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.     

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.     

Elimination of DNA sequences during macronuclear differentiation in Tetrahymena thermophila,as detected by in situ hybridization

Previous studies have indicated that certain sequences in the micronuclear genome are absent from the somatic macronucleus of Tetrahymena (Yao and Gorovsky, 1974; Yao and Gall, 1979; Yao, submitted). The present study used in situ hybridization to follow the elimination process during the formation of the new macronucleus. Micronuclear-specific DNA cloned in recombinant plasmids was labelled with ³H and hybridized to cytological preparations of T. thermophila at various stages of conjugation. Despite a smaller size and lower DNA content, the micronucleus has more hybridization than the mature macronucleus. Hybridization initially increased in the anlage (newly developing macronucleus) to reach a maximal level right after the old macronuclei had disappeared. The hybridization in the anlage then decreased to a significant extent prior to the first cell division. The results suggest that the micronuclear-specific sequence is first replicated a few rounds before it is eliminated from the anlage, and the elimination process occurs without nuclear division.     

Nuclear Roles in the Post-Conjugant Development of the Ciliate Euplotes aediculatus. III. Roles of Old Macronuclear Fragments in Nuclear and Cortical Development1

Following conjugation of the hypotrichous ciliate Euplotes aediculatus, the posterior fragments of the old (prezygotic) macronucleus persist until after the first vegetative division. These fragments remain viable during exconjugant development as shown by their ability to regenerate should the cell's new macronucleus be damaged. It thus seemed possible that these parental nuclear fragments might participate in the development of the new macronucleus and/or the crucial post-conjugant cortical reorganization that restores the exconjugant cell's ability to feed. This idea was tested by damaging the posterior fragments with various doses of microbeam ultraviolet (UV) light and assessing the results of such treatment on subsequent cortical and nuclear development. When the posterior fragments of the macronucleus were irradiated at the beginning of cortical morphogenesis, the new macronucleus in 1/3 to 1/2 of the cells assumed a “folded” appearance but did not mature. These cells did not undergo cortical reorganization. Cells irradiated at earlier stages did not detectably develop an oral apparatus; their new macronucleus remained arrested at the spherical anlage stage. The results show that the posterior fragments of the parental macronucleus are necessary for normal nuclear and cortical development. These old nuclear fragments appear to influence the growing macronuclear anlage directly and probably the cortex as well. There also appears to be an information flow from the non-irradiated partner of a persistently joined exconjugant doublet to its irradiated counterpart, enabling normal anlage and cortex development in the irradiated cell.     

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.     

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.     

Death harmony played by nucleus and mitochondria: nuclear apoptosis during conjugation of tetrahymena

Tetrahymena programmed nuclear death or nuclear apoptosis is a unique process during conjugation in which only the parental macronucleus is eliminated from the progeny cytoplasm, and other nuclei such as new micro- and macronuclei are unaffected. The nuclear death process consists of three successive steps: chromatin cleavage into high-molecular mass DNA, oligonucleosomal laddering concomitant with nuclear condensation, and complete degradation of the nuclear DNA. Following the first step of the death process, the parental macronucleus is engulfed by a large autophagosome in which many mitochondria are incorporated. Those sequestered mitochondria simply break down and release endonuclease similar to mammalian endonuclease G that is responsible for the generation of the DNA ladder, leading to the conclusion that mitochondria play a crucial role in the execution of the death program. Thus, the parental macronucleus is subject to final death by autophagy in collaboration with caspase-like enzymes, resulting in the ultimate outcome of nuclear resorption.     

伍氏游仆虫接合过程中老大核后碎块的去向

在伍氏游仆虫(Euplotes woodruffi)接合后体发育过程中,已呈退化状态的老大核后碎块,在细胞第二次形态发生时,逐渐恢复其正常形态结构。T形新大核原基向后延伸而与恢复正常形态的老大核后碎块紧密靠拢。此时在光镜下观察,很容易误认为二者已融合为一。但在接合后体分裂之前,老大核后碎块再次瓦解,T形大核原基缩短成棒状而与老大核后碎块分开,此时二者界限分明。细胞分裂后,残存的老大核后碎块停留于后子虫中,最后被吸收。几个关键时期大核原基和老大核后碎块DNA含量的测定,也证明新老大核不融合。本文还讨论了老大核后碎块在有性过程中的功能。     

Site-specific methylation of adenine in the nuclear genome of a eucaryote, Tetrahymena thermophila.

DNA in the polyploid macronucleus of the ciliated protozoan Tetrahymena thermophila contains the modified base N6-methyladenine. We identified two GATC sites which are methylated in most or all of the 45 copies of the macronuclear genome. One site is 2 kilobases 5' to the histone H4-I gene, and the other is 5 kilobases 3' to the 73-kilodalton heat shock protein gene. These sites are de novo methylated between 10 and 16 h after initiation of conjugation, during macronuclear anlage development. The methylation states of these two GATC sites and four other unmethylated GATC sites do not change in the DNA of cells cultured under conditions which change the activity of the genes, including logarithmic growth, starvation, and heat shock.     

Death Harmony Played by Nucleus and Mitochondria: Nuclear Apoptosis During Conjugation of Tetrahymena

Tetrahymena programmed nuclear death or nuclear apoptosis is a unique process during conjugation in which only the parental macronucleus is eliminated from the progeny cytoplasm, and other nuclei such as new micro- and macronuclei are unaffected. The nuclear death process consists of three successive steps: chromatin cleavage into high-molecular mass DNA, oligonucleosomal laddering concomitant with nuclear condensation, and complete degradation of the nuclear DNA. Following the first step of the death process, the parental macronucleus is engulfed by a large autophagosome in which many mitochondria are incorporated. Those sequestered mitochondria simply break down and release endonuclease similar to mammalian endonuclease G that is responsible for the generation of the DNA ladder, leading to the conclusion that mitochondria play a crucial role in the execution of the death program. Thus, the parental macronucleus is subject to final death by autophagy in collaboration with caspase-like enzymes, resulting in the ultimate outcome of the nuclear resorption.     

Nuclear Behavior of Euplotes woodruffi During Conjugation

SYNOPSIS. During conjugation of E. woodruffi , the micro-nucleus divides repeatedly four times prior to synkaryon formation and twice thereafter. The first division resembles an ordinary somatic mitosis, resulting in the formation of two daughter nuclei in each conjugant. Both products of this division enter the second division which corresponds to the heterotypic division of other ciliates, characterized by a parachute stage. Following this stage sixteen bivalents appear and separate into dyads and pass to the poles. During the following divisions individualized chromosomes do not appear but only certain chromatin elements comparable to those seen in the somatic and preliminary divisions. These divide and pass to the poles. All daughter nuclei of the second division enter and complete the third division. Only two of the products of the third division enter the final pregamic division while the rest degenerate. Exchange of pronuclei and their fusion leads to synkaryon formation. The conjugants then separate and in each exconjugant the synkaryon divides twice in rapid succession. Of the four products one condenses to become the functional micronucleus, another enlarges rapidly to become the macronuclear anlage while the remaining two degenerate and disintegrate. The old macronucleus breaks into irregular and polymorphic bodies. As the macronuclear anlage enlarges the remnants of the old macronucleus reorganize and fuse with the macronuclear anlage to form a characteristic vegetative macronucleus.     

抗沉默因子Asf1调控嗜热四膜虫细胞核的稳定性

组蛋白H3/H4的分子伴侣Asf1(anti-silencing factor 1),参与依赖DNA复制及不依赖DNA复制的核小体装配,同时参与转录调控、基因沉默以及DNA损伤修复等过程. 在不同生物中,Asf1具有功能的保守性和多样性．嗜热四膜虫ASF1（TTHERM_00442300）基因编码的蛋白质含有保守的N端结构域和酸性的C端结构域．N端结构域同源序列进化树分析表明,Asf1进化与物种进化一致．实时荧光定量PCR表明,ASF1在四膜虫营养生长、饥饿及有性生殖时期均有表达,且在有性生殖4～6 h转录水平达到最高．免疫荧光定位分析表明,HA-Asf1在营养生长时期以及有性生长时期定位于功能大核和小核中,而在凋亡的大核中信号消失．过表达ASF1导致大核及小核变大,抑制细胞增殖．敲减ASF1后会导致大核形态异常,小核缺失．结果表明,ASF1表达对细胞核的形态和结构维持发挥重要的调控作用．     

Conjugation-Specific Genes in the Ciliate Euplotes crassus: Gene Expression from the Old Macronucleus

ABSTRACT. Following mating or conjugation, the hypotrichous ciliate Euplotes crassus undergoes a massive genome reorganization process. While the nature of the rearrangement events has been well studied, little is known concerning proteins that carry out such processes. As a means of identifying such proteins, differential screening of a developmental cDNA library, as well as construction of a cDNA subtraction library, was used to isolate genes expressed only during sexual reproduction. Five different conjugation-specific genes have been identified that are maximally expressed early in conjugation, during the period of micronuclear meiosis, which is just prior to macronuclear development and the DNA rearrangement process. All five genes are retained in the mature macronucleus. Micronuclear, macronuclear, and cDNA clones of one gene ( conZ47 ) have been sequenced, and the results indicate that the gene encodes a putative DNA binding protein. In addition, the presence of an internal eliminated sequence in the micronuclear copy of the conZ47 gene indicates that this conjugation-specific gene is transcribed from the old macronucleus.     

Conjugation in the spirotrich ciliate Halteria grandinella (Müller, 1773) Dujardin, 1841 (Protozoa, Ciliophora) and its phylogenetic implications

The conjugation of Halteria grandinella was studied in protargol preparations. The isogamontic conjugants fuse partially with their ventral sides to a homopolar pair. The first maturation division generates dramatic transformations: (i) the partners obtain an interlocking arrangement; (ii) the number of bristle kineties decreases from seven to four in each partner; and (iii) the right conjugant loses its buccal membranelles, the left the whole adoral zone. The remaining collar membranelles arrange around the pair's anterior end and are shared by both partners; finally, the couple resembles a vegetative specimen in size and outline. The vegetative macronucleus fragments before pycnosis. The micronucleus performs three maturation divisions, but only one derivative each performs the second and third division. The synkaryon divides twice, producing a micronucleus, a macronucleus anlage, and two disintegrating derivatives. Scattered somatic kinetids occur during conjugation, but disappear without reorganization. An incomplete oral primordium originates in both partners. The conjugation of Halteria grandinella resembles in several respects that of hypotrich spirotrichs; however, the majority of morphological, ontogenetical, and ultrastructural features still indicates an affiliation with the oligotrich and choreotrich spirotrichs. Accordingly, the cladistic analysis still contradicts the genealogy based on the sequences of the small subunit rRNA gene.     

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.     

Observations on the Life-cycle of a New Race of Blepharisma undulans from India

SYNOPSIS. A full account of the nuclear changes during binary fission and conjugation in a local race of Blepharisma is presented in this paper. The macronucleus consists of 2 nodes connected by a strand. Number of micronuclei varies from 6 to 18. During binary fission, condensation of macronucleus is followed by elongation and thinning of the middle region which finally breaks. Daughter nuclei later attain the typical vegetative form. Notably, during binary fission some micronuclei appear to complete their mitoses by the time the macronucleus attains the condensed form, while others lag behind and exhibit practically every stage of mitosis.
During conjugation, from 6 to 10 micronuclei undergo the first pregamic division, the same number through the second division, and two products of the second division take part in the third division. The rest degenerate. Division products of the nuclei in the paraoral region take part in synkaryon formation. The synkaryon undergoes either 2 or 3 divisions. In the former case, of the 4 products, 2 become the macronuclear anlagen, one the micronucleus and the fourth degenerates. In the latter case, of the 8 products, 3 to 4 become the macronuclear anlagen and the rest become micronuclei. Chromatin elimination has been observed during the division of the macronuclear anlage, followed by an extra metagamic fission of the cell.
Comparison with two other races from India and an American race indicates considerable diversity in the structure and behaviour of the nuclear apparatus in different races of Blepharisma undulans.     

An essential role for the DNA breakage-repair protein Ku80 in programmed DNA rearrangements in Tetrahymena thermophila

Programmed DNA rearrangements are important processes present in many organisms. In the ciliated protozoan Tetrahymena thermophila, DNA rearrangements occur during the sexual conjugation process and lead to the deletion of thousands of specific DNA segments and fragmentation of the chromosomes. In this study, we found that the Ku80 homologue, a conserved component of the nonhomologous end-joining process of DNA repair, was essential for these two processes. During conjugation, TKU80 was highly expressed and localized to the new macronucleus, where DNA rearrangements occur. Homokaryon TKU80-knockout mutants are unable to complete conjugation and produce progeny and are arrested at the two-micronuclei/two-macronuclei stage. Analysis of their DNA revealed failure to complete DNA deletion. However, the DNA-cutting step appeared to have occurred, as evidenced by the presence of circularized excised DNA. Moreover, chromosome breakage or de novo telomere addition was affected. The mutant appears to accumulate free DNA ends detectable by terminal deoxynucleotidyl transferase dUTP nick end labeling assays that led to the degradation of most DNA in the developing macronucleus. These findings suggest that Tku80p may serve an end-protective role after DNA cleavage has occurred. Unexpectedly, the large heterochromatin structures that normally associate with DNA rearrangements failed to form without TKU80. Together the results suggest multiple roles for Tku80p and indicate that a Ku-dependent DNA-repair pathway is involved in programmed DNA rearrangements in Tetrahymena.