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1.
Histone synthesis and deposition into specific classes of nuclei has been investigated in starved and conjugating Tetrahymena. During starvation and early stages of conjugation (between 0 and 5 hr after opposite mating types are mixed), micronuclei selectively lose preexisting micronuclear-specific histones α, β, γ, and H3F. Of these histones, only α appears to accumulate in micronuclear chromatin through active synthesis and deposition during the mating process. Curiously, α is not observed (by stain or label) in young macronuclear anlagen (4C, 10 hr of conjugation). Thus, young macronuclear anlagen are missing all of the histones which are known to be specific to micronuclei of vegetative cells. By 14–16 hr of conjugation, we observe active synthesis and deposition of macronuclear-specific histones, hv1, hv2, and H1, into new macronuclear anlagen (8C). Thus macronuclear differentiation seems well underway by this time of conjugation. It is also in this time period (14–16 hr) that we first detect significant amounts of micronuclear-specific H1-like polypeptides β and γ in micronuclear extracts. These polypeptides do not seem to be synthesized during this period, which suggests that β and γ are derived from a precursor molecule(s). Since these micronuclear-specific histones do not appear in micronuclear chromatin until after other micronuclei have been selected to differentiate as macronuclei, we suspect that micronuclear differentiation is also an important process which occurs in 10–16 hr mating cells. Our results also suggest that proteolytic processing of micronuclear H3S into H3F (which occurs in a cell cycle dependent fashion during vegetative growth) is not operative during most if not all of conjugation. Thus micronuclei of mating cells contain only H3S which also seems consistent with the fact that some micronuclei differentiate into new macronuclei (micronuclear H3S is indistinguishable from macronuclear H3). Interestingly, the only H3 synthesized and deposited into the former macronucleus of mating cells is the relatively minor macronuclear-specific H3-like variant, hv2. These results demonstrate that significant histone rearrangements occur during conjugation in Tetrahymena in a manner consistent with the fact that during conjugation some micronuclei eventually differentiate into new macronuclei. Our results suggest that selective synthesis and deposition of specific histones (and histone variants) plays an important role in the nuclear differentiation process in Tetrahymena. The disappearance of specific histones also raises the possibility that developmentally regulated proteolytic processing of specific histones plays an important (and previously unsuspected) role in this system. 相似文献
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SYNOPSIS. Relationships between the cell cycle and the beginning of conjugation were analyzed for 3 hypotrichs: Diophrys scutum, Oxytricha bifaria, and Euplotes crassus. The first 2 species enter conjugation with micronuclei in G1; the latter species with a micronucleus in G2. The 1st micronuclear division of conjugating E. crassus is mitotic. Thus meiotic DNA replication occurs when the cells of each species have already entered the mating process. Cells from asynchronous populations start conjugation with their macronuclei primarily in G1 or more rarely at the beginning of the S stage in a percentage significantly different from that expected on the basis of random mating among all cells in the population. Also, macronuclear replication, when already begun, was blocked in cells undergoing conjugation. Therefore only the G1 or the very early S stages of the cell cycle are compatible with conjugation in the 3 analyzed species. 相似文献
4.
Scheduled and unscheduled DNA synthesis during development in conjugating Tetrahymena 总被引:10,自引:0,他引:10
Autoradiography has been used to confirm and to extend previous microspectrophotometric studies (Doerder and DeBault, 1975) on the timing of DNA synthesis during conjugation in Tetrahymena thermophila. The majority of DNA synthesis occurs at the expected periods preceding gamete formation and the two postzygotic divisions and during macronuclear development. DNA in new macronuclei is endoreplicated in an extremely discontinuous fashion. Under starvation conditions, the first endoreplication (2C to 4C) occurs immediately after the second postzygotic division when both new macronuclei and new micronuclei replicate. The second endoreplication (4C to 8C) does not occur until after separation of conjugants. If mating cells are kept under prolonged starvation conditions (20-24 hr), refeeding induces a partially synchronous division, after which an unexpectedly high percentage of cells incorporate tritiated thymidine into both macro- and micronuclei. Two previously undescribed periods of DNA synthesis were observed in the micronuclei of conjugating Tetrahymena. The first occurs during the early stages of meiotic prophase, before full crescent elongation. The second takes place in an extended period corresponding to macronuclear anlagen development, before conjugants have separated. CsCl gradient analyses indicate that, in micronuclear fractions, only main band DNA is being synthesized in both of these periods. However, in macronuclear fractions from both stages, a significant fraction (approximately 20%) of the DNA being synthesized has the buoyant density of ribosomal DNA. The finding that macro- and micronuclear DNA can be synthesized simultaneously in a single cell, both during conjugation and after refeeding starved exconjugants, raises interesting questions of how macro- or micronuclear-specific histones are targeted to the appropriate nuclei. 相似文献
5.
KAZUYUKI MIKAMI 《The Journal of eukaryotic microbiology》1996,43(1):43-48
ABSTRACT. The germinal micronucleus divides six times during conjugation of Paramecium caudatum : this includes two meiotic divisions and one mitosis of haploid nuclei during mating, and three mitoses of a fertilization nucleus (synkaryon). Microsurgical removal of the macronucleus showed that micronuclei were able to divide repeatedly in the absence of the macronucleus, after metaphase of meiosis I of the micronucleus and also after synkaryon formation. When the macronucleus was removed after the first division of synkaryon, in an extreme case the synkaryon divided five times and produced 32 nuclei, compared to three divisions and eight nuclei produced in the presence of the macronucleus. Treatment with actinomycin D (100 μ /ml) inhibited the morphological changes of the macronucleus during conjugation and induced a multimicronucleate state in exconjugants. However, in other cells, it induced production of a few giant micronuclei. We conclude that the micronucleus is able to undergo repeated divisions at any stage of conjugation in the absence of the macronucleus once the factor(s) for induction of the micronuclear division has been produced by the macronucleus. The macronucleus may also produce a regulatory factor required to stop micronucler division. 相似文献
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K Mikami 《Developmental biology》1992,149(2):317-326
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. 相似文献
8.
ABSTRACT. The nuclear apparatus of H. vermiculare consists of a single moniliform macronucleus and about 25 micronuclei. the micronuclei are about 3 μm in diameter and characterized by a meshwork of thick condensed chromatin. Mitosis is intranuclear and acentric as in all other ciliates. In metaphase, interpolar and chromosomal microtubules are abundant and the length of the micronuclei increases to about 5 μm. In late anaphase, interzonal microtubules become prominent and the spindle elongates to about 50 μ. In meta- and anaphase, the microtubules of the spindle are attached to the polar vesicles, and in anaphase, chromosomes become attached to it. In contrast to most other eukaryotes, micronuclear mitosis is not strictly bound to cell division in H. vermiculare. While most of the micronuclei divide prior to cytokinesis, others retain their interphasic shape or degenerate. In addition, some micronuclei divide in the interdivision period, i.e. between two successive divisions of the cell and macronucleus. Mating cells of H. vermiculare become joined to each other in the cilia-free region covering the cytostome. In the course of conjugation, the cell membranes and the underlying oral filamentous sheaths of both cells fuse, thus uniting the endoplasm of both cells in the mouth region. Synaptonemal complexes in the meiotic chromosomes are more distinct in H. vermiculare than in most other dilates. the micrographs presented here depict dearly the central filament, transverse elements, and other substructures. 相似文献
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C. David Allis Josephine K. Bowen George N. Abraham Claiborne V.C. Glover Martin A. Gorovsky 《Cell》1980,20(1):55-64
Micronuclei of Tetrahymena thermophila contain two electrophoretically distinct forms of histone H3. The slower migrating micronuclear species, H3S, is indistinguishable from macronuclear H3 by electrophoretic analyses in three gel systems and by partial proteolytic peptide mapping. The faster species, H3F, is unique to micronuclei. Pulse-chase experiments with radioactive amino acids show that H3S is a precursor to H3F. We present evidence that the in vivo processing of H3S into H3F requires cell growth and/or division and may occur regularly each generation at a specific point in the cell cycle. The processing event must occur after H3F is deposited on micronuclear chromatin, since both H3S and H3F can be isolated from sucrose gradient-purified mononucleosomes (Allis, Glover and Gorovsky, 1979). Partial proteolytic peptide mapping coupled with 3H-N-ethylmaleimide labeling suggest that the processing event involves a proteolytic cleavage from the amino terminal end of H3F. Automated sequence analyses of 14C-lysine-labeled macronuclear H3 together with either 3H-lysine-labeled H3S or H3F demonstrated that H3F is derived from H3S by a proteolytic cleavage which removes six residues from the amino terminus. These observations represent the first demonstration of a physiologically regulated proteolytic processing event in histone metabolism. 相似文献
11.
Each cell of Paramecium caudatum has a germinal micronucleus. When a bi-micronucleate state was created artificially by micronuclear transplantation, both micronuclei divided for at least 2 cell cycles after nuclear transplantation. However, this bi-micronucleate state was unstable and reduced to a uni-micronucleate state after several fissions. Although the number of micronuclei was usually 1 during the vegetative phase, 4 presumptive micronuclei differentiated after conjugation. At the first post-conjugational fission, only 1 of the 4 micronuclei divided, indicating that there is tight regulation of micronuclear number in exconjugants. Micronuclei that did not divide at the first post-conjugational fission may persist through the first and second post-conjugational cell cycles. The decision to divide appears to be separate from the decision to degenerate, as evidenced by division of a remaining micronucleus upon removal of the dividing micronucleus at the first division. Degeneration of micronuclei in exconjugants differs from that of haploid nuclei after meiosis. Nutritional state affected micronuclear degeneration. Under well-fed conditions, the micronuclei destined to degenerate lost the ability to divide earlier than after starvation treatment, suggesting that micronuclear degeneration is an "apoptotic" phenomenon, probably under the control of the new macronuclei (macronuclear anlagen). 相似文献
12.
A. VASANTHI BHANDARY 《The Journal of eukaryotic microbiology》1959,6(4):333-339
SYNOPSIS. The Indian race of Blepharisma undulans described in this paper measures 150 μ in length. The macronucleus consists of 5–7 nodes, all of equal size. During binary fission, condensation of macronucleus is followed by its elongation and a thinning of the middle region which breaks with the division of the animal. It later attains the typical vegetative form.
During conjugation 7 or 8 micronuclei pass through the first pregamic division, 5 to 7 through the second pregamic division and one product of the second division takes part in the third division. The rest degenerate. At the same time, the macronucleus also starts degenerating. After the synkaryon has divided twice, the conjugating pairs separate. Of the 4 products, 3 become macronuclear anlagen and one, micronuclear anlage.
The micronuclei divide asynchronously both during binary fission and during conjugation. There is apparently considerable diversity in the structure and behaviour of the macronucleus and micronuclei in the different races of Blepharisma undulans. 相似文献
During conjugation 7 or 8 micronuclei pass through the first pregamic division, 5 to 7 through the second pregamic division and one product of the second division takes part in the third division. The rest degenerate. At the same time, the macronucleus also starts degenerating. After the synkaryon has divided twice, the conjugating pairs separate. Of the 4 products, 3 become macronuclear anlagen and one, micronuclear anlage.
The micronuclei divide asynchronously both during binary fission and during conjugation. There is apparently considerable diversity in the structure and behaviour of the macronucleus and micronuclei in the different races of Blepharisma undulans. 相似文献
13.
Macronuclei of Tetrahymena thermophila contain a typical H1 which has been shown to be missing from micronuclei. Instead, micronuclei contain three unique polypeptides, alpha, beta, and gamma, which are associated with linker regions of micronuclear chromatin. In this report polyclonal antibodies raised against macronuclear H1 are shown to react with alpha, beta, and gamma by immunoblotting analyses. This result suggests that these polypeptides share some common structural feature(s). Also consistent with this result is the finding that both macro- and micronuclei in growing and mating cells stain positively with H1 antibodies by in situ indirect immunofluorescence. However, these analyses demonstrate that the level of linker histone is greatly reduced in the micronucleus of starved cells and in young macronuclear anlagen. These results are in agreement with earlier biochemical studies and together provide strong evidence that dramatic changes in linker histone accompany nuclear differentiation (and dedifferentiation) in Tetrahymena. 相似文献
14.
MARTIN A. GOROVSKY 《The Journal of eukaryotic microbiology》1973,20(1):19-25
The macro- and micronucleus of Tetrahymena pyriformis are formed from a common diploid synkaryon during conjugation. Shortly after the 2nd postzygotic division, distinct morphologic and physiologic differences develop between the 2 nuclei. Micronuclei remain small, presumably diploid, and electronmicroscopic observations indicate that micronuclear DNA is contained in a dense, fibrous, chromosome-like coil. Macronuclei contain considerably more DNA than micronuclei, and the DNA of the macronucleus is found largely in the chromatin bodies typical of ciliate nuclei. The functional differences between macro- and micronuclei in vegetative cells also are striking. The template activity of DNA in the micronucleus is highly restricted compared to that in the macronucleus. Micronuclei synthesize and contain little RNA, and do not contain either nucleoli or ribonucleoprotein granules. Macronuclei, on the other hand, synthesize and contain large amounts of RNA and have many nucleoli and ribonucleoprotein granules. Macro- and micronuclei also have distinct differences in the timing of DNA synthesis during the cell cycle and in the timing and mechanism of nuclear division. Finally, during conjugation the macronucleus becomes pycnotic and disappears while the micronucleus undergoes meiosis and fertilization, ultimately giving rise to new macro- and new micronuclei. In short, the macro- and micronuclei of Tetrahymena provide an excellent system for studying the molecular mechanisms by which the same (or related) genetic information is maintained in different structural and functional states. Methods have been devised to isolate and purify macro- and micronuclei of Tetrahymena in the hope of correlating differences in the nucleoprotein composition of these nuclei with differences in their structure and function. The DNAs of macro- and micronuclei have been found to differ markedly in their content of a methylated base, N6-methyl adenine, and major differences in the histones of the 2 nuclei have been observed. Macronuclei contain histones similar to those found in vertebrate nuclei, while 2 major histone fractions seem to be missing in micronuclei. In addition, histone fraction F2A1 which is found in multiple, acetylated forms in macronuclei, is present only as a single, unacetylated form in micronuclei. 相似文献
15.
Kazuyuki Mikami 《Genesis (New York, N.Y. : 2000)》1992,13(3):223-228
Paramecium caudatum loses the ability to form food vacuoles at the crescent stage of the micronucleus from 5 to 6 hr after the initiation of conjugation and regains it immediately after the third division of the zygotic nucleus. To assess the micronuclear function in the development of the oral apparatus after coniugation, prezygotic micronuclei was removed from cells at various stages of conjugation, and their ability to form food vacuoles were examined. (1) When all of the prezygotic micronuclear derivatives were eliminated before the stage of formation of the zygotic nucleus, the exconjugant did not regain its ability. (2) When a zygotic nucleus or postzygotic nuclei were removed, in some cases the cell formed as many food vacuoles as did nonoperated cells after conjugation, while in other operated cells the number of food vacuoles was subnormal. (3) When a micronucleus from a cell at vegetative phase (G1) was transplanted into a cell of an amicronucleate mating pair at the stage between 8 and 9 hr after the initiation of conjugation, the implanted cell regained the ability to form food vacuoles. However, no cell regained the ability when the implantation was carried out within 1 hr after the separation of the mates. The results show that the micronucleus plays an indispensable role in the development of the oral apparatus at the stages of exchange of gametic nuclei and fertilization and that the micronucleus transplanted from asexual cells can fulfill this function. On the other hand, removal of the macronucleus from exconjugants showed that the maternal macronucleus also has an indispensable function in regaining the ability to form food Vacuoles. © 1992 Wiley-Liss, Inc. 相似文献
16.
Mating-Type Dependent Cell Pairing in Oxytricha hymenostoma: Macronuclear Fusion and Lack of Meiosis
FULVIO ESPOSITO ROSALBA BANCHETTI NICOLA RICCI 《The Journal of eukaryotic microbiology》1978,25(2):238-241
SYNOPSIS In a hologamy-like process observed in Oxytricha hymenostoma , 2 cells belonging to complementary mating types fused and appeared to contribute equally to the macronuclei of the organism resulting from the fusion. The process was characterized by the absence of micronuclear division and by fusion of the macronuclei of the 2 partners. True conjugation involving micronuclear meiosis was not observed in O. hymenostoma. The biologic significance and the possible adaptive role of the hologamy-like process are discussed. 相似文献
17.
Accumulation of low molecular weight DNA and changes in chromatin structure in HeLa cells treated with human fibroblast interferon 总被引:2,自引:0,他引:2
R J Suhadolnik Y Sawada J Gabriel N L Reichenbach E E Henderson 《The Journal of biological chemistry》1984,259(8):4764-4769
The addition of human fibroblast interferon (IFN-beta) (100 units/ml) at the S/G2 boundary of the cell cycle of synchronously grown HeLa cells is characterized by the accumulation of newly synthesized low molecular weight DNA and changes in chromatin assembly. In addition, there is a 3-fold stimulation in the incorporation of tracer amounts of [3H]thymidine, but not [3H] deoxyguanosine, into DNA and a 2-fold increase in the incorporation of [3H]dTTP into the DNA of isolated nuclei. Fluorescence-activated cell sorting by laser flow cytometry revealed that IFN-beta-treated cells were delayed in entering and passing through the S phase. The inhibition of proliferation of HeLa cells treated with IFN-beta is characterized by a 3-fold accumulation of newly synthesized DNA of Mr less than 56 X 10(6) compared to untreated cells as determined by alkaline sucrose gradient centrifugation. The newly synthesized DNA in IFN-beta-treated cells was replicative and not repair DNA. The observation that IFN-beta inhibits the processing of newly synthesized low molecular weight DNA into normal DNA might be explained by the intracellular accumulation of S-adenosylhomocysteine in IFN-beta-treated HeLa cells (de Ferra, F., and Baglioni, C. (1983) J. Biol. Chem. 258, 2118-2121) which could change the soluble ribonucleotide and deoxyribonucleotide pool and ultimately affect DNA processing. Interferon may also affect processing of DNA by interfering with normal chromatin assembly. Evidence for the effect of IFN-beta on chromatin assembly is provided; we have observed a more condensed structure in IFN-beta treated cells by circular dichroism spectroscopy. Simultaneous with the affect on chromatin assembly, there is a 70% decrease in poly(ADP-ribosylation) of either histone and/or non-histone proteins. The loss of coordination between the pool size for DNA synthesis, decreased postsynthetic modifications of chromatin, and normal chromatin formation may explain the inability of the cell to differentiate and to continue cell division. 相似文献
18.
Cytoplasmic exchange between conjugating cells of Paramecium caudatum has been implicated by mating experiments using wild-type and behavioral mutant cells. To observe macromolecular transport between mating cells, we cloned and expressed the P. caudatum histone H2B gene as a fusion protein attached to an enhanced yellow fluorescent protein (YFP) named PcVenus. Significant fluorescent signals derived from histone H2B-PcVenus were detected throughout the macro- and micronuclei of transformant cells after microinjection of the expression vector. The normal growth and high mating reactivity of the transformants indicated that H2B-PcVenus functioned normally. Seven hours after a transformant cell expressing histone H2B-PcVenus was mated with an untransformed complementary mating-type cell, fluorescence derived from histone H2B-PcVenus was emitted from the macronuclei of the untransformed cell. About 48 h later, the fluorescent signal was detected not only in the macro- and micronuclei of untransformed cells but also in the macronuclear anlagen of both mating cells. This suggests that conjugant cells share parental histones during meiosis and subsequent DNA rearrangement. Single-cell RT-PCR analysis demonstrated the presence of H2B-PcVenus mRNA in untransformed cells 15 and 24 h after conjugation. We concluded that at least the mRNA of histone H2B-PcVenus was transferred from the transformed, to the untransformed cell during conjugation. 相似文献
19.
Rad51p, the eukaryotic homolog of the prokaryotic recA protein, catalyzes strand exchange between single- and double-stranded DNA and is involved in both genetic recombination and double-strand break repair in the ciliate Tetrahymena thermophila. We have previously shown that disruption of the Tetrahymena RAD51 somatic macronuclear locus leads to defective germline micronuclear division and that conjugation of two somatic rad51 null strains results in an early meiotic arrest. We have constructed Tetrahymena strains that are capable of RAD51 expression from their parental macronuclei and are homozygous, rad51 nulls in their germline micronuclei. These rad51 null heterokaryons complete all of the early and middle stages of conjugation, including meiosis, haploid nuclear exchange, zygotic fusion, and the programmed chromosome fragmentations, sequence eliminations, and rDNA amplification that occur during macronuclear development. However, the rad51 null progeny fail to initiate the first vegetative cell division following conjugal development. Coincident with the developmental arrest is a disproportionate amplification of rDNA, despite the maintenance of normal total DNA content in the developing macronuclei. Fusion of arrested rad51 null exconjugants to wild-type cells is sufficient to overcome the arrest. Cells rescued by cytoplasmic fusion continue to divide, eventually recapitulating the micronuclear mitotic defects described previously for rad51 somatic nulls. 相似文献
20.
Günter Cleffmann 《Chromosoma》1980,78(3):313-325
In exponentially growing Tretrahymena thermophila the DNA content of the following structures was determined by cytophotometry: macronuclei of sister cells immediately after division; micronuclei; extranuclear chromatin in dividing cells and postdividers. Further, the development of macro-nuclear DNA amount in successive cell generations was determined. It was found that chromatin elimination is a frequent process reducing DNA content by about 4% per fission. This chromatin disappears within 20 min after division. The quantity of DNA extruded is highly variable and is different from the micronuclear DNA amount or multiples of it. The frequency of generations with two replication rounds as well as those without replication is estimated to be in the range of 2% each. These findings together with the qualitative difference between micro- and macronuclear DNAs suggest that the macronucleus of Tetrahymena is not entirely composed of complete genomes and that parts of the genetic material must be treated specifically for different sequences either during extrusion or during replication. 相似文献