scholarly journals MACROMOLECULAR EVENTS LEADING TO CELL DIVISION IN TETRAHYMENA PYRIFORMIS AFTER REMOVAL AND REPLACEMENT OF REQUIRED PYRIMIDINES

1965 ◽  
Vol 25 (2) ◽  
pp. 9-19 ◽  
Author(s):  
Ivan L. Cameron
Keyword(s):  
Cell Division ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Nuclear Dna ◽  
Early Period ◽  
Tetrahymena Pyriformis ◽  
Cell Number ◽  
Cellular Protein ◽  
Cellular Content ◽  
Endogenous Protein

Tetrahymena pyriformis were brought to a non-growing state by removal of pyrimidines from their growth medium. During pyrimidine deprivation cell number increased 3- to 4 fold, and this increase was accompanied by one or more complete cycles of macronuclear DNA replication. Autoradiographic studies show that endogenous protein and RNA were turning over throughout starvation and that RNA breakdown products were used to support the DNA synthesis that occurred during the early period of starvation. However, after 72 hours of starvation all DNA synthesis and cell division had ceased. Feulgen microspectrophotometry shows the macronuclei of these cells to have been stopped at a point prior to DNA replication (G1 stage). After pyrimidine replacement the incorporation of H3-uridine, H3-adenosine, and H3-leucine was measured by the autoradiographic grain counting method. The results indicate that RNA synthesis began to increase almost immediately, but that there was a lag of almost an hour before an increase in protein synthesis. In agreement with the autoradiographic data, chemical data also show that cellular content of RNA began to increase shortly after pyrimidine replacement but that cellular protein content did not increase until about one hour later. Pulse labeling of the cells with H3-thymidine at intervals after pyrimidine replacement shows that labeled macronuclei first began to appear at 150 minutes; that 98 per cent of the macronuclei were in DNA synthesis at 240 to 270 minutes; and that the percentage then began to decrease from 300 to 390 minutes, at which time only 25 per cent of the macronuclei were labeled. Cellular content of DNA did not increase for at least 135 minutes after pyrimidine replacement; however, just before the first cells divided (360 minutes) the DNA content had doubled. After pyrimidine replacement the cells first began to divide at 360 minutes, and 50 per cent had divided at 420 minutes; however, all cells had not divided until 573 minutes. This technique of chemical synchronization of cells in mass cultures makes feasible detailed biochemical analysis of events leading to nuclear DNA replication and cell division.

Cytofluorimetric analysis of nuclear DNA during meiosis, fertilization and macronuclear development in the ciliate Tetrahymena pyriformis, syngen 1

1975 ◽  
Vol 17 (3) ◽  
pp. 471-493 ◽  
Author(s):  
F.P. Doerder ◽  
L.E. Debault
Keyword(s):  
Cell Division ◽  
Dna Synthesis ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Tetrahymena Pyriformis ◽  
Cell Cycles ◽  
Macronuclear Development ◽  
Sister Chromatids ◽  
Nuclear Development ◽  
S Period

Fluorescence cytophotometry was used to study nuclear DNA content and synthesis patterns during meiosis, fertilization and macronuclear development in the ciliated protozoon, Tetrahymena pyriformis, syngen 1. It was found that cells entered conjugation with a G1 (45C) macronucleus and a G2 (4C) micronucleus. During meiosis the micronucleus was reduced to 4 haploid nuclei, each with a 1C amount of DNA; each meiotic product then replicated to 2C, but only the nucleus next to the attachment membrane in each conjugant divided to form the two 1C gametic nuclei. The gametic nuclei replicated to 2C prior to fertilization; hence there was no S-period in the 4C fertilization nucleus (synkaryon). The first postzygotic division products immediately entered an S-period to become 4C, and at the second postzygotic division, each of the two 4C nuclei in each conjugant divided to form one 2C micronucleus and one 2C macronuclear Anlage. The macronuclear Anlagen began DNA synthesis immediately and were about 8C at the completion of conjugation; the micronuclei did not undergo rapid DNA doubling and measured between 2C and 3C when the conjugants separated. The old macronucleus did not participate in any S-period during conjugation and began to decompose after the second postzygotic division; it contained an average of 24C at the end of conjugation. From this sequence of nuclear divisions a pattern emerges that, unless a general cytoplasmic signal for DNA synthesis is suppressed, DNA synthesis always occurs in micronuclear division products immediately following separation of sister chromatids. Nuclear development continued in the first two cell cycles after conjugation. In exconjugants (the first cycle), macronuclear Anlagen underwent two rounds of DNA synthesis to become 32C and both micronuclei also underwent DNA synthesis. However, prior to the first cell division, one micronucleus and the old macronucleus completely disintegrated, and at the first cell division the remaining 4C micronucleus divided and one macronuclear Anlage was distributed to each resulting caryonide. At the end of the second cell cycle, the dividing macronucleus of each caryonide contained about 128C. These results relate to the question of ploidy of macronuclear subunits. It is argued that the G1 macronucleus contains 22 or 23 diploid subunits, each subunit being a copy of the diploid micronuclear genome. It is suggested that unequal macronuclear division relates to the question of subunit ploidy by playing a role in the phenomenon of macronuclear assortment.

Benzyladenine-stimulation of nuclear DNA synthesis and cell division in germinating maize

1991 ◽  
Vol 1 (2) ◽  
pp. 113-117 ◽  
Author(s):  
J. Reyes ◽  
L. F. Jiménez-García ◽  
M. A. Gonzalez ◽  
J. M. Vázquez-Ramos
Keyword(s):  
Cell Division ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Mitotic Index ◽  
Nuclear Dna ◽  
Fold Increase ◽  
Mechanisms Of Action ◽  
Dna Metabolism ◽  
Stimulation Of

AbstractWe have studied by means of cytology and autoradiography the effect of benzyladenine (BA, a synthetic cytokinin) on DNA metabolism during early maize germination.The data indicate that BA stimulates nuclear DNA replication. The doubling of the amount of nuclear DNA in BA-treated axes occurs earlier than in nontreated axes, and there is a three-fold increase in the mitotic index at 24 h of germination. These results provide further corroboration for the suggestion that the stimulation of DNA synthesis observed relates to a nuclear replicative type of synthesis. Possible mechanisms of action of BA are discussed.

Replication of Macronuclear DNA in the Cytoplasm of Tetrahymena Pyriformis

1974 ◽  
Vol 14 (2) ◽  
pp. 289-300
Author(s):  
H. A. ANDERSEN
Keyword(s):  
Cell Division ◽  
Dna Replication ◽  
Nuclear Dna ◽  
Tetrahymena Pyriformis ◽  
Cell Generation ◽  
The Other ◽  
Previous Generation ◽  
Cytoplasmic Dna ◽  
Continue Growth ◽  
S Period

Previous experiments showed that a synchronous population of Tetrahymena could divide even though DNA replication was blocked during the latter half of the preceding S-period by addition of methotrexate plus uridine (M + U). Furthermore, it was found that the DNA fraction which was in replication at the time of inhibition became localized in the cytoplasm following elimination from the nucleus at the time of division. When the inhibitory treatment (M + U) was removed prior to or at the time of the cell division the cells were found to engage in new DNA replication and continue growth. Two questions arose from these studies. First, is the DNA replication normal following release from M + U? Second, what is the fate of the cytoplasmic DNA? In the present paper DNA replication has been studied using incorporation of 5-bromodeoxyuridine and centrifugation of the labelled DNA in CsCl gradients. It is concluded that the DNA which finished replication prior to the effect of the M + U treatment replicates again during the S-period of the next cell generation. On the other hand, the DNA fraction which was stalled in replication and subsequently eliminated from the nucleus also replicates in the cytoplasm in the next generation but during G2 period, out of phase with the undamaged nuclear DNA. The cytoplasmic DNA replication appeared to be a continuation of the replication initiated in the nucleus in the previous generation.

Changes of the DNA content of differentiating and adult macronuclei of the ciliate Loxodes magnus (Karyorelictida)

1980 ◽  
Vol 44 (1) ◽  
pp. 375-394
Author(s):  
N.N. Bobyleva ◽  
B.N. Kudrjavtsev ◽  
I.B. Raikov
Keyword(s):  
Cell Division ◽  
Dna Replication ◽  
Hydrochloric Acid ◽  
Dna Synthesis ◽  
Acid Hydrolysis ◽  
Gene Amplification ◽  
Dna Content

The DNA content of isolated micronuclei, differentiating macronuclei (macronuclear Anlagen), and adult macronuclei of Loxodes magnus was measured cytofluorimetrically in preparations stained with a Schiff-type reagent, auramine-SO2, following hydrochloric acid hydrolysis. The DNA content of the youngest macronuclear Anlagen proved to be the same as that of telophasic micronuclei (2 c). The Anlagen thus differentiate from micronuclei which are still in G1. The quantity of DNA in the macronuclear Anlagen thereafter rises to the 4-c level, simultaneously with DNA replication in the micronuclei which immediately follows mitosis. In non-dividing animals most micronuclei are already in G2. Adult macronuclei here contain on average 1.5 times more DNA than the micronuclei; their DNA content is about 5–6 c (in some individual nuclei, up to 10 c). These data are consistent with autoradiographic evidence indicating a weak DNA synthesis in the macronuclei of Loxodes and make likely the existence of partial DNA replication (e.g. gene amplification) in the macronuclei. The DNA content of adult macronuclei isolated from dividing animals proved to be significantly smaller than that of macronuclei isolated from non-dividing specimens of the same clone. In 3 clones studied, the former value amounted on average to 71–79, 78 and 95% of the latter, respectively. This drop of DNA content cannot be explained by ‘dilution’ of the old macronuclei with newly formed ones. The quantity of DNA in adult macronuclei thus seems to undergo cyclical changes correlated with cytokinesis, despite the fact that, in Loxodes magnus, the macronuclei themselves never divide and are simply segregated at every cell division. The macronuclei of Loxodes can be termed paradiploid or hyperdiploid.

scholarly journals LIMITED DNA SYNTHESIS IN THE ABSENCE OF PROTEIN SYNTHESIS IN PHYSARUM POLYCEPHALUM

1966 ◽  
Vol 31 (3) ◽  
pp. 577-583 ◽  
Author(s):  
J. E. Cummins ◽  
H. P. Rusch
Keyword(s):  
Protein Synthesis ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Physarum Polycephalum ◽  
Nuclear Dna ◽  
S Phase ◽  
Early Prophase ◽  
Late Prophase ◽  
Removal Experiments ◽  
Inhibitor Of Protein Synthesis

Actidione (cycloheximide), an antibiotic inhibitor of protein synthesis, blocked the incorporation of leucine and lysine during the S phase of Physarum polycephalum. Actidione added during the early prophase period in which mitosis is blocked totally inhibited the initiation of DNA synthesis. Actidione treatment in late prophase, which permitted mitosis in the absence of protein synthesis, permitted initiation of a round of DNA replication making up between 20 and 30% of the unreplicated nuclear DNA. Actidione treatment during the S phase permitted a round of replication similar to the effect at the beginning of S. The DNA synthesized in the presence of actidione was replicated semiconservatively and was stable through at least the mitosis following antibiotic removal. Experiments in which fluorodeoxyuridine inhibition was followed by thymidine reversal in the presence of actidione suggest that the early rounds of DNA replication must be completed before later rounds are initiated.

Stimulation of cell division and DNA replication inPrototheca richardsiby passage through larval amphibian guts

Parasitology ◽  
1993 ◽  
Vol 107 (2) ◽  
pp. 119-124 ◽  
Author(s):  
T. J. C. Beebee ◽  
A. L.-C. Wong
Keyword(s):  
Cell Division ◽  
Dna Replication ◽  
Growth Inhibition ◽  
Dna Synthesis ◽  
Free Living ◽  
Leucine Uptake ◽  
Amphibian Larvae ◽  
Larval Amphibian ◽  
Stimulation Of

SUMMARYPrototheca richardsi, an unpigmented heterotrophic alga, causes growth inhibition in amphibian larvae and has proved refractory to culturein Vitro.P. richardsireplication is dependent on regular passaging through tadpole digestive systems; uptake of thymidine by free-livingProtothecacells and incorporation into DNA are very low by comparison with leucine uptake and incorporation into protein, but DNA synthesis is detectable in cells isolated from tadpole intestines. DNA replication was elicited 6–8 h after ingestion in protothecans fed to tadpoles and subsequently re-isolated from them, providing that the tadpoles were fed subsequent to the ingestion. It appears that passaging through tadpole intestines provides an essential stimulus to maintaining an active cell division cycle inP. richardsi.

scholarly journals Nonperiodic Activity of the Human Anaphase-Promoting Complex–Cdh1 Ubiquitin Ligase Results in Continuous DNA Synthesis Uncoupled from Mitosis

2000 ◽  
Vol 20 (20) ◽  
pp. 7613-7623 ◽  
Author(s):  
Claus Storgaard Sørensen ◽  
Claudia Lukas ◽  
Edgar R. Kramer ◽  
Jan-Michael Peters ◽  
Jiri Bartek ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Kinase Inhibitor ◽  
Cyclin B1 ◽  
S Phase ◽  
Anaphase Promoting Complex

ABSTRACT Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccharomyces cerevisiae andDrosophila spp., triggers exit from mitosis and during G1 prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference with the APC-Cdh1 dissociation at the G1/S transition resulted in an inability to accumulate a surprisingly broad range of critical mitotic regulators including cyclin B1, cyclin A, Plk1, Pds1, mitosin (CENP-F), Aim1, and Cdc20. Unexpectedly, although constitutively assembled APC-Cdh1 also delayed G1/S transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27Kip1 cyclin-dependent kinase inhibitor. Consequently, failure to inactivate APC-Cdh1 beyond the G1/S transition not only inhibited productive cell division but also supported slow but uninterrupted DNA replication, precluding S-phase exit and causing massive overreplication of the genome. Our data suggest that timely oscillation of the APC-Cdh1 ubiquitin ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells.

A human nuclear protein with sequence homology to a family of early S phase proteins is required for entry into S phase and for cell division

1994 ◽  
Vol 107 (1) ◽  
pp. 253-265 ◽  
Author(s):  
I.T. Todorov ◽  
R. Pepperkok ◽  
R.N. Philipova ◽  
S.E. Kearsey ◽  
W. Ansorge ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Amino Acid ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Nuclear Protein ◽  
Amino Acid Level ◽  
S Phase ◽  
Significant Similarity

Molecular cloning and characterisation of a human nuclear protein designated BM28 is reported. On the amino acid level this 892 amino acid protein, migrating on SDS-gels as a 125 kDa polypeptide, shares areas of significant similarity with a recently defined family of early S phase proteins. The members of this family, the Saccharomyces cerevisiae Mcm2p, Mcm3p, Cdc46p/Mcm5p, the Schizosaccharomyces pombe Cdc21p and the mouse protein P1 are considered to be involved in the onset of DNA replication. The highest similarity was found with Mcm2p (42% identity over the whole length and higher than 75% over a conservative region of 215 amino acid residues), suggesting that BM28 could represent the human homologue of the S. cerevisiae MCM2. Using antibodies raised against the recombinant BM28 the corresponding antigen was found to be localised in the nuclei of various mammalian cells. Microinjection of anti-BM28 antibody into synchronised mouse NIH3T3 or human HeLa cells presents evidence for the involvement of the protein in cell cycle progression. When injected in G1 phase the anti-BM28 antibody inhibits the onset of subsequent DNA synthesis as tested by the incorporation of bromodeoxyuridine. Microinjection during the S phase had no effect on DNA synthesis, but inhibits cell division. The data suggest that the nuclear protein BM28 is required for two events of the cell cycle, for the onset of DNA replication and for cell division.

Effects of DMSO on Vacuole Formation, Contractile Vacuole Function, and Nuclear Division in Tetrahymena Pyriformis GL

1974 ◽  
Vol 16 (1) ◽  
pp. 39-47
Author(s):  
J. R. NILSSON
Keyword(s):  
Cell Division ◽  
Nuclear Division ◽  
Physiological State ◽  
Tetrahymena Pyriformis ◽  
Cell Number ◽  
Contractile Vacuole ◽  
Vacuole Formation ◽  
Control Value ◽  
Dividing Cells

Increasing concentrations of dimethyl sulphoxide (DMSO) affect vacuole formation in Tetrahymena, as measured quantitatively by the uptake of carmine particles. The rate of vacuole formation decreased to about 50% of the control value in 5.0% DMSO (v/v) and to zero in 7.5%. At the latter concentration, the inhibition was expressed immediately; however, the effect of 1-h exposure was reversible after removal of DMSO by washing. In vivo observations revealed abnormal function of the contractile vacuole in 7.5% DMSO, while cell motility and cell division appeared to be unaffected. Although cell division occurred there was little or no increase in cell number, as studied over a cell generation time. Feulgen preparations showed that nuclear division was inhibited and that cell division resulted in one anucleate and one nucleate daughter cell. This effect was also observed in some dividing cells at lower concentrations of DMSO. The effect of DMSO on Tetrahymena was dependent not only on the concentration of the compound but also on the physiological state of the cells.

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