Selective Autolysis of Nuclei as a Source of DNA Precursors in Paramecium aurelia Exconjugants*

The fate of labeled DNA in macronuclear fragments of starving Paramecium aurelia exconjugants was studied by quantitative autoradiography. Labeled material originally contained in DNA of macronuclear fragments is incorporated into macronuclear anlagen. During the starvation period the mean number of macronuclear fragments per cell decreased exponentially while there was an approximately exponential increase in the volume of macronuclear anlagen. Fragments appeared to be selectively and individually autolyzed. Labeled material originally contained in fragments was largely if not completely conserved through 108 hr of starvation during which more than 90% of the fragments were lost. Soluble labeled material was detectable after autolysis of fragments began, but finally almost all labeled material was incorporated into macronuclear anlagen.     

Mutation affecting cell separation and macronuclear resorption during conjugation in Tetrahymena thermophila: Early expression of the zygotic genotype

A new recessive conjugation lethal mutation was found in Tetrahymena thermophila which was named mra for macronuclear resorption arrest. Other events affected by the mra mutations are separation of pairs, DNA replication in the macronuclear anlagen, and resorption of one of the two micronuclei. In wild-type crosses 50% of the pairs had separated by 12 hr after mixing two mating types and had completed resorption of the old macronucleus 1–2 hr later. In contrast most mra conjugants did not separate even by 24 hr after mixing and the old relic (condensed) macronucleus was seen in over 90% of them. After addition of 10mM calcium to the conjugation medium, the mra conjugants did separate but they still failed to complete resorption of the old macronucleus and to replicate macronuclear anlagen DNA in the exconjugants. The calcium induced separation of the mra conjugants occurred later than the separation of control pairs. During normal conjugation cell separation occurs before the first expression of known macronuclear genes and prior to processing of the macro-nuclear DNA. Therefore, the mra phenotype infers that separation of conjugants requires a signal which is produced by the macronuclear anlagen at an unusually early time. © 1992 Wiley-Liss, Inc.     

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.     

Mutation affecting cell separation and macronuclear resorption during conjugation in Tetrahymena thermophila: early expression of the zygotic genotype.

A new recessive conjugation lethal mutation was found in Tetrahymena thermophila which was named mra for macronuclear resorption arrest. Other events affected by the mra mutations are separation of pairs, DNA replication in the macronuclear anlagen, and resorption of one of the two micronuclei. In wild-type crosses 50% of the pairs had separated by 12 hr after mixing two mating types and had completed resorption of the old macronucleus 1-2 hr later. In contrast most mra conjugants did not separate even by 24 hr after mixing and the old relic (condensed) macronucleus was seen in over 90% of them. After addition of 10 mM calcium to the conjugation medium, the mra conjugants did separate but they still failed to complete resorption of the old macronucleus and to replicate macronuclear anlagen DNA in the exconjugants. The calcium induced separation of the mra conjugants occurred later than the separation of control pairs. During normal conjugation cell separation occurs before the first expression of known macronuclear genes and prior to processing of the macronuclear DNA. Therefore, the mra phenotype infers that separation of conjugants requires a signal which is produced by the macronuclear anlagen at an unusually early time.     

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.     

Positional control of nuclear differentiation in paramecium

Nuclear reorganization, which results in the differentiation between macronuclear anlagen and micronuclei during autogamy or conjugation in Paramecium tetraurelia, was compared in wild-type cells and in two mutants, mic44 and kin241, which form abnormal numbers of macronuclear anlagen and micronuclei. Our observations show that all macronuclear anlagen derive from the nuclei positioned at the posterior pole of the cell at the second postzygotic division. This posterior localization is transient and correlated with a marked change in cell shape and decrease of cell length. These results suggest that cytoplasmic or cortical factors precisely located in the posterior pole are essential to trigger macronuclear differentiation and that the control of nuclear positioning is dependent upon precise modifications of cell shape.     

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.     

Effects of K+ and the K+ Ionophore Valinomycin on the Post-Conjugational Nuclear Differentiation of Paramecium caudatum

For determination of the effect of K⁺ on macro- and micronuclear differentiation Paramecium caudatum exconjugants were transferred to medium with various concentrations of Valinomycin and/or K⁺ at the critical stage of nuclear differentiation. The differentiation was not disturbed by transfer to medium containing 1.5 mM to 50 mM KCl. Injection of KCl solution at the critical stage also did not affect differentiation of the macronucleus appreciably. But change of the KCl concentration in the medium at the critical stage interrupted of normal development of the macronucleus.
Macro- and micronuclear differentiations after conjugation are known to be determined by the antero-posterior localization of postzygotic micronuclei. This nuclear localization is achieved by elongation of mitotic spindles and marked shortening of the cell length at the time of micronuclear division. Successive measurements of cell length at 25°C showed that cells began to shorten 1.5 hr after mating-pair separation, reaching to half the initial length about 2.5 hr after the separation, and then returning to recover their initial length within about 50 min. In a solution of K⁺ (50 mM) plus Valinomycin (1μg/ml or more), cell shortening was inhibited. It is not known whether elongation of mitotic spindles at the time of critical nuclear division was disordered by this treatment, but the macronuclear anlagen developed in the treated cells. Thus shortening in the cell length is not indispensable for nuclear differentiation.     

DNA synthesis, methylation and degradation during conjugation in Tetrahymena thermophila.

We have investigated the timing of DNA synthesis, methylation and degradation during macronuclear development in the ciliate, Tetrahymena thermophila. DNA synthesis was first detected in the anlagen early in macronuclear development, but the majority of DNA synthesis occurred later, after pair separation. Anlagen DNA was first detectably methylated at GATC sites 3-5 hours after its synthesis. Once initiated, de novo methylation was rapid and complete, occurring between 13.5 and 15 hours of conjugation. The level of methylation of GATC sites was constant throughout the remainder of conjugation, and was similar to that in mock-conjugated cells. Degradation of DNA in the old macronucleus and DNA synthesis in the anlagen began at about the same time. Upon pair separation, less than 20% of old macronuclear DNA remained. A small percentage of nucleotides prelabeled prior to conjugation were recycled in the developing anlagen.     

Macronuclear DNA organization and transcription in Paramecium primaurelia

Macronuclear DNA was isolated from Paramecium primaurelia, stock 168. Although the macronucleus is polyploid to the extent of 840C, in other respect the DNA appears to be simply organized, having neither satellite sequences nor substantial amounts of intermediately repetitive sequence. The sequence complexity of macronuclear DNA is quite low for a eukaryote cell, being approximately 19 times more complex than the genome of Escherichia coli. In addition, the GC content is low (25%) and the isolated DNA molecules have lengths mostly in the range 0.2–5 μm. In these various respects, the macronuclear DNA of Paramecium is similar to that of other ciliates. A clone of Paramecium cultured under controlled conditions contains polyadenylated RNA sequences which are homologous to 5–8% of the macronuclear DNA. Sequence complexity analysis indicates that the polyadenylated RNA contains two abundance classes of molecules, one present at low frequency and transcribed from approximately 10⁴ genes, the other at 100 times greater concentration and transcribed from about 100 genes. The relevance of these results to the control of gene expression in Paramecium is discussed.     

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.     

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.     

Synthesis of ribosomal DNA in conjugating Tetrahymena.

In the ciliate protozoan Tetrahymena thermophila, a single integrated gene coding for ribosomal RNA in the micronucleus is amplified during the sexual cycle to yield many copies of extrachromosomal palindromic rDNA in the macronucleus. Hybridization of newly synthesized DNA with rRNA has shown that extensive rDNA synthesis takes place early in the sexual cycle of Tetrahymena. The number of genes synthesized during this period is sufficient to account for gene amplification. A later period of rDNA synthesis occurs when new macronuclear anlagen are beginning to develop. This synthesis may represent preferential polyploidization of already amplified rDNA.     

冠突伪尾柱虫(Pseudourostyla cristata)接合生殖的研究 Ⅰ.核器演化

在25℃条件下,冠突伪尾柱虫接合生殖全程历时10天左右。接合生殖过程中的核器演化包括:①数十枚老的大核逐步瓦解。电镜观察表明,老的大核是以一种类似于食物泡消化的方式被吸收的,并在此过程中伴有大量溶酶体出现。②仅8枚左右小核中的一枚参与新核器的发生。首先,位于胞口后部的一枚小核膨大并进行一次预备分裂,接着发生三次成熟分裂。每一接合体内形成一枚雄原核和一枚雌原核。雄原核互向对方迁移并与其雌原核融合成为合子核。合子核分裂两次,四枚子核之一发育为大核原基,另一枚发育为小核原基,其余两枚退化。预备分裂和前两次成熟分裂各自产生的两枚子核中,仅一枚进入下一次分裂,另一枚解体消失。在第一次成熟分裂前期,“降落伞”的形成和发展经历着复杂的结构变化,持续一小时以上。③大核原基经过长时间的发育,伴有多线染色体的形成和解体等一系列变化,方达成熟状态。成熟的大核原基以伸长断裂、分叉断裂和哑铃形缢缩三种方式进行分裂,小核原基亦随之分裂,逐步形成具60枚左右大核、8枚左右小核的正常营养体。其后,大核融合,开始配后第一次无性分裂。值得注意的是,大核原基发育到将成熟时,最初的迹象是染色质向大核原基中央集结成团,染色质团与核膜之间充满着匀质的核液。当中央染色质团伸长时,又将     

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.     

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.     

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.     

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.