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.     

The Full Schedule of Macronuclear DNA Synthesis is Not Required for Cell Division in Paramecium tetraurelia1 1

Macronuclear DNA synthesis normally continues until late in the cell cycle in Paramecium; however, blockage of macronuclear DNA synthesis after 0.72 in the cell cycle does not alter the occurrence or timing of the subsequent cell division. When DNA synthesis is blocked after cells have reached the transition point, macronuclear DNA content at the following division is reduced to about 75% of the normal level. The point at which macronuclear DNA synthesis is no longer required for division corresponds to the beginning of micronuclear mitosis and the early stages of oral morphogenesis.     

Commitment to Division in Ciliate Cell Cycles

Near the end of the cell cycle, ciliates commit irreversibly to cell division. The point of commitment occurs at the time of oral polykinetid assembly and micronuclear anaphase. The commitment is a checkpoint which requisites a threshold cell mass/ DNA ratio and stomatogenesis. It is also a physiological transition point, involving cdk protein kinases similar to those of other eukaryotes. Both P34 kD and P36 kD kinases, similar to the S. pombe cdc2 kinases, have been described to have activity as monomers. Subsequent to commitment to division, dramatic cytoskeletal modifications occur for separation of organelles, cortex morphogenesis and cytokinesis. Numerous mutants affecting cytoskeletal function associated with the division process have been obtained in several species. Of these, only the ccl mutant in Paramecium affects cell cycle progression prior to commitment to division. The material reviewed is used to speculate about the mechanisms of regulation of pre-fission morphogenesis and cell division related processes in ciliates.     

The Dynamics of Micronuclear Mitosis in the Living Ciliate Spirostomum teres as Revealed by Polarization Microscopy1

ABSTRACT. Micronuclear mitosis in living Spirostomum teres has been studied by sensitive polarization microscopy, and the dynamic aspects of micronuclear division are described. The small, spherical, interphase micronuclei lie in form-fitting depressions in the macronuclear surface. Nuclear division begins with the rounding and slight swelling of the macronucleus and, coincidentally, the micronuclei move out of the depressions and away from the macronucleus, increase in size, and become weakly birefringent. As mitosis proceeds, the micronuclei increase in uniaxial birefringence and elongate to form irregular ovoids that convert to angular structures displaying principal axes of positive birefringence so divergent as to appear oriented at a right angle to one another. Micronuclei maintain this appearance for as long as 60 min and then abruptly change to rectangular-shaped structures, increase in uniaxial birefringence, and begin anaphase elongation. The somewhat dumbbell-shaped micronuclei lengthen at the constant rate of 2.0 μm/min to reach lengths >70 μm. It appears that little half-spindle shortening occurs during spindle elongation. Accompanying the changes in micronuclear spindle length are changes in birefringence. Just before elongation begins, presumably metaphase, the micronucleus is uniformly and intensely birefringent. At the magnifications employed, a chromosome plate is not clearly visible as a region of reduced birefringence. As elongation begins, the putative half-spindles are more birefringent than is the interzone, a condition that is maintained until the spindles have achieved ～30% elongation, at which time a region of increased birefringence develops at the center of the interzone. This pattern persists for a very short time, then gives way to a uniform birefringence of the entire separation spindle that is maintained until elongation is completed. The rate of micronuclear spindle elongation, changes in micronuclear dimensions, and corresponding changes in birefringence are discussed with respect to possible mechanisms of mitosis.     

A temperature-sensitive mutation affecting cilia regeneration, nuclear development, and the cell cycle of Tetrahymena thermophila is rescued by cytoplasmic exchange.

A temperature-sensitive mutation was isolated that blocks cilia regeneration and arrests growth in Tetrahymena thermophila. Protein and RNA synthesis and ATP production appeared to be largely unaffected at the restrictive temperature, suggesting that the mutation is specific for cilia regeneration and growth. At the restrictive temperature, mutant cells arrested at a specific point in the cell cycle, after macronuclear S phase and shortly before micronuclear mitosis. Arrested cells did not undergo nuclear divisions, DNA replication, or cytokinesis, so the mutation appears to cause true cell cycle arrest. Surprisingly, the mutation does not appear to affect micronuclear mitosis directly but rather some event(s) prior to micronuclear mitosis that must be completed before cells can complete the cell cycle. The cell cycle arrest was transiently complemented by wild-type cytoplasm exchanged during conjugation with a wild-type cell. Each starved, wild-type cell apparently contained enough rescuing factor to support an average of six cell divisions. Thus, this mutation affects assembly and/or function of at least one but not all of the microtubule-based structures in T. thermophila.     

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.     

Preconjugant cell interaction and cell cycle in the ciliate Euplotes crassus. Switching from the vegetative to the sexual stage.

Cells of Euplotes crassus can mate during the entire extent of G₁ as well in the very early macronuclear S phase. An arrest to cell cycling that occurs during mating is triggered by the ciliary agglutination that takes place between complementary cells, after a waiting period and before the cell bodies fuse into conjugal pairs. Cycling arrest is reversible. Sexually developing cells returned to nutrient conditions are able to reinitiate macronuclear DNA synthesis, although after a lag period. The synchronous reinitiation of synthesis is interpreted as a consequence of reprogramming events that occur as cells switch from the vegetative to the sexual stage of the life cycle.     

Abnormal micronuclear telomeres lead to an unusual cell cycle checkpoint and defects in Tetrahymena oral morphogenesis

Telomere mutants have been well studied with respect to telomerase and the role of telomere binding proteins, but they have not been used to explore how a downstream morphogenic event is related to the mutated telomeric DNA. We report that alterations at the telomeres can have profound consequences on organellar morphogenesis. Specifically, a telomerase RNA mutation termed ter1-43AA results in the loss of germ line micronuclear telomeres in the binucleate protozoan Tetrahymena thermophila. These cells also display a micronuclear mitotic arrest, characterized by an extreme delay in anaphase with an elongated, condensed chromatin and a mitotic spindle apparatus. This anaphase defect suggests telomere fusions and consequently a spindle rather than a DNA damage checkpoint. Most surprisingly, these mutants exhibit unique, dramatic defects in the formation of the cell's oral apparatus. We suggest that micronuclear telomere loss leads to a “dynamic pause” in the program of cortical development, which may reveal an unusual cell cycle checkpoint.     

Organization of the Euplotes crassus Micronuclear Genome1

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

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.     

Kinesin‐14 is Important for Chromosome Segregation During Mitosis and Meiosis in the Ciliate Tetrahymena thermophila

Ciliates such as Tetrahymena thermophila have two distinct nuclei within one cell: the micronucleus that undergoes mitosis and meiosis and the macronucleus that undergoes amitosis, a type of nuclear division that does not involve a bipolar spindle, but still relies on intranuclear microtubules. Ciliates provide an opportunity for the discovery of factors that specifically contribute to chromosome segregation based on a bipolar spindle, by identification of factors that affect the micronuclear but not the macronuclear division. Kinesin‐14 is a conserved minus‐end directed microtubule motor that cross‐links microtubules and contributes to the bipolar spindle sizing and organization. Here, we use homologous DNA recombination to knock out genes that encode kinesin‐14 orthologues (KIN141, KIN142) in Tetrahymena. A loss of KIN141 led to severe defects in the chromosome segregation during both mitosis and meiosis but did not affect amitosis. A loss of KIN141 altered the shape of the meiotic spindle in a way consistent with the KIN141's contribution to the organization of the spindle poles. EGFP‐tagged KIN141 preferentially accumulated at the spindle poles during the meiotic prophase and metaphase I. Thus, in ciliates, kinesin‐14 is important for nuclear divisions that involve a bipolar spindle.     

Histone rearrangements accompany nuclear differentiation and dedifferentiation in Tetrahymena

Histone synthesis and deposition into specific classes of nuclei has been investigated in starved and conjugating Tetrahymena. During starvation and early stages of conjugation (between 0 and 5 hr after opposite mating types are mixed), micronuclei selectively lose preexisting micronuclear-specific histones α, β, γ, and H3^F. Of these histones, only α appears to accumulate in micronuclear chromatin through active synthesis and deposition during the mating process. Curiously, α is not observed (by stain or label) in young macronuclear anlagen (4C, 10 hr of conjugation). Thus, young macronuclear anlagen are missing all of the histones which are known to be specific to micronuclei of vegetative cells. By 14–16 hr of conjugation, we observe active synthesis and deposition of macronuclear-specific histones, hv1, hv2, and H1, into new macronuclear anlagen (8C). Thus macronuclear differentiation seems well underway by this time of conjugation. It is also in this time period (14–16 hr) that we first detect significant amounts of micronuclear-specific H1-like polypeptides β and γ in micronuclear extracts. These polypeptides do not seem to be synthesized during this period, which suggests that β and γ are derived from a precursor molecule(s). Since these micronuclear-specific histones do not appear in micronuclear chromatin until after other micronuclei have been selected to differentiate as macronuclei, we suspect that micronuclear differentiation is also an important process which occurs in 10–16 hr mating cells. Our results also suggest that proteolytic processing of micronuclear H3^S into H3^F (which occurs in a cell cycle dependent fashion during vegetative growth) is not operative during most if not all of conjugation. Thus micronuclei of mating cells contain only H3^S which also seems consistent with the fact that some micronuclei differentiate into new macronuclei (micronuclear H3^S is indistinguishable from macronuclear H3). Interestingly, the only H3 synthesized and deposited into the former macronucleus of mating cells is the relatively minor macronuclear-specific H3-like variant, hv2. These results demonstrate that significant histone rearrangements occur during conjugation in Tetrahymena in a manner consistent with the fact that during conjugation some micronuclei eventually differentiate into new macronuclei. Our results suggest that selective synthesis and deposition of specific histones (and histone variants) plays an important role in the nuclear differentiation process in Tetrahymena. The disappearance of specific histones also raises the possibility that developmentally regulated proteolytic processing of specific histones plays an important (and previously unsuspected) role in this system.     

RAD51 is required for propagation of the germinal nucleus in Tetrahymena thermophila

RAD51, the eukaryote homolog of the Escherichia coli recA recombinase, participates in homologous recombination during mitosis, meiosis, and in the repair of double-stranded DNA breaks. The Tetrahymena thermophila RAD51 gene was recently cloned, and the in vitro activities and induction of Rad51p following DNA damage were shown to be similar to that of RAD51 from other species. This study describes the pattern of Tetrahymena RAD51 expression during both the cell cycle and conjugation. Tetrahymena RAD51 mRNA abundance is elevated during macronuclear S phase during vegetative cell growth and with both meiotic prophase and new macronuclear development during conjugation. Gene disruption of the macronuclear RAD51 locus leads to severe abnormalities during both vegetative growth and conjugation. rad51 nulls divide slowly and incur rapid deterioration of their micronuclear chromosomes. Conjugation of two rad51 nulls leads to an arrest early during prezygotic development (meiosis I). We discuss the potential usefulness of the ciliates' characteristic nuclear duality for further analyses of the potentially unique roles of Tetrahymena RAD51.     

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.     

Gene-sized macronuclear DNA molecules are clustered in micronuclear chromosomes of the ciliate Oxytricha nova.

Following the sexual phase of its life cycle, the hypotrichous ciliate Oxytricha nova transforms a copy of its chromosomal micronucleus into a macronucleus containing short, linear DNA molecules with an average size of 2.2 kilobase pairs. In addition, more than 90% of the DNA sequences in the micronuclear genome are eliminated during this process. We have examined the organization of macronuclear DNA molecules in the micronuclear chromosomes. Macronuclear DNA molecules were found to be clustered and separated by less than 550 base pairs in two cloned segments of micronuclear DNA. Recombinant clones of two macronuclear DNA molecules that are adjacent in the micronucleus were also isolated and examined by DNA sequencing. The two macronuclear DNA molecules were found to be separated by only 90 base pairs in the micronuclear genome.     

Micronuclear and Macronuclear Sequences of a Euplotes crassus Gene Encoding a Putative Nuclear Protein Kinase

The sequences of a 1.8-kbp macronuclear DNA molecule (V3), and the majority of its micronuclear counterpart, are reported. The macronuclear V3 DNA molecule contains an open reading frame that is interrupted by a single intron, while the micronuclear copy is interrupted by four internal eliminated sequences, one of which is located within the intron. The predicted protein product of the macronuclear V3 gene is a 471-amino acid polypeptide that is very similar to a group of protein-serine/threonine kinases from both plant and animal species, some of whose members appear to be involved in cell cycle or growth control.     

Division and Formation of the Macronuclei of Keronopsis rubra*

The hypotrichous ciliate Keronopsis rubra has ～10 micronuclei and ～100 small macronuclei. DNA synthesis proceeds synchronously in all macronuclei in the 2nd half of the cell cycle which takes about 24 hr at room temperature. A G₂ phase is virtually absent, each nucleus dividing as soon as the replication band has passed over it. The micronuclear S phase falls within macronuclear G₁ and is followed by immediate division. Comparative cytophotometric measurements of Feulgen-stained preparations indicate that the DNA content of G₁ macronuclei is scattered widely in a skewed normal distribution, with a peak corresponding to the DNA content of a G₁ micronucleus. Measurements of dividing macronuclei indicate unequal distribution of DNA between daughter nuclei and lead to the conclusion that the units of assortment must be smaller than whole genomes unless the micronucleus is polyploid. After conjugation, a large macronuclear anlage with threads resembling split prophase chromosomes is formed. The threads condense and pass singly into the cytoplasm where they are thought to give rise to the numerous small macronuclei of the vegetative cells.     

Genetic Characterization and Use of a Restriction Fragment Length Variant in the Hypotrichous Ciliate Euplotes crassus1

Two forms of a macronuclear DNA molecule differing in the presence or absence of a restriction endonuclease recognition site have been detected in the hypotrichous ciliate Euplotes crassus. Through a series of genetic crosses the two forms were shown to be allelic, being derived from a single micronuclear genetic locus. This restriction fragment length variant (RFLV) was used as a genetic marker to determine that the migratory and stationary pronuclei generated during mating can be genetically non-identical. In addition, the RFLV was used to investigate the efficiency of processing of the alternate alleles during macronuclear development and their subsequent transmission during vegetative growth. Little or no bias in the processing and/or amplification of the two alleles was observed during macronuclear development. During vegetative growth, however, changes in the relative amounts of the two alleles were observed.