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 Differentiation in Paramecium caudatum: Analysis by the Monoclonal Antibody against a Micronuclear Antigen

I obtained the monoclonal antibody 93A against a micronuclear antigen of the ciliate Paramecium caudatum . Immunocytochemical observations showed that the antigen 93A appeared in some portion of the micronucleus in every stage of life cycle. In dividing micronuclei, the antigen appeared mainly in their both poles and in fibrous structures between the poles. These results suggest that the micronuclear antigen 93A may be a component of microtubule organizing center and spindles. During nuclear differentiation in P. caudatum , four among eight postzygotic micronuclei differentiate new macronuclear anlagen and one becomes a new micronucleus and the remaining three degenerate. The micronuclear antigen 93A appeared in all of the eight nuclei in the early stage of macronuclear differentiation but then disappeared in the four macronuclear anlagen and eventually persisted only in the new micronucleus, showing that the newly developing macronuclear anlagen lose the micronuclear antigen 93A during their differentiation.     

Nuclear Differentiation in Exconjugants of Paramecium caudatum: Role of Nuclear Division in Differetiation

It has been known that, immediately after the third division of fertilization nucleus (synkaryon), nuclei localized near the posterior region of exconjugant are to be macronuclear anlagen and those near the anterior region are to be presumptive micronuclei in Paramecium caudatum. One of such posterior nuclei was transplanted into amicronucleate cell at vegetative phase in this work. The implanted nuclei were able to divide at every fission. Their DNA content was nearly equal to or less than ordinary micronuclei during vegetative phase. When conjugation was induced between clones obtained and amicronucleates, macronuclear anlagen developed from the division products of implanted nuclei and thereafter derivative caryonides were true to the marker gene of implanted nuclei. The results indicate that there was no intrinsic difference between nuclei localized anteriorly and those situated posteriorly in exconjugant. Differentiation of nuclei into macronucleus may be irreversible at the stage of anteroposterior localization of the nuclei. The role of nuclear division in differentiation may be only to transport the daughter nuclei into the cytoplasm/cortex differentiated anteroposteriorly.     

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.     

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.     

Interactions between newly developed macronuclei and maternal macronuclei in sexually immature multinucleate exconjugants of Paramecium caudatum

The macronucleus of Paramecium caudatum controls most cellular activities, including sexual immaturity after conjugation. Exconjugant cells have two macronuclear forms: (1) fragments of the maternal macronucleus, and (2) the new macronuclei that develop from the division products of a fertilization micronucleus. The fragments are distributed into daughter cells without nuclear division and persist for at least eight cell cycles after conjugation. Conjugation between heterokaryons revealed that the fragmented maternal macronuclei continued to express genetic information for up to eight cell cycles. When the newly developed macronucleus was removed artificially within four cell cycles after conjugation, the clones regenerated the macronuclear fragments (macronuclear regeneration; MR) and showed mating reactivity, because they were sexually mature. However, when the new macronucleus was removed during later stages, many MR clones did not show mating reactivity. In some extreme cases, immaturity continued for more than 50 fissions after conjugation, as seen with normal clones that had new macronuclei derived from a fertilization micronucleus. These results indicate that the immaturity determined by the new macronucleus is not annulled by the regenerated maternal macronucleus. Mature macronuclear fragments may be "reprogrammed" in the presence of the new macronucleus, resulting in their expression of "immaturity."     

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.     

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

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

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.     

A Nuclear Protein Involved in Apoptotic-like DNA Degradation in Stylonychia: Implications for Similar Mechanisms in Differentiating and Starved Cells

Ciliates are unicellular eukaryotic organisms containing two types of nuclei: macronuclei and micronuclei. After the sexual pathway takes place, a new macronucleus is formed from a zygote nucleus, whereas the old macronucleus is degraded and resorbed. In the course of macronuclear differentiation, polytene chromosomes are synthesized that become degraded again after some hours. Most of the DNA is eliminated, and the remaining DNA is fragmented into small DNA molecules that are amplified to a high copy number in the new macronucleus. The protein Pdd1p (programmed DNA degradation protein 1) from Tetrahymena has been shown to be present in macronuclear anlagen in the DNA degradation stage and also in the old macronuclei, which are resorbed during the formation of the new macronucleus. In this study the identification and localization of a Pdd1p homologous protein in Stylonychia (Spdd1p) is described. Spdd1p is localized in the precursor nuclei in the DNA elimination stage and in the old macronuclei during their degradation, but also in macronuclei and micronuclei of starved cells. In all of these nuclei, apoptotic-like DNA breakdown was detected. These data suggest that Spdd1p is a general factor involved in programmed DNA degradation in Stylonychia.     

Characterization of a family of repetitive sequences that is eliminated late during macronuclear development of the hypotrichous ciliate Stylonychia lemnae

A repetitive element from the hypotrichous ciliate Stylonychia lemnae was characterized by restriction and hybridization analysis. This repetitive element is present in about 5,000–7,000 copies per haploid genome in the micronucleus and the macronuclear anlagen. Its DNA sequence is very conserved, but the length of the repetitive sequence blocs is variable. In some cases, it is associated with telomeric sequences and macronucleus–homologous sequences. Restriction analysis of genomic micronuclear and macronuclear anlagen DNA and in situ hybridization showed that the repetitive sequences are amplified during the formation of polytene chromosomes. They are localized in many bands of the polytene chromosomes and are eliminated during the degradation of the polytene chromosomes. Possible functions of the repetitive sequences during macronuclear differentiation are discussed. Dev. Genet. 21:201–211, 1997.© 1997 Wiley-Liss, Inc.     

Delayed degradation of parental macronuclear DNA in programmed nuclear death of Paramecium caudatum

In the ciliated protozoan Paramecium caudatum, a parental macronucleus that is fragmented into some 40-50 pieces during conjugation does not degenerate immediately, but persists until the eighth cell cycle after conjugation. Here we demonstrate that the initiation of the parental macronuclear degeneration occurs at about the fifth cell cycle. The size of parental macronuclear fragments continued to increase between the first and fourth cell cycle, but gradually decreased thereafter. By contrast, a new macronucleus grew and reached a maximum size by the fourth cell cycle, suggesting that the new macronucleus matured by that stage. Southern blot analysis revealed that parental macronuclear DNA was degraded at about the fifth cell cycle. The degradation was supported by acridine orange staining, indicating degeneration of the macronuclear fragments. Prior to the degradation, the fragments once attached to the new macronucleus were subsequently liberated from it. These observations lead us to conclude that once a new macronucleus has been fully formed by the fourth cell cycle, the parental macronuclear fragments are destined to degenerate, probably through direction by new macronucleus. Considering the long persistence of the parental macronucleus during the early cell cycles after conjugation, the macronuclear fragments might function in the maturation of the imperfect new macronucleus. Two possible functions, a gene dosage compensation and adjustment of ploidy level, 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.     

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.     

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.     

Genomic exclusion in Tetrahymena thermophila: A cytogenetic and cytofluorimetric study

Genomic exclusion is an aberrant form of conjugation of Tetrahymena thermophila in which the genome of a defective conjugant is excluded from the genotype of the exconjugant progeny. This paper is concerned with the cytogenetic and nucleocytoplasmic events of genomic exclusion in senescent clones A*III and C*. In crosses between A*III or C* and strain B, functional, haploid gametic nuclei are formed only in the strain B cell. In some instances one of the gametic nuclei divides prior to transfer of the migratory gametic nucleus, and both products then undergo DNA synthesis. Two alternative cytogenetic pathways are followed after transfer of the migratory nucleus. In the first, the conjugants separate without further micronuclear divisions. This pathway was most common in A*III genomic exclusion. In exconjugants the former gametic nuclei undergo both DNA synthesis and (presumably) intranuclear separation of centromeres to restore micronuclear diploidy. The old macronucleus of each exconjugant is retained without autolysis. This class of exconjugant survives and contributes genes to future sexual progeny. In the second cytogenetic pathway the gametic nuclei divide and macronuclear anlagen are formed, as in normal conjugation. This pathway was more common in C* genomic exclusion. The initial DNA content of the anlagen ranges from haploid to diploid. Following two to three rounds of DNA synthesis, further macronuclear development ceases and the anlagen appear to undergo autolysis. The old macronucleus condenses and also undergoes autolysis, as in normal conjugation. Except for rare C* exconjugants, in which macronuclear development is completed, anlagen-bearing genomic exclusion exconjugants die. Death may be caused by aneuploidy, errors in the timing or receptivity to signals for autolysis, or the inability of anlagen-bearing exconjugants to feed. Anlagenbearing conjugants are frequently abnormal with respect to the number of anlagen and micronuclei. Most of the anomalies can be explained by postulating errors in the timing of both developmental signals and nuclear divisions. Rare conjugants in which gametic nuclei divide but do not give rise to macronuclear anlagen are also observed. In these instances, the old macronuclei condense and undergo autolysis. Destruction of the old macronucleus therefore is independent of the presence of macronuclear anlagen and requires cell pairing in order to be initiated.     

COMPARTMENTALIZATION OF THE DEVELOPING MACRONUCLEUS FOLLOWING CONJUGATION IN STYLONYCHIA AND EUPLOTES

The development of the macronucleus following conjugation in the hypotrichous ciliates Euplotes and Stylonychia has been examined with the electron microscope. Banded polytene chromosomes can be seen in thin sections of the macronuclear anlagen during the early periods of exconjugant development. As the chromosomes reach their maximum state of polyteny, sheets of fibrous material appear between the chromosomes and transect the chromosomes in the interband regions. Individual bands of the polytene chromosomes thus appear to be isolated in separate compartments. Subsequently, during the stage when the bulk of the polytenic DNA is degraded (1), these compartments swell, resulting in a nucleus packed with thousands of separate spherical chambers. Individual chromosomes are no longer discernible. The anlagen retain this compartmentalized condition for several hours, at the end of which time aggregates of dense material form within many of the compartments. The partitioning layers disperse shortly before replication bands appear within the elongating anlagen, initiating the second period of DNA synthesis characteristic of macronuclear development in these hypotrichs. The evidence presented here suggests that the "chromatin granules" seen in the mature vegetative macronucleus represent the material of single bands of the polytene chromosomes seen during the earlier stages of macronuclear development. The possibility is also discussed that the degradation of DNA in the polytene chromosomes may be genetically selective, which would result in a somatic macronucleus with a different genetic constitution than that of the micronucleus from which it was derived.