Amitotic division of the macronucleus in Tetrahymena thermophila: DNA distribution by genomic unit

The macronucleus of the ciliate Tetrahymena cell contains euchromatin and numerous heterochromatins called chromatin bodies. During cell division, a chromatin aggregate larger than chromatin body appears in the macronucleus. We observed chromatin aggregates in the dividing macronucleus in a living T. thermophila cell, and found that these were globular in morphology and homogeneous in size. To observe globular chromatin clearly, optimal conditions for making it compact were studied. Addition of Mg ion, benomyl and oryzalin, microtubule inhibitors, to cell suspension was effective. Globular chromatin appeared when the micronuclear anaphase began at the cell cortex, and disappeared long after cell separation. Using living cells with a small macronucleus at early log phase, we counted the number of globular chromatin per nucleus and measured the DNA content of globular chromatin in the macronucleus which was stained with Hoechst 33342 by using ImageJ. The number of globular chromatin per nucleus was reduced by half after division, indicating the globular chromatin is a distribution unit of DNA. A globular chromatin contained similar DNA content as that of the macronuclear genome. We developed methods for inducing and isolating a cell with an extremely small macronucleus with a DNA amount of one globular chromatin. These cells grew, divided, and give clones, suggesting that the macronuclear genome is not dispersed within the macronucleus and the globular chromatin may be a macronuclear genome. We named this globular chromatin "macronuclear genome unit" (MGU).     

Localization of microtubules during macronuclear division in Tetrahymena and possible involvement of macronuclear microtubules in 'amitotic' chromatin distribution

The ciliated protozoa Tetrahymena contains two nuclei, a micronucleus and a macronucleus. In the vegetatively growing cell, the macronucleus divides amitotic while the micronucleus divides by mitosis. It has been indicated that microtubules are involved in macronuclear division and microtubules are observed to exist in the dividing macronucleus. To clarify the localization and the organization of microtubules in the amitotic dividing macronuclei, we used immunofluorescent staining technique. The microtubules were observed in the cytoplasm and macronucleus. The microtubules were organized and dynamically changed their distribution throughout the macronuclear division. We suggest a possibility that these microtubules are involved in 'amitotic' distribution of chromatin throughout the macronuclear division.     

The Nuclear Apparatus of the Ditransversal Ciliate Homalozoon vermiculare (Ciliophora,Rhabdophora) during Interphase and Division. I. The Macronucleus1

ABSTRACT. The nuclear apparatus of Homalozoon vermiculare consists of a single moniliform macronucleus and about 25 micronuclei. The number of macronuclear segments depends (i) on the number of divisions of individual segments during the interphase and (ii) on the number of segments that arise prior to cytokinesis from the (temporary) filiform macronucleus. Precytokinetic changes of the macronucleus involve the fusion of individual segments followed by contraction and subsequent elongation of the entire macronucleus. The chromatin bodies uncoil into fine fibrils during macronuclear contraction. At the time when the division furrow appears, the macronucleus starts to renodulate. The interphase segment contains a more or less reticulated chromatin body partly attached to the nuclear envelope and about 30 polymorphous nucleoli. The latter consist of the pars granulosa, the pars fibrosa, and an additional fibrillar component. The nucleoli undergo drastic changes prior to division and the granular component disappears completely during macronuclear condensation. On the average, the macronucleus contains a 3,400-fold amount of DNA compared with a haploid micronucleus, but the intraspecific differences in the DNA content of the entire macronucleus are extremely large. In contrast, DNA content and size of an individual segment of the macronucleus are precisely regulated during interphase.     

Electron microscopic studies on the macronuclear development in the ciliate Chilodonella uncinata.

The development of macronuclear anlage in the ciliate Chilodonella uncinata has been studied through electron microscopy. The ultrastructure of macro- and micronuclei is described for comparison. During the first stage of development, when the DNA content of the macronuclear anage increases from 2 C to 32 C, chromosomes are visible as distinct osmiophilic bodies inside the anlage. At the end of the initial polyploidization phase, the chromosomes despiralize, giving rise to long filamentous structures 25 to 50 nm in diameter. The latter show a singular banding pattern, with dense bands 12 to 25 nm thick alternating regularly with less dense interbands. Such an organization has not yet been observed in any other type of nucleus. These filamentous structures have been interpreted as highly despiralized oligotenic chromosomes. During the final stage of macronuclear development, these structures condense into thin chromatin strands and small dense granules; the number of granules increases progressively as the chromatin strands disappear. These small granules very likely fuse amongst themselves to form the chromatin granules of the vegetative macronucleus. No evidence has yet been found for a fragmentation of chromatin in this ciliate, but this problem needs further study. The old macronucleus maintains a normal ultrastructure until a late stage of development of the macronuclear anlage, becoming pycnotic only towards the very end of the latter process.     

Replicon size and rate of DNA replication in the macronucleus of Tetrahymena pyriformis]

The size of replication units (or replicons) measured in Tetrahymena pyriformis GL macronuclear DNA reaches 20--30 microns, according to the two independent methods: DNA fiber autoradiography, and alkaline isokinetic sucrose gradient centrifugation. The synthesis of new DNA fragments--replicons and their subsequent assembly are separated by time intervals (30 min). The rate of DNA synthesis for one fork averaged 0.6--0.7 microns/min. These data were obtained for cells of cultures being both in the expotential phase of growth, and those synchronized by starvation-refeeding. The generation time of T. pyriformis cells, calculated by the increase of the part of labeled nuclei, is almost 2 hours; the synthesis lasts 1 hour. Total amount of replication units in polyploid (polygenomic) Tetrahymena macronucleus is about 3000. Their initiation during S-period is presumably asynchronous.     

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.     

Reorganization of microtubules in the amitotically dividing macronucleus of tetrahymena

We developed a modified immunofluorescence protocol that permitted visualization of microtubules inside the macronucleus of the ciliate Tetrahymena. Although the amitotically dividing macronucleus lacks a spindle, an elaborate system of microtubules is assembled inside the macronucleus and between the macronucleus and the cortex. Microtubules could not be detected inside the interphase macronuclei. The early stage of macronuclear division was associated with the assembly of short macronuclear microtubules that localized randomly. The intramacronuclear microtubules were subsequently organized in a radial manner. During elongation of the macronucleus, the distribution of macronuclear microtubules changed from radial to parallel. During constriction of the macronucleus, dense and tangled macronuclear microtubules were detected at the region of nuclear constriction. In the cytosol, microtubules were linking the macronucleus and cell cortex. During recovery after drug-induced depolymerization, microtubules reassembled at multiple foci inside the macronucleus in close proximity to the chromatin. We propose that these microtubules play roles in chromatin partitioning, macronuclear constriction, and positioning of the macronucleus in relation to the cell cortex.     

Evidence for Chromosomal Macronuclear Substructures in Tetrahymena*†

SYNOPSIS.
Under the growth conditions employed, the G₁ macronucleus of Tetrahymena pyriformis HSM contains 7.4 × 10^-12 g DNA, the G₂ micronucleus 0.42 × 10^-12 g. DNA content from the Tetrahymena thermophila macronucleus did not significantly differ from that of HSM, but the micronucleus contained about twice as much DNA as the micronucleus of the HSM cells. The T. thermophila macronucleus contained on average enough DNA for ˜ 35 haploid micronuclear copies. A new spreading technic allowed separation of macronuclear substructures from cells of late G₂ to early G₁. Photometric determination of DNA content of 345 individual structures suggested the existence of 5 different-sized macronuclear structures with a DNA content corresponding to 2, 4, 8, and 16 × the basic values. Comparison of the DNA content of these structures with (a) mitotic micronuclear chromosomes and (b) meiotic micronuclear chromosomes of T. thermophila cells suggests that the 5 basic values of macronuclear structures derive from structures of micronuclear chromosomes. The micronuclear chromosomes of T. pyriformis may be oligotenic. It is suggested that these results further our understanding of macronuclear organization.     

Microtubular Structures in Macronuclei of Synchronously Dividing Tetrahymena pyriformis

SYNOPSIS. When heat-synchronized cultures of Tetrahymena pyriformis , amicronucleate strain GL, were examined by electron microscopy, intramacronuclear microtubules were observed in dividing cells. These tubules have a diameter of 180–230 A and occur either singly or packed together in bundles. They are predominantly associated with outpocketings and invaginations of the nucleus. Sections as well as negatively stained preparations of isolated macronuclear envelopes indicate that the microtubules are inserted at the inner nuclear membrane.
The findings suggest that microtubules of the spindle type participate in the process of macronuclear division.     

Cytochemical studies on the problem of macronuclear subnuclei in tetrahymena

DNA amounts in macronuclei and micronuclei of Tetrahymena pyriformis were measured by Feulgen microspectrophotometry. Assuming that the unreplicated micronucleus is diploid, the unreplicated macronucleus was found to contain approximately 45 times the haploid DNA amount. The relationship of these findings to the 45 independently assorting genetic subunits characterized by Allen and Nanney and their collaborators is discussed. The pattern of DNA synthesis in macro- and micronuclei during the cell cycle is also described.     

Chromatin structure in somatic nucleus of ciliate <Emphasis Type="Italic">Didinium nasutum</Emphasis>

The structural organization of macronuclear chromatin of the ciliate Didinium nasutum was studied. The macronuclear genome of D. nasutum is represented by DNA molecules of subchromosomal size. At interphase, macronuclear chromatin is organized into chromatin of 100–200-nm clumps. Some of these clumps form short, thick fibers that consist of several chromatin clumps. Using the differential staining of nucleic acids on ultrathin sections, we revealed perichromatin fibers and granules on the surface of many chromatin clumps. A 3D model of the spatial distribution of chromatin clumps in the macronucleus was built based on serial ultrathin sections and peculiar features of chromatin spatial organization were studied.     

The effect of trifluoroperazine on microtubules,nuclear division and the nuclear membranes of the ciliate Nyctotherus ovalis

Exposure of the dividing ciliate Nyctotherus ovalis to the tranquilizer trifluoroperazine (TFP; 10 M) leads to the complete disassembly of kinetochore microtubules in the metaphase micronucleus. Interpolar microtubules located underneath the micronuclear envelope at anaphase and telophase stembody microtubules are more resistant to TFP. However, stembodies of drug-exposed ciliates are much shorter than in the controls. In their centre they contain only a reduced number of widely separated microtubules, indicating that assembly of new tubules or elongation of existing microtubules at this site, which appears essential for further separation of the future daughter nuclei, is blocked by TFP. Although microtubules polymerized in the macronucleus during its elongation include a set of tubules made up of more than 13 protofilaments, comparable to the micronuclear stembody microtubules, they are much more sensitive towards drug treatment. Macronuclear tubules become completely depolymerized resulting in failure of nuclear stretching. Already elongated macronuclei can still become constricted in their centre which suggests that microtubules are not involved in this process. Disassembly and higher sensitivity of macronuclear compared with micronuclear microtubules may be explained by a different composition and behaviour of nuclear membranes towards TFP in the two types of nuclei. While the micronuclear envelope may be only partially destroyed where it is facing the macronucleus, the inner membrane of the macronuclear envelope is severely affected by drug treatment. It shows a multitude of infoldings accompanied by attachment of chromatin to it. Cytoplasmic microtubules which proved resistant to other depolymerizing drugs become partly disassembled during TFP treatment.     

SIMULTANEOUS SYNTHESIS OF HISTONE AND DNA IN SYNCHRONOUSLY DIVIDING TETRAHYMENA PYRIFORMIS

Histone and DNA syntheses have been studied in synchronously dividing Tetrahymena pyriformis GL. During the heat treatment necessary to synchronize cultures of this amicronucleate protozoan, the DNA content of the already polyploid macronucleus increases. When the cells begin synchronous division, their DNA content is reduced in a stepwise process which is closely paralleled by reduction of macronuclear histone content. During cell division, the contents of DNA and histone decrease by slightly more than twofold, and in the subsequent S phase, DNA and histone increase simultaneously to 85% of the values expected if all chromosomes were to double. The first step in the process of reduction of DNA and histone contents is their decrease in excess of twofold, and this is accomplished by removal of extrusion bodies from the nuclei of dividing cells. The second step is a mechanism which allows, in effect, only 70% of the chromatin in the average nucleus to duplicate. Such partial duplication suggests that both histone and DNA syntheses in synchronous Tetrahymena depend upon a regulatory mechanism, the mediating elements of which are localized in only certain chromosomes.     

A development-specific histone H3 localizes to the developing macronucleus of Euplotes

During the process of macronuclear development, the ciliate Euplotes crassus undergoes extensive programmed DNA rearrangement. Previous studies have identified a gene, H3(P), that is expressed only during sexual reproduction and is predicted to encode a variant histone H3 protein. In the current study, an antiserum to the H3(P) protein has been generated. The antiserum has been used to demonstrate that H3(P) is maximally expressed during the polytene chromosome stage of macronuclear development. Moreover, H3(P) is localized to the developing macronucleus, but not other nuclei present within the cell. Additional studies indicate that at least one additional variant histone is also present within the developing macronucleus. The results indicate that there are significant changes in nucleosome composition within the developing macronucleus, and provide additional support for the notion that changes in chromatin structure play a role in the DNA rearrangement processes of macronuclear development. genesis 26:179-188, 2000.     

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.     

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。     

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 chromatin of Blepharisma japonicum compared to that of Tetrahymena pyriformis

Abstract The macronuclear chromatin of the ciliate Blepharisma japonicum was studied by electrophoretic and immunological techniques as well as by micrococcal nuclease digestion and circular dichroism spectroscopy. Under these experimental conditions the macronuclear chromatin of B. japonicum was compared to that of Tetrahymena pyriformis . Data obtained through this analysis showed that the macronuclear chromatin of B. japonicum is structurally more relaxed than that of T. pyriformis . A perchloric acid soluble polypeptide, referred to as P3, is the only polypeptide of B. japonicum chromatin to appear immunologically related to the H1 histone of T. pyriformis . It is suggested that this P3 polypeptide in B. japonicum should be considered functionally equivalent to the T. pyriformis H1 histone, even though it differed from it both in terms of molecular mass and relative concentration.     

Fine structure and cytochemistry of the nuclei of the primitive ciliateTracheloraphis crassus (Karyorelictida)

Summary The nuclei ofTracheloraphis crassus were studied using light and electron microscopy combined with Bernhard's RNP staining and pronase digestion. The nuclear apparatus of this species consists of a longitudinal row of 11–43 macronuclei and 4–16 micronuclei. Like in all karyorelictids, the macronuclei are unable to divide and become segregated during cytokinesis; their number is supplemented in every cell cycle by differentiation of several new macronuclei from micronuclei.Each adult macronucleus contains a single compact endonuclear aggregate of several large chromocenters, readily destained with EDTA, and several RNP containing nucleoli. There is continuity between the material of the chromocenters and the decondensed DNP fibrils in the nuclear matrix. The nucleoli contain NORs in the form of fibrillar centers. The endonuclear aggregate includes also groups of RNP granules which are especially resistant to EDTA destaining. A microfibrillar sphere, usually localized at the periphery of the aggregate, contacts one or several nucleoli. The sphere is not bleached with EDTA, and only its periphery becomes digested with pronase. The macronuclear matrix consists of both protein fibrils and pronase-resistant fibrils, the latter being localized at the nuclear periphery.Developing macronuclear primordia contain loose strands of decondensed chromatin; only later they form chromocenters and nucleoli.The micronuclei reproduce by mitosis with typical chromosomes (2n=66). During interphase, they are filled with condensed chromatin which can be bleached with EDTA; they form no nucleoli. Ring-like lamellae, existing in the cavities of the chromatin mass, stain for RNA (after Bernhard) and are pronase-sensitive. These lamellae resemble the kinetochore material conserved during interphase in another karyorelictid ciliate,Trachelocerca geopetiti.     

The DNA of ciliated protozoa.

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.