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At least 19 recordsLinked to original sources
PubMed1996

Macronucleus structure and macronucleus development in hypotrichous ciliates.

In the course of macronuclear development of the hypotrichous ciliates all genetic information not required for normal growth of the cell is removed from the new macronucleus. This differentiation process involves DNA-splicing, excision of transposons, DNA-fragmentation, selective gene amplification and telomere addition. Since many of the processes observed during macronuclear development, such as DNA-transposition, DNA-rearrangement or selective DNA-amplification, may occur in differentiating cells of higher organisms, this biological system provides an unusual opportunity to study the ways in which DNA-sequences can be manipulated in a differentiating cell.

Animals↗
PubMed2000

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.

Animals↗
PubMed2001

Lysosomal enzymes in the macronucleus of Tetrahymena during its apoptosis-like degradation.

A key characteristic of apoptosis is its regulated nuclear degradation. Apoptosis-like nuclear degradation also occurs in the ciliated unicellular organism, Tetrahymena thermophila. Chromatin of the macronucleus undergoes massive condensation, a process that can be blocked by caspase inhibitors. The nucleus becomes TUNEL-positive, and its DNA is cleaved into nucleosome-sized fragments. In a matter of hours the macronucleus is completely degraded, and disappears. The condensed nucleus sequesters acridine orange, which means that it might become an acidic compartment. We therefore asked whether lysosomal bodies fuse with the condensed macronucleus to form an autophagosome. We monitored acid phosphatase (AP) activity, which is associated with lysosomal bodies but is not found in normal nuclei. We find that after the macronucleus condenses AP activity is localized in cap-like structures at its cortex. Later, after the degrading macronucleus loses much of its DNA, acid phosphatase deposits appear deeper within the nucleus. We conclude that although macronuclear elimination is initiated by an apoptosis-like mechanism, its final degradation may be achieved through autophagosomy.

Acid Phosphatase↗
PubMed1992

Behavior of germinal micronuclei under control of the somatic macronucleus during conjugation in Paramecium caudatum.

In conjugating pairs of Paramecium caudatum, the micronuclear events occur synchronously in both members of the pair. To find out whether micronuclear behavior is controlled by the somatic macronucleus or by the germinal micronucleus, and whether or not synchronization of micronuclear behavior is due to intercellular communication between conjugating cells, the behavior of the micronucleus was examined after removal of the macronuclei from either or both cells of a mating pair at various stages of conjugation. When macronuclei were removed from both cells of a pair, micronuclear development was arrested 1 to 1.5 hr after macronuclear removal. When the macronucleus of a micronucleate cell mating with an amicronucleate cell was removed later than 3 to 3.5 hr of conjugation, that is, an early stage of meiotic prophase of the micronucleus, micronuclear events occurred normally in the operated cell. These results suggest that most micronuclear events are under the control of the macronucleus and that the gene products provided by the macronucleus are transferable between mating cells. One such product is required for induction of micronuclear division and is provided just before metaphase of the first meiotic division of the micronucleus. This factor is effective at a lower concentration in the cytoplasm and/or is more transferable between mating cells than the factors required for other stages. This factor, which seems to be present at least until the stage of micronuclear disintegration, is able to induce repeated micronuclear division as long as it remains active. The factor can act on a micronucleus which has not passed through a meiotic prophase. Moreover, the results suggest the existence of a second factor which is provided by the macronucleus after the first meiotic division that inhibits further micronuclear division.

Animals↗
PubMedDate not supplied

Clonal aging in Paramecium tetraurelia. II. Evidence of functional changes in the macronucleus with age.

The contribution of the macronucleus of Paramecium tetraurelia to the long term proliferation potential of a cell line was tested using a nuclear transplantation protocol. Macronuclei from young or old wild-type cells were injected into genetically marked host cells of a standard clonal age. The subsequent proliferation to clonal death of successfully and stably transformed hybrids was compared to the proliferation of injected but untransformed lines (injection controls). Young macronuclear donor material significantly prolonged the proliferation of the hybrid cell lines over that of the injection controls, but old donor material caused only a slight increase in post-injection proliferation of the hybrids. Total cell proliferation was also considered. Comparison of the total life spans of injected, non-transformed lines with uninjected host controls demonstrates that the injection itself has no significant effect upon proliferation potential. The mean life spans of uninjected donor controls and the mean total life spans of the transplanted macronuclear material (donor age at injection plus subsequent hybrid proliferation) are similar, regardless of the age of the donor at the time of transplantation. These results suggest that there is an age-related decrease in the ability of a macronucleus to support subsequent cell growth and division. The results also show that the proliferation potential of the donor macronucleus does not appear to be changed to any great degree by transplantation into a host cell of different clonal age. The macronucleus thus "remembers its age" after transplantation. Coupled with an absence of any detectable cytoplasmic effects upon aging during vegetative growth, these results argue in favor of a macronuclear determination of the proliferation potential of a cell line. The identification of a macronuclear basis for clonal aging in P. tetraurelia should permit a better directed approach for further research in this area.

Cell Division↗
PubMed2003

De novo cytosine methylation in the differentiating macronucleus of the stichotrichous ciliate Stylonychia lemnae.

Dramatic DNA reorganization and elimination processes occur during macronuclear differentiation in ciliates. In this study we analyzed whether cytosine methylation of specific sequences plays a functional role during DNA rearrangement. Three classes of sequences, macronuclear-destined sequences (MDSs, pCE7), members from a large family of transposon-like elements and micronuclear-specific sequences (pLJ01), differing in their structure and future destiny during nuclear differentiation, were studied in the micronucleus, the developing macronucleus and, when present, in the mature macronucleus. While the MDSs become processed to a 1.1 and 1.3 kb gene-sized macronuclear DNA molecule, the family of transposon-like elements represented by MaA81 becomes removed late in the course of polytene chromosome formation. The micronuclear-specific sequence pLJ01 is eliminated together with bulk micronuclear DNA during degradation of polytene chromosomes. No methylated cytosine could be detected in the vegetative macronucleus and no difference in methylation pattern was observed either between micronucleus and developing macronucleus in MDSs or in a micronuclear-specific sequence. However, a significant percentage of the cytosines contained in the transposon-like element becomes methylated de novo in the course of macronuclear differentiation. This is the first demonstration that cytosine methylation in specific sequences occurs during macronuclear differentiation and may provide a first step towards understanding epigenetic factors involved in DNA processing.

Animals↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed1992

Interstitial telomeres are hotspots for illegitimate recombination with DNA molecules injected into the macronucleus of Paramecium primaurelia.

DNA molecules injected into the macronucleus of Paramecium primaurelia replicate either as free linear telomerized or chromosome integrated molecules. In the present study we show that when a 1.77 kb BamHI DNA fragment harbouring the his3 gene of Saccharomyces cerevisiae was microinjected into the macronucleus, a fraction of the molecules are integrated into the chromosome via an illegitimate recombination process. The injected molecules were mostly inserted at their extremities at multiple points in the genome by replacing the Paramecium sequences. However, insertion sites were not totally at random. Roughly 30% of the molecules were integrated next to or in telomeric repeats. These telomeric repeats were not at the extremities of chromosomes but occupy an internal or interstitial position. We argue that such sites are hotspots for integration as the probability of random insertion near or in an interstitial telomeric site, of which there are 25-60 in a macronucleus is between 5 x 10(-4) and 3 x 10(-5).

Animals↗
PubMed1995

Effects of nullisomic chromosome deficiencies on conjugation events in Tetrahymena thermophila: insufficiency of the parental macronucleus to direct postzygotic development.

Conjugation fails postzygotically after mating of Tetrahymena cells that have wild-type parental macronuclei but harbor noncomplementing nullisomic parental germline deficiencies. Failures begin shortly after formation of the new macronuclear precursor (anlage) and completion of the first step in elimination of the parental macronucleus (pycnosis). Conjugants fail to complete pair separation, to eliminate one new micronucleus, and to amplify anlage DNA, and they eventually die. Some deficiencies block resorption of the pycnotic parental macronucleus, but we find no evidence for its regeneration. Some deficiencies cause aberrant anlage DNA loss. Those that do not cause DNA loss are epistatic to those that do, indicating that normal anlage development requires the dependent function of at least two types of genes. The possibility that these genes are involved in developmentally regulated anlage DNA rearrangements is discussed. Each observed conjugation defect indicates insufficiency of the parental macronucleus to direct postzygotic development and can be explained by the deficiency of essential conjugation genes that are expressed from the anlage. The failure of nullisomic conjugants to complete pair separation indicates a requirement for gene products, expressed from the early anlage or its precursors, soon after anlage first differentiate.

Animals↗
PubMed1985

Infection and maintenance of Holospora obtusa, a macronucleus-specific bacterium of the ciliate Paramecium caudatum.

The gram-negative bacterium Holospora obtusa is a macronucleus-specific symbiont of the ciliate Paramecium caudatum, which invades the host cell via a food vacuole, infects its macronucleus and grows exclusively in the nucleus. From infection experiments, we showed that a property of the macronucleus that is necessary for it to be recognized and infected by H. obtusa is commonly provided by P. caudatum, P. multimicronucleatum and 14 species of the P. aurelia complex, but not by P. jenningsi, P. bursaria, P. trichium, P. duboscqui, Didinium nasutum, Blepharisma japonicum, Pseudourostyla levis, seven species of Euplotes or Tetrahymena thermophila. Furthermore, it was also shown that the bacteria that infect the macronuclei of P. multimicronucleatum and the P. aurelia species complex always disappear from the nuclei within 5 days and the infected bacteria are maintained stably in the host nuclei in only 13 out of 22 strains of P. caudatum. The results indicate that the species specificity of the habitat of H. obtusa is not simply a matter of its ability to penetrate the host nuclear membrane but depends on unknown factors that exist only in certain strains of P. caudatum.

Bacteriological Techniques↗
PubMed1955

Observations on the fine structure of the macronucleus of Tokophrya infusionum.

The macronucleus in Tokophrya infusionum is composed of numerous Feulgen-positive chromatin bodies (about 0.5 micro in diameter) which appear in thin sections as a dense spongework, homogeneous throughout. The same appearance characterizes metaphase chromosomes of higher forms. Some chromatin bodies of the macronucleus were found to possess a highly organized structure in certain old organisms. This structure appears in cross-sections as a honeycomb and in longitudinal sections as parallel lines about 120 A in diameter evenly spaced (about 230 A). As far as is known this is the first time a regular structure has been found in bodies of chromosomal character at the dimensional level presently explored by electron microscopy. The demonstration that OsO(4) can preserve order in chromatin material is another significant aspect of these findings.

Cell Nucleus↗
PubMed1992

DNA elimination and its relation to quantities in the macronucleus of Tetrahymena.

The macronucleus of Tetrahymena contains a large number of DNA molecules of subchromosomal size. They belong to about 270 species each one occurring at an average number of 45 copies. Macronuclei divide unequally and nothing is known of segregation control. This and the elimination and degradation of DNA during macronuclear amitosis make the clonal stability of macronuclei a problem of qualitative and quantitative control on a subchromosomal level. We studied the contribution of DNA elimination to the quantitative composition of the macronucleus cytophotometrically in single cells of different strains. This was done under standard conditions and under conditions known to influence the amount of macronuclear DNA. The following results were found: Elimination of DNA occurs at almost every division. The size of the elimination body is highly variable but still positively correlated with the macronuclear DNA content. In T. thermophila the amount of eliminated DNA is 2.5% of the G2 content and is not dependent on the growth state. It varies with species, amounting to as much as 8% in T. pigmentosa. During conditions which increase the macronuclear DNA content, very little DNA is eliminated. On the other hand, large amounts are eliminated under other conditions causing the macronuclear DNA content to decrease. DNA to be eliminated at division is synthesized at the same time as bulk DNA. We developed a computer program which helps us study the effects of DNA elimination and unequal divisions upon the copy numbers of subchromosomal DNA classes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1980

Isolation and mapping of the rRNA genes in the macronucleus of Oxytricha fallax.

The DNA in the macronucleus of the protozoan Oxytricha, unlike like that of typical eukaryotes, exists as short, gene-sized molecules. Within the macronucleus the rRNA genes are contained in molecules 7,380 nucleotide pairs in length. This rDNA has been substantially purified by selective denaturation of non-ribosomal DNA followed by S1 nuclease digestion. Results from restriction nuclease digestion and rRNA:DNA hybridization who that the rDNA is a linear, non-palindromic molecule which contains one gene each for the 19s and 25s rRNAs. A total of less than 600 base pairs of DNA lies between the 19s and 25s genes or at the 3' end of the 25s gene. The non-coding portion of the ribosomal DNA is almost entirely limited to an approximately 1,400 base pair region at the 5' end of the molecule.

Animals↗
PubMed1983

Antibodies against Z DNA react with the macronucleus but not the micronucleus of the hypotrichous ciliate stylonychia mytilus.

Using indirect immunofluorescence, we studied the reaction of antibodies specific for left-handed Z DNA with the nuclei of the hypotrichous ciliate Stylonychia mytilus. In the vegetative cell, the macronucleus reacts strongly with these antibodies, but no reaction can be detected with micronuclei. However, an antibody that binds to denatured and right-handed B DNA reacts with both types of nuclei. No reaction of the anti-Z DNA antibody is seen in the macronuclear replication band. Digestion of macronuclei with DNAase I leads to a decrease in the anti-Z DNA antibody reaction. Some stages of the developing macronucleus were also investigated. No reaction is seen at the polytene chromosome stage, but following DNA elimination the nucleus is seen to react with the antibody.

Animals↗
PubMed1985

Functional intron+ and intron- rDNA in the same macronucleus of the ciliate Tetrahymena pigmentosa.

Diallelic clones of Tetrahymena pigmentosa containing equal amounts of intron+ and intron- rDNA in the macronucleus were constructed. The macronucleus of the resulting strains divides amitotically during vegetative growth and the diallelic genotype is therefore unstable. The coexistence of the two alleles was followed in the total culture and in single cells during their vegetative segregation and it was observed that replication was non-preferential with respect to the two alleles. The diallelic clones were also used to demonstrate that intron-containing rDNA was transcribed and the transcript processed in the presence of corresponding intron- rDNA. The results are discussed in the light of the 'non-function' idea for ribosomal RNA introns.

Alleles↗
PubMed1984

Recombination and assortment in the macronucleus of Tetrahymena thermophila: a theoretical study by computer simulation.

The compound nature of the macronucleus of Tetrahymena thermophila presents multiple opportunities for recombination between genes on the same macronuclear chromosome. Such recombinants should be detectable through their assortment at subsequent amitotic macronuclear divisions. Thus, a macronucleus that is initially AB/ab should produce recombinant assortees of the genotypes Ab/aB. Computer simulation shows that, when the recombination frequency is two or fewer times per cell cycle, recombinant assortees are produced at experimentally measurable frequencies of less than 40%. At higher recombination frequencies, linked genes appear to assort independently. The simulations also show that recombination during macronuclear development can be distinguished from recombination in subsequent cell cycles only if the first appearance of recombinant assortees is 100 or more fissions after conjugation. The use of macronuclear recombination and assortment as a means of mapping macronuclear genes is severely constrained by the large variances in assortment outcomes; with experimentally small sample sizes, such mapping is impossible.

Animals↗
PubMedDate not supplied

Evolution of amitosis of the ciliate macronucleus: gain of the capacity to divide.

Ciliates exhibit nuclear dimorphism, i.e. they have a germline micronucleus and a somatic macronucleus. Macronuclei are differentiated from mitotic sisters of micronuclei. The macronuclei of "higher ciliates" are polyploid and divide acentromerically ("amitotically"); they differentiate once per life cycle. By contrast, Karyorelict (KR) ciliate macronuclei are nearly diploid and cannot divide; they must differentiate at every cell cycle. Diverse lines of evidence are presented to support the hypothesis that ancestral ciliate macronuclei were incapable of division (as in living karyorelict ciliates) and that higher ciliates gained, perhaps independently more than once, the ability to divide the macronucleus. Selective pressures that could have driven the evolution and macronuclear division and two plausible step-wise pathways for the evolution of macronuclear division are proposed. These hypotheses are relevant to our understanding of amitosis mechanisms, evolution of nuclear dimorphism, and phylogenetic classification of ciliates.

Animals↗
PubMedDate not supplied

Conjugation-specific genes in the ciliate Euplotes crassus: gene expression from the old macronucleus.

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 (conZA7) 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 conZA7 gene indicates that this conjugation-specific gene is transcribed from the old macronucleus.

Amino Acid Sequence↗