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 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.     

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.     

Developmental expression of macronuclear specific antigen in Paramecium caudatum.

We obtained a monoclonal antibody (MA-1) specific for macronuclei of the ciliate Paramecium caudatum and P. dubosqui. Immunoblotting showed that the antigen was a polypeptide of 50 kilodalton (kDa). During the process of nuclear differentiation in P. caudatum, the MA-1 antigens appeared in the macronuclear anlagen immediately after four out of eight post zygotic nuclei differentiated morphologically into the macronuclear anlagen. Afterwards, the antigens could be detected in the macronucleus through the cell cycle, and disappeared when the macronucleus began to degenerate in exconjugant cells. These results suggest that the antigens may play a role in the differentiation and function of the macronucleus.     

Macronuclear differentiation in conjugating pairs of Tetrahymena treated with the antitubulin drug nocodazole

During Tetrahymena conjugation gamic nuclei (pronuclei) are produced, reciprocally exchanged, and fused in each mate. The synkaryon divides twice; the two anterior nuclei develop into new macronuclei while the two posterior nuclei become micronuclei. The postzygotic divisions were blocked with the antitubulin drug nocodazole (ND). Then pronuclei (gamic nuclei) developed directly into macronuclear anlagen (primordial macronuclei), inducing amicronucleate cells with two anlagen, or, rarely, cells with one anlagen and one micronucleus. ND had a similar effect on cells that passed the first postzygotic division inducing amicronucleate cells with two anlagen, while cells treated with ND at the synkarya stage produced only one large anlage. Different intracytoplasmic positioning of the nuclei treated with ND (pronuclei, synkarya and two products of the first division) shows that most of cell cytoplasm is competent for inducing macronuclear development. Only posteriorly positioned nuclei--products of the second postzygotic division--remain micronuclei. The total cell DNA content, measured cytophotometrically in control and in ND-induced amicronucleate conjugant cells with one and two anlagen, was similar in all three samples at 12 h of conjugation. Eventually, at 24 h this content was about 2 pg (8 C) per anlagen both in nonrefed control and in amicronucleate exconjugants. Therefore "large" nuclei developing in the presence of ND were true macronuclear anlagen.     

Developmental expression of macronuclear specific antigen in Paramecium caudatum

We obtained a monoclonal antibody (MA-1) specific for macronuclei of the ciliate Paramecium caudotum and P. dubosqui. Immunoblotting showed that the antigen was a poly-peptide of 50 kilodalton (kDa). During the process of nuclear differentiation in P. caudatum, the MA-1 antigens appeared in the macronuclear anlagen immediately after four out of eight post zygotic nuclei differentiated morphologically into the macro-nuclear anlagen. Afterwards, the antigens could be detected in the macronucleus through the cell cycle, and disappeared when the macronucleus began to degenerate in exconjugant cells. These results suggest that the antigens may play a role in the differentiation and function of the macronucleus. © 1992 Wiley-Liss, Inc.     

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.     

冠突伪尾柱虫（Pseudourostyla cristata）实验再生研究

冠突伪尾柱虫(Pseudvurostyla cristata) 含约70枚大核。我们用显微手术横切G1期细胞,得前后两块相等断片;分别培养。60小时后,断片再生完成。在再生过程中,随细胞体积增大,大核数目也增加。大核的数目和细胞体积存在着一定的均衡关系。在细胞无性分裂过程中,许多大核改组后,融合成一个融合大核。这个融合大核具两个仔虫的大核数目和DNA量。我们用显微手术得到含融合大核的后断片。在后断片再生后恢复的虫体内,我们发现本应分配到两个仔虫中去的大核数目,被限制在一个虫体的大核数目上。这说明了细胞质可以影响和调节大核的数目。并还证明了这种虫体大核DNA量较正常虫的大核DNA量约多一倍。其中大部分虫体分裂时,大核不经改组就开始融合和分裂;从而使DNA量回复正常。同讨还发现小部分虫体通过排出大核多余核物质方式来调节大核DNA量。这些现象说明了细胞核质之间存在着一种调节相对平衡和相互协调的机制。     

Regulation of histone acetylation during macronuclear differentiation in Tetrahymena: evidence for control at the level of acetylation and deacetylation

During the postzygotic period of the sexual cycle (conjugation) in the ciliated protozoan, Tetrahymena, daughter products from a single micronuclear mitotic division develop into new macronuclei (anlagen) or new micronuclei depending upon their cytoplasmic location. In this study we have monitored the status of histone acetylation in synchronous populations of developing nuclei isolated from conjugating cells. Particular attention has been paid to the level of histone acetylation in new macronuclei following their differentiation from micronuclei. Like micronuclei isolated from vegetative cells (Vavra et al., 1982), micronuclei from conjugating cells (5 hr, 10-12 hr, and 15-16 hr) contain little if any acetylated histone and incorporate little postsynthetic acetate under any of our experimental conditions. In contrast, young new macronuclei (4C, 10-12 hr) incorporate significant amounts of acetate in vitro and in vivo provided that sodium butyrate is included during the labeling period. These results suggest that 4C anlagen contain both active acetylase and deacetylase activities even though the actual steady state level of acetylation found in these nuclei is low, more like that of micronuclei. At later stages of macronuclear maturation (8C, 15-16 hr), inner histones are hyperacetylated in a manner similar to parental, fully differentiated macronuclei. Furthermore, 8C anlagen incorporate acetate well even in the absence of sodium butyrate. Taken together these results suggest that endogenous deacetylase enzymes become either down-regulated and/or the rate of histone acetylases increases markedly during macronuclear differentiation.     

Nuclear Roles in the Post-Conjugant Development of the Ciliate Euplotes aediculatus II. Experimentally Induced Regeneration of Old Macronuclear Fragments1

Following conjugation in ciliates, the usual fate of the old pre-conjugant macronucleus is resorption. In some species, however, old macronuclei, or their fragments, have the ability to reform functional vegetative macronuclei when new macronuclear anlagen are defective. The present work on Euplotes shows that if anlagen are allowed to carry out their essential roles in early exconjugant development, including influence on cortical reorganization such that feeding can resume, they can then be permanently damaged by UV-microbeam irradiation and regeneration of old macronuclear fragments can occur. E. aediculatus exconjugants were anlage-irradiated at 40–60 hr of development and the irradiated cells cultured individually and fed. Squashes revealed enlargement and anteriorward migration of the persistent (posterior) macronuclear fragments. The first post-conjugant fission of such cells was delayed (times ranged 6–43 days) and did not seem to involve the damaged anlagen, which remained rudimentary, did not divide along with the cells, and were subsequently resorbed. It appeared that cell fission was supported by the fragments of the old macronuclei, which either divided or partitioned themselves between the two daughter cells. Mating tests performed on early clones derived from irradiated exconjugants revealed ample conjugation competence; intraclonal conjugation in such clones was also apparent. The absence of the immature period seen in normal exconjugants provides further evidence that the clones arose from cells with regenerated macronuclei.     

Studies on the Macronuclei of Doublet Paramecium tetraurelia: Distribution of Macronuclei and DNA at Fission*

SYNOPSIS. Doublet Paramecium tetraurelia would be expected to contain 2 macronuclei if their nuclear complement were strictly analogous to that of singlets. However, most doublets are unimacronucleate. It is shown in this study that dimacronucleate cells are present only in young clones. Unimacronucleate cells arise either through abnormalities in the determination and distribution of macronuclear anlagen during the first cell cycle after conjugation, or from dimacronucleate cells through abnormal division and segregation of macronuclei during the fission process. When a change in the number of macronuclei occurs through abnormalities in the division and segregation of daughter macronuclei, the daughter cells produced typically have DNA contents more similar than those expected from either random segregation of daughter macronuclei, or from the normal segregation pattern in ciliates in which changes in the number of macronuclei in progeny cells do not occur. This suggests that part of the regulation process of macronuclear DNA content in Paramecium may occur through control of the segregation pattern of daughter macronuclei.     

Macronuclear Regeneration and Cell Division in Paramecium caudatum

SYNOPSIS. In Paramecium caudatum , occurrence of macronuclear regeneration is closely related to the time of feeding after conjugation. Macronuclear regeneration is induced with a high frequency when conjugating pairs are transferred into fresh culture medium. Feeding immediately after conjugation induces early cell division and 3 or more fissions occur without macronuclear division because of the inability of the macronuclear anlagen to divide. In the cells lacking normal macronuclear anlagen, old macronuclear fragments undergo regeneration and form vegetative macronuclei.     

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.     

Morphology and development of the macronuclei of the ciliates Stylonychia mytilus and Euplotes aediculatus

Some stages of macronuclear anlagen development, known from earlier investigations (see Fig. 1), were studied in detail. The results are: a) The giant chromosomes of Stylonychia mytilus are not somatically paired, but are connected end-to-end to form one or a few composite chromosomes. When they later disintegrate, the bands become isolated granules. b) Spectrophotometric measurements show that during the DNA-poor stage which follows the disintegration of the chromosomes, the macronuclear anlagen of Euplotes have a DNA content of 21 c, while the syncaryotic (deriving from syncarya) and hemicaryotic (deriving from haploid hemicarya) anlagen of Stylonychia have the DNA content of diploid micronuclei (2c). Nevertheless the syncaryotic anlagen of Stylonychia and Euplotes initially develop two nucleoli at the end of this stage, the hemicaryotic anlagen of Stylonychia only one. From this it is concluded that the genes of one giant chromosome band stay together in one granule, c) Labeled DNA from the giant chromosomes which remains in the anlagen during the DNA-poor stage is distributed approximately equally to the daughter nuclei during the first few fissions of the exconjugants.-Autoradiographic experiments showed that the DNA of the macronuclei of Stylonychia that is duplicated at one time in a replication band is not duplicated simultaneously during the next DNA-duplication. The DNA duplications during the second polyploidization stage of the macronuclear anlagen development are exceptions, because the mixing of the macronuclear DNA which occurs before every fission does not occur during the second polyploidization stage.—The pseudomicronuclei which sometimes are formed from the macronuclei in emicronucleated strains of Stylonychia contain numerous elements which are much smaller than the chromosomes.—The macronucleus of Stylonychia is very insensitive to irradiation with X-rays.—The results lead to the following hypothesis: The macronuclei of the two hypotrich ciliates contain unconnected chromomeres or small aggregates which are distributed at random to the two daughter nuclei during the divisions.Research supported by the Deutsche Forschungsgemeinschaft.     

Polytene chromosomes and nucleic acid metabolism during macronuclear development in Euplotes

Polytene chromosomes in two species of Euplotes, E. woodruffi and E. eurystomus, have been described during the macronuclear development following conjugation. In these two species, the giant chromosomes appear briefly in the macronuclear anlagen and disappear completely later. DNA synthesis begins concomitantly with the appearance of the giant chromosomes and reaches a peak at the maximum stage of polyteny. Shortly thereafter DNA begins to break down and the breakdown products leave the macronuclear anlagen, reducing the DNA content in the anlagen to the amount present at the earlier stages of the polytene development of the chromosomes. A later phase of DNA synthesis occurs in the anlagen with the appearance of replication bands comparable to the bands which double the DNA in the somatic macronucleus. These replication bands initiate several rounds of DNA synthesis which finally lead to the development of the vegetative macronucleus. RNA synthesis occurs uniformly on the giant chromosomes and no special RNA producing puffs or other regions are noticed on them.Research supported by American Cancer Society grant E 434 to David M. Prescott and by the Deutsche Forschungsgemeinschaft to Dieter Ammermann.     

Characterization of macronuclear DNA in five species of ciliates

Macronuclear DNA of four hypotrichous and one holotrichous ciliate species was characterized by biochemical techniques. The renaturation kinetics of the macronuclear DNAs of all five species were similar. Repetitive sequences occur only in an amount below 2%. Although the DNA content of the macronuclei of the species differs considerably, the kinetic complexity of the macronuclear DNA is rather uniform (around 3 × 10¹⁰ daltons, i.e., 4–11 x the E. coli genome). Only in the macronuclei of the hypotrichous species the DNA exists as gene-sized fragments.Dedicated to Professor W. Beermann on the occasion of his 60th birthday     

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.     

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.