Causal relations among cell cycle processes in Tetrahymena pyriformis. An analysis employing temperature-sensitive mutants

Utilization of temperature-sensitive mutants of Tetrahymena pyriformis affected in cell division or developmental pathway selection has permitted elucidation of causal dependencies interrelating micronuclear and macronuclear replication and division, oral development, and cytokinesis. In those mutants in which cell division is specifically blocked at restrictive temperatures, micronuclear division proceeds with somewhat accelerated periodicity but maintains normal coupling to predivision oral development. Macronuclear division is almost totally suppressed in an early acting mutant (mola) that prevents formation of the fission zone, and is variably affected in other mutants (such as mo3) that allow the fission zone to form but arrest constriction. However, macronuclear DNA synthesis can proceed for about four cycles in the nondividing mutant cells. A second class of mutants (psm) undergoes a switch of developmental pathway such that cells fail to enter division but instead repeatedly carry out an unusual type of oral replacement while growing in nutrient medium at the restrictive temperature. Under these circumstances no nuclei divide, yet macronuclear DNA accumulation continues. These results suggest that (a) macronuclear division is stringently affected by restriction of cell division, (b) micronuclear division and replication can continue in cells that are undergoing the type of oral development that is characteristic of division cycles, and (c) macronuclear DNA synthesis can continue in growing cells regardless of their developmental status. The observed relationships among events are consistent with the further suggestion that the cell cycle in this organism may consist of separate clusters of events. with a varying degree of coupling among clusters. A minimal model of the Tetrahymena cell cycle that takes these phenomena into account is suggested.     

Regulation of macronuclear DNA content in Paramecium tetraurelia

The macronucleus of Paramecium divides amitotically, and daughter macronuclei with different DNA contents are frequently produced. If no regulatory mechanism were present, the variance of macronuclear DNA content would increase continuously. Analysis of variance within cell lines shows that macronuclear DNA content is regulated so that a constant variance is maintained from one cell generation to the next. Variation in macronuclear DNA content is removed from the cell population by the regulatory mechanism at the same rate at which it is introduced through inequality of macronuclear division. Half of the variation in macronuclear DNA content introduced into the population at a particular fission by inequality of division is compensated for during the subsequent period of DNA synthesis. Half of the remaining variation is removed during each subsequent cell cycle. The amount of variation removed in one cell cycle is proportional to the postfission variation. The cell's power to regulate DNA content is substantially greater than that required to compensate for the small differences that arise during division of wild-type cells. For example, a constant variance was still maintained when the mean difference between sister cells was increased to ten times its normal level in a mutant strain. The observations are consistent with a replication model that assumes that each cell synthesizes an approximately constant amount of DNA which is independent of the initial DNA content of the macronucleus. It is suggested that the amount of DNA synthesized may be largely determined by the mass of the cell.     

Timing of micronuclear mitosis and its relation to commitment to division inParamecium tetraurelia

This study examines the timing of micronuclear mitosis during the vegetative cell cycle and shows that mitosis begins early in the division process and coincides approximately with the earliest stages of oral morphogenesis (about 0.6 in the cell cycle in synchronous cell samples). The cc1 mutation blocks cell cycle progression prior to the point of commitment to division. Although the cc1 mutation blocks macronuclear DNA synthesis under restrictive conditions, it does not block micronuclear DNA synthesis. However, absence of functional cc1 gene product leads to blockage of micronuclear mitosis prior to completion of anaphase. This point coincides with commitment to division and is also the point at which oral morphogenesis is blocked in cc1 cells. The tim-ings of the transition points for micronuclear mitosis and oral morphogenesis in cc1 cells are closely associated in both synchronous cell samples and in asynchronous cultures. © 1992 Wiley-Liss, Inc.     

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.     

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.     

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.     

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.     

Control of cell division in Paramecium tetraurelia. Effects of abrupt changes in nutrient level on accumulation of macronuclear DNA and cell mass

In the cell cycle of Paramecium there are three points of interaction between cell growth-related processes and the processes of macronuclear DNA replication and cell division: initiation of DNA synthesis, regulation of the rates of growth and DNA accumulation, and initiation of cell division. This study examines the regulation of the latter two processes by analysis of the response of each to abrupt changes in nutrient level brought about either by transferring dividing cells from a steady-state chemostat culture to medium with unlimited food, or by transferring well-fed dividing cells to exhausted medium. The rates of DNA accumulation and cell growth respond quickly to changes in nutrient level. The amounts of these cell components accumulated during the cell cycle following a shift in nutrient level are typical of those occurring during equilibrium growth under post-shift conditions. Commitment to division occurs at a fixed interval prior to fission that is similar in well-fed and nutrient-limited cells. Initiation of cell division in Paramecium is associated with accumulation of a threshold DNA increment, whose level is largely independent of nutritive conditions. The amount of DNA accumulated during the cell cycle varies with nutritional conditions because the rates of growth and DNA accumulation are affected by nutrient level; slowly growing cells accumulated relatively little DNA during the fixed interval between commitment to cell division and fission.     

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.     

Cell cycle mutation in Paramecium tetraurelia discriminates between sexual and vegetative functions

The temperature-sensitive mutation cc1 blocks a number of cell cycle processes in Paramecium including macronuclear DNA synthesis, oral morphogenesis, and the later stages of micronuclear mitosis. Oral morphogenesis and micronuclear mitosis also occur in the sexual pathway. This study shows that cc1 cells can proceed through conjugation or autogamy under restrictive conditions; neither stomatogenesis nor micronuclear mitosis is blocked. Fertilization and macronuclear determination occur normally, but DNA synthesis in macronuclear anlagen is blocked. Therefore, this mutation discriminates between oral replacement during meiosis and vegetative prefission stomatogenesis, and between mitotic spindle elongation during the pregamic and postzygotic divisions and spindle elongation during the vegetative cell cycle. These results point to a fundamental regulatory difference between morphogenesis in the vegetative and sexual pathways. © 1994 Wiley-Liss, Inc.     

Deciliation interferes with cell-cycle progression in Tetrahymena

The impact of ciliary regeneration upon cell-cycle progression of the ciliate Tetrahymena was studied. It was found that cell division ceases during ciliary regeneration, and starts again about 4 h after deciliation. Deciliation of an asynchronously multiplying culture results in a rapid interruption of DNA synthesis, followed by resumption 1 h later. This was shown by pulse-labelling the cells with [3H]thymidine at various times after deciliation. Cytophotometric determinations of the macronuclear DNA content substantiated these observations, since the average DNA content per cell remained constant within the first hour of regeneration, confirming the labelling experiments, after which it rose. At its maximum, the average DNA content was more than doubled as compared with the beginning of the experiment. This indicates that a substantial proportion of the regenerating cells performed two rounds of DNA replication prior to cell division. The massive drop in the average DNA content during the fifth hour after deciliation indicates that the culture becomes partly synchronized for cell division by the deciliation procedure. The division synchrony results from a greater delay of the next cell division when G2 cells are deciliated than occurs in G1 cells. This was shown by deciliating cultures of Tetrahymena thermophila cells in the respective stages of the cell cycle, which had been partly synchronized by elutriator centrifugation. Thus, deciliation followed by ciliary regeneration causes a varying degree of retardation in progression through the cell cycle, being greatest for G2 cells and least for G1 cells.     

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.     

Erythropoietic progenitors capable of colony formation in culture: response of normal and genetically anemic W-W-V mice to manipulations of the erythron

The macromolecular reguirements for the initiation and maintenance of macronuclear DNA replication were studied in heat synchronized Tetrahymena pyriformis GL-C. Previous work had established that macronuclear S periods could occur in a consecutive fashion without intervening cell divisions during a multiple heat shock treatment, as well as immediately following the synchronized cell divisions. Cycloheximide treatment prior to or during the S period which follows the first synchronized cell division resulted in abolition of the initiation of DNA synthesis or an almost immediate cessation of DNA synthesis in progress. Temporary inhibition of DNA synthesis occurred when cycloheximide was added late in the S period. Treatment with actinomycin D was found to block the initiation of DNA synthesis but did not appreciably affect the continuation of the S period. It was concluded that RNA synthesis was required for the initiation but not the maintenance of DNA replication, whereas protein synthesis was necessary for both processes. The dependency of the initiation of an S period on prior RNA and protein synthesis was also shown to exist when a second consecutive S period was initiated without a preceding cell division. Treatment with actinomycin or cycloheximide prior to a supernumerary S period during a multiple heat shock treatment completely abolished the initiation of DNA synthesis. In T. pyriformis the synthesis of RNA and protein related to the initiation of the S period is tightly coupled to each cycle of DNA replication.     

TIF1 Represses rDNA replication initiation, but promotes normal S phase progression and chromosome transmission in Tetrahymena

The non-ORC protein, TIF1, recognizes sequences in the Tetrahymena thermophila ribosomal DNA (rDNA) minichromosome that are required for origin activation. We show here that TIF1 represses rDNA origin firing, but is required for proper macronuclear S phase progression and division. TIF1 mutants exhibit an elongated macronuclear S phase and diminished rate of DNA replication. Despite this, replication of the rDNA minichromosome initiates precociously. Because rDNA copy number is unaffected in the polyploid macronucleus, mechanisms that prevent reinitiation appear intact. Although mutants exit macronuclear S with a wild-type DNA content, division of the amitotic macronucleus is both delayed and abnormal. Nuclear defects are also observed in the diploid mitotic micronucleus, as TIF1 mutants lose a significant fraction of their micronuclear DNA. Hence, TIF1 is required for the propagation and subsequent transmission of germline chromosomes. The broad phenotypes associated with a TIF1-deficiency suggest that this origin binding protein is required globally for the proper execution and/or monitoring of key chromosomal events during S phase and possibly at later stages of the cell cycle. We propose that micro- and macronuclear defects result from exiting the respective nuclear S phases with physically compromised chromosomes.     

Inhibition of an early event in the cell division cycle of Escherichia coli by FL1060, an amidinopenicillanic acid.

Analysis of exponential and synchronous cultures of Escherichia coli B/r after the addition of FL1060 indicates a block point for division by this agent some 15 to 20 min before the end of the preceding cell division cycle, a time corresponding to the beginning of the C period of the cell division cycle. Morphological examination of FL1060-treated synchronous cultures of E. coli /r was consistent with inhibition by FL1060 of a very early event in the cell division cycle. This event appears to be essential for normal cell surface elongation in a rod configuration. Temporary treatment of synchronous cultures of E. coli B/r with FL1060 resulted in division delay, the extent of which was a function of the duration of exposure to FL1060. However, even after relatively long times of FL1060 treatment the delayed divisions were still synchronous. Although FL1060 had no direct effect on deoxyribonucleic acid (DNA) synthesis, the synchronous delayed division occuring after temporary treatment with FL1060 were accompanied by a delay in the attainment of resistance of cell division to inhibitors of DNA, ribonucleic acid, and protein synthesis. These results suggest aht an FL1060-sensitive event initiates at the beginning of the C period of the cell division cycle of E. coli and is responsible for normal cell elongation. This cell elongation pathway procedes independently of DNA synthesis, but there is an interaction between this pathway and termination of a round of DNA replication in which a normal rod configuration is necessary to allow a signal for cell division to be generated upon completion of DNA replication.     

Scheduled and unscheduled DNA synthesis during development in conjugating Tetrahymena

Autoradiography has been used to confirm and to extend previous microspectrophotometric studies (Doerder and DeBault, 1975) on the timing of DNA synthesis during conjugation in Tetrahymena thermophila. The majority of DNA synthesis occurs at the expected periods preceding gamete formation and the two postzygotic divisions and during macronuclear development. DNA in new macronuclei is endoreplicated in an extremely discontinuous fashion. Under starvation conditions, the first endoreplication (2C to 4C) occurs immediately after the second postzygotic division when both new macronuclei and new micronuclei replicate. The second endoreplication (4C to 8C) does not occur until after separation of conjugants. If mating cells are kept under prolonged starvation conditions (20-24 hr), refeeding induces a partially synchronous division, after which an unexpectedly high percentage of cells incorporate tritiated thymidine into both macro- and micronuclei. Two previously undescribed periods of DNA synthesis were observed in the micronuclei of conjugating Tetrahymena. The first occurs during the early stages of meiotic prophase, before full crescent elongation. The second takes place in an extended period corresponding to macronuclear anlagen development, before conjugants have separated. CsCl gradient analyses indicate that, in micronuclear fractions, only main band DNA is being synthesized in both of these periods. However, in macronuclear fractions from both stages, a significant fraction (approximately 20%) of the DNA being synthesized has the buoyant density of ribosomal DNA. The finding that macro- and micronuclear DNA can be synthesized simultaneously in a single cell, both during conjugation and after refeeding starved exconjugants, raises interesting questions of how macro- or micronuclear-specific histones are targeted to the appropriate nuclei.     

A CDC2-Related Kinase is Associated with Macronuclear DNA Synthesis in Paramecium tetraurelia

ABSTRACT. Cell division in higher eukaryotes is mainly controlled by CDK (serine/threonine) protein kinases and by other components of these kinase complexes. Previously, we showed homologous kinases also occur in the ciate Paramecium tetraurelia . In this report, a polyclonal antibody was produced against a GST fusion protein which contained the N-terminal third of the previously isolated CDC2FTA protein. It recognized a 36 kDa polypeptide on western blots and did not cross-react with the related 34 kDa polypeptide. This 36 kDa polypeptide showed no affinity for yeast P13^suc1 protein. The CDC2PTA level was invariant throughout the cell cycle and decreased slightly when cells entered stationary phase. Indirect immunofluorescence showed CDC2PTA localized in the macronucleus, but not in micronuclei. Native CDC2FTA was immunoprecipitated and the kinase activity was assayed using histone HI as substrate in elutriation synchronized samples. The kinase activity was high during the early stages of the cell cycle and reached a peak at 2.5 h after elutriation, which corresponds to the time of initiation of macronuclear DNA synthesis. This suggests CDC2PTA kinase may be associated with the regulation of macronuclear DNA synthesis. These results allow us to draw concrete comarisons for the first time between the role of CDK in ciliates and higher eukaryotes.     

Regulation of cell cycle events in asymmetrically dividing cells: functions required for DNA initiation and chain elongation in Caulobacter crescentus

To study the regulation of cell cycle events after asymmetric cell division in Caulobacter crescentus, we have identified functions that are required for DNA synthesis in the stalked cell produced at division and in the new stalked cell that develops from the swarmer cell 60 min after division. The initiation of DNA synthesis in the two progeny cells is dependent upon at least two common functions. One of these is a requirement for protein synthesis and the other is a gene product identified in a temperature-sensitive cell cycle mutant. DNA chain elongation requires a third common function. The characteristic pattern of DNA synthesis in C. crescentus appears to be controlled in part by the expression of these functions in the two stalked cells at different times after cell division. The age distribution for Caulobacter cells in an exponential population has been calculated (Appendix by Robert Tax) and used to analyze some of the results.     

Downward regulation of macronuclear DNA content in Paramecium tetraurelia : Effects of excess DNA on the subsequent DNA and protein content and the cell growth rate

Paramecium cells were selected which received the entire parental macronucleus at fission and thus started the cell cycle with twice the normal post-fission DNA content. During each of the subsequent two cell cycles the cells synthesized approximately as much DNA as did control cells. The amount of excess macronuclear DNA was consequently halved during each cell cycle. The minimum pre-fission DNA content was just larger than the mean post-replication DNA amount, confirming that a similar amount of DNA, approximately equal to the mean post-fission DNA content of the non-selected population, was synthesized in macronuclei, regardless of the post-fission DNA content. These observations confirm a model for DNA content regulation previously devised for Paramecium and are inconsistent with DNA content regulation schemes proposed for other ciliates. The increased DNA content has no effect either on the subsequent total protein content of pre-fission cells, or on the rate of cell growth. This suggests that the rate of cell growth is limited by the size of the cell when the macronuclear gene-dosage is equal to or greater than that in normal cells. The results also suggest that the amount of DNA synthesized within an interfission period is also limited by the size of the cell and is proportional to the cell mass. Paramecium does not require a fixed nucleocy oplasmic ratio as a pre-condition either for cell division, or, by inference, for initiation of DNA synthesis.