjugants at an early stage of macronuclear development (oval macronuclear anlagen) did not reconjugate frequently whereas exconjugants at a late stage of macronuclear development (rod-like macronuclear anlagen) reconjugated frequently. In all cases, the micronucleus underwent normal meiosis and other nuclear changes. After reconjugation, a new macronuclear anlage and a new micronucleus were formed normally, so that there were two kinds of macronuclear anlagen in the exconjugants, an old and a new. The old rod-shaped anlage did not disappear after the differentiation of a new one, but it was broken up into several fragments. While the survival rate after normal conjugation was 78%, it was 0–20% after reconjugation. These results suggest that the micronuclei of exconjugants can act as germ nuclei even at a very early stage and that reconjugation, unlike conjugation, is harmful to the cell.     

Maternal effect mutations affecting macronuclear determination and development in Paramecium tetraurelia

Gene mutations that interfere with macronuclear development in Paramecium were obtained by selecting lines that failed to produce normal macronuclear anlagen following the second autogamy after mutagenesis. The mutants fell into several complementation groups. There was one case of apparent intragenic noncomplementation among the eight mutants examined. In the stronger mutants, macronuclear anlagen were not formed, and all four mitotic products of the posfzygotic divisions of the synkaryon remained as micronuclei. Under semirestrictive conditions, cells often contained a single anlage, suggesting that determination of anlagen was a discrete event for each nucleus. The missing anlagen trait was recessive and associated with a strong maternal effect. The phenocritical period of one of the stronger alleles, aal^a, began at the second postzygotic division and ended with the first morphological differentiation of macronuclear anlagen. Nuclear migration in this mutant was abnormal. Under restrictive conditions, the posterior products of the second postzygotic division reached a posterior-most position, which was 8% of cell length more anterior than that of the most posterior nuclei in wild-type cells. Under permissive conditions, the pattern of migration was intermediate between that of wild-type cells and mutants under fully restrictive conditions. The patterns of nuclear migration were consistent with the nuclear growth kinetics.     

Induction of Blocks in Nuclear Divisions and Overcondensation of Meiotic Chromosomes with Cycloheximide During Conjugation of Tetrahymena thermophila

During conjugation, the micronucleus of Tetrahymena thermophila undergoes five consecutive nuclear divisions: meiosis, third prezygotic division (pregamic mitosis) and two postzygotic mitoses of the synkaryon. The four products of the synkaryon differentiate into macronuclear anlagen and new micronuclei and the old macronucleus is resorbed. The protein synthesis inhibitor cycloheximide, applied during conjugation, induced several developmental blocks. Pairs shifted to the drug during early meiotic prophase (stages I–III) were arrested at prophase. Cycloheximide applied to cells at pachytene (stages IV-VI) to metaphase arrested the conjugants at the stage of modified prometaphase/metaphase with overcondensed, swollen bivalents. In contrast to other systems, in the presence of cycloheximide, separation of chromatids, decondensation of chromosomes and exit from metaphase I were inhibited in both diploid and haploid cells. Pairs shifted to the drug after metaphase I were arrested at postmeiotic interphase after completing one nuclear cycle. The same rule applied to the subsequent cycle; then cells were arrested at the stage of pronuclei, and those pairs with functional pronuclei and synkarya were arrested at the stage of two products of the first postzygotic division (pronuclei were not arrested in nuclear transfer and karyogamy). Only pairs with two products of the first postzygotic division were arrested at the same stage after the cycloheximide treatment. Pairs shifted to cycloheximide during the second postzygotic division were arrested in development of macronuclear anlagen and resorption of old macronuclei. The postmeiotic conjugants pulse-treated with cycloheximide (2 h) yielded heterokaryons retaining parental macronuclei (i.e. they exhibited macronuclear retention).     

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.     

Differential DNA Amplification and Copy Number Control in the Hypotrichous Ciliate Euplotes crassus

ABSTRACT. During macronuclear development in hypotrichous ciliated protozoans, several thousand macronuclear DNA molecules are amplified several-hundred fold. We investigated the regulation of this amplification by determining the copy numbers of three different macronuclear DNA molecules in the hypotrichous ciliate Euplotes crassus. Two of the macronuclear DNA molecules were present in approximately 1,000 copies per cell, while the third was present in approximately 6,500 copies per cell. These reiteration levels were achieved either during macronuclear development, or shortly thereafter, and were maintained during vegetative growth. The most abundant macronuclear DNA molecule is present as a single-copy sequence in the micronuclear genome. Thus, its high copy number results from differential amplification. These results indicate that DNA amplification during macronuclear development is regulated individually for each macronuclear DNA molecule.     

DNA replication in macronuclei of Tetrahymena pyriformis GL

DNA replication in macronuclei of Tetrahymena pyriformis GL has been studied to discriminate between hypotheses developed for the interpretation of intraclonal differentiation in ciliated protozoa (the diploid subnuclear, and the ‘master’-‘slave’ hypotheses). Tetrahymena cells were grown in a heavy ¹⁵N-³H medium and then transferred to a light ¹⁴N-¹⁴C medium. DNA was isolated after various periods following this transfer and studied in equilibrium CsCl density gradient centrifugation. Time-related changes in the DNA buoyant density pattern were investigated. The data obtained are interpreted to mean that all DNA in macronuclei of asynchronously growing Tetrahymena at exponential phase replicates semiconservatively once in a cell cycle. These data are in good agreement with the findings of Andersen & Zeuthen obtained on synchronous Tetrahymena cultures in the presence of BUdR.These results are not consistent with the ‘master’-‘slave’ hypothesis. The diploid subnuclear hypothesis is not in accord with other experimental evidence. An alternative hypothesis has been proposed concerning the nature of the macronuclear units and the process of determination. The two main points of this hypothesis are: (a) macronuclear units are diploid genome fragments (‘nucleosomes’); (b) determination is a process of haploidization by ‘allelic splitting’ at a definite macronuclear fission. Consistency with experimental data is discussed and some predictions of the hypothesis are given.     

The Kinetic Complexity of Paramecium Macronuclear Deoxyribonucleic Acid

DNA extracted from macronuclei of axenically cultured Paramecium aurelia has been characterized with regard to its kinetic complexity. Renaturation of macronuclear DNA from this protozoon appeared to follow 2nd order kinetics and revealed the presence of 2 components: a main component comprising ～96% of the genome which renatured slowly and a minor component comprising ～4% of the genome which renatured at a rate ～3000 faster than the main component. The value of the kinetic complexity of the main component has been estimated at 3.8 × 10¹⁰ daltons and that of the minor component at 1.45 × 10⁷ daltons. It is suggested that the macronucleus contains ～840 diploid copies of the slowly renaturing component; for each copy of the latter there are ～100 copies of the fast renaturing component.     

冠突伪尾柱虫有性生殖期间皮膜发育的核控制

通过显微手术去小核建立多个冠突伪尾柱虫（Pseudourostyla cristata)无小细胞系,并诱导它们与有小核细胞进行接合生殖,以评估小核及其衍生的大核原基在有性生殖期间对皮膜形态发生的影响,当无小核接合体从有小核配偶获得1枚配子核后,接合双方不仅能平行地继续核器演化,而且使第1次皮膜改组能够同步进行和正常发育,说明小核在有性周期中除了生殖功能外仍保留着某些控制皮膜发育的体功能,虽然大部分接合后体的大核原基在DNA贫乏期停止发育,但少数接合后体能够超越这一时期,并启动第2次皮膜改组和顺利完成其后续的有性发育全程,表明指令发动第2次皮膜发育的信号来自DNA贫乏期后以排出一核物质团块为标志的大核原基。     

Membrane dielectric responses of bufalin-induced apoptosis in HL-60 cells detected by an electrorotation chip

A non-invasive electrorotation (ROT) technique was used to monitor the apoptosis-induced changes in HL-60 cells. The specific membrane capacitance of the cells fell from 15.6 ± 0.9 mF/cm² to 6.4 ± 0.6 mF/cm² after 48 h treatment with 10 nM bufalin, a component of bufadienolides in traditional Chinese medicine, Chan Su. However, the average membrane conductance remained almost constant during the first 24 h of treatment and then increased afterwards. Apoptosis was verified by a DNA fragmentation assay and scanning electron microscopy. The results demonstrate that the ROT technique gives a quantitative analysis of the toxic damage by chemicals to cells and can be exploited in the testing and development of new pharmaceuticals and active cell agents. Chengjun Huang and Ailiang Chen contributed equally to this work.     

Partial Synchronization of Cell Division in Suspension Cultures of Haplopappus gracilis

Four different chemicals were tested in their ability to synchronize cell division in asynchronous cell cultures of Haplopappus gracilis. Twentyfour-hour treatments with 5-amino uracil resulted in a peak in the mitotic index about 14–16 hours after the end of the treatment. The increase in the frequency of mitoses was about three times that of the control. Hydroxyurea, at a concentration of 3 mM, gave after a treatment period of 12–24 hours an increase in the frequency of mitoses which appeared about 10 hours after the treatment. The mitotic index was about 35 per cent, which is 4 times that of the control. 5-Fluorodeoxyuridine (FUdR) at a concentration of 2 × 10^?7M gave a mitotic burst about 16 hours after treatment. At that time about 15 per cent of the cells were dividing which was about twice that of the control. The block was reversed with 4 × 10^?5M thymidine. Thymidine at a high concentration caused a reduction in the frequency of mitoses during the treatment. After 15 to 16 hours in a thymidine free medium a mitotic peak appeared with a doubling of the frequency of mitoses in treated cells. Cytological studies showed that parlicularly hydroxyurea but also 5-aminouracil and 5-fluorodeoxyuridine produced gaps and fragments at the concentrations which gave cell synchronization.     

Nuclear differentiation and nucleic acid synthesis in well-fed exconjugants of Paramecium aurelia

Paramecium aurelia exconjugants contain new macronuclear anlagen and numerous fragments of the old pre-zygotic macronucleus. Macronuclear anlagen develop during the first two cell cycles after conjugation. During this time their volume increases from about 11 m³ to about 3700 m³ and more than 10 doublings of DNA content occur. The rate of DNA synthesis is between two and three times as great as in the vegetative macronucleus. — In macronuclear fragments, however, DNA synthesis is suppressed. The rate of DNA synthesis in macronuclear fragments during the extended first cell cycle after conjugation (11 1/2 hr. vs. 5 1/2 hr. for the vegetative cell cycle) is only about one-third of the rate in vegetative macronuclei and there is only a 65% increase in the mean DNA content of fragments. The rate of fragment DNA synthesis continues to decrease during each of the subsequent two cell cycles. — Unlike the rate of DNA synthesis, the rate of RNA synthesis per unit of DNA is similar in macronuclear anlagen, macronuclear fragments and fully developed macronuclei. Macronuclear fragments continue to synthesize RNA at the normal rate long after the new macronuclei are fully developed. Fragments contribute about 80% of all RNA synthesized during the first two cell cycles after conjugation. RNA synthesis begins very early in the development of macronuclear anlagen and nucleolar material appears during the first half-hour of anlage development. — Chromosome-like structures were never observed during anlage development and there was no evidence of two periods of DNA synthesis separated by a DNA poor stage as has been observed in several hypotrichous Ciliates.