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.

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.

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.

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.

scholarly journals Sensitivity of the Cell Division Cycle to α-Amanitin In ALLIUM ROOT TIPS

1974 ◽  
Vol 14 (3) ◽  
pp. 461-473
Author(s):  
C. DE LATORRE ◽  
M. E. FERNANDEZ-GOMEZ ◽  
G. GIMENEZ-MARTIN ◽  
A. GONZALEZ-FERNANDEZ
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Normal Structure ◽  
Cell Division Cycle ◽  
The Other ◽  
Root Tips ◽  
Meristematic Cells ◽  
Normal Duration ◽  
S Period ◽  
Nucleolar Rna

The effect of α-amanitin on the cell cycle in Allium cepa meristematic cells was studied: the G1 and G2 periods are prolonged respectively to 1.9 and 1.7 times the normal duration; the S-period is lengthened very little; and the prophase of mitosis is increased to twice the normal duration. It is postulated that real differences in the activity of the non-nucleolar RNA poly-merase might exist in the course of the cell division cycle and that they would account for the higher sensitivities shown by G1, G2 and prophase. On the other hand, the interphase nucleolus responds by segregation in the first few hours of α-amanitin treatment, but recovers its normal structure in continued presence of the drug; and nucleolar reorganization is inhibited in the first few hours in recently formed cells, but the process is subsequently speeded up to attain the same value 4 h after the treatment was begun as in untreated cells.

scholarly journals EVIDENCE FOR A TEMPORAL INCOMPATIBILITY BETWEEN DNA REPLICATION AND DIVISION DURING THE CELL CYCLE OF TETRAHYMENA

1972 ◽  
Vol 53 (3) ◽  
pp. 624-634 ◽  
Author(s):  
William R. Jeffery
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Replication ◽  
Heat Shock ◽  
Dna Synthesis ◽  
Shock Treatment ◽  
Coupling Mechanism ◽  
Complete Suppression ◽  
Heat Shock Treatment ◽  
S Period

The mechanism of coordination between DNA replication and cell division was studied in Tetrahymena pyriformis GL-C by manipulation of the timing of these events with heat shocks and inhibition of DNA synthesis. Preliminary experiments showed that the inhibitor combination methotrexate and uridine (M + U) was an effective inhibitor of DNA synthesis. Inhibition of the progression of DNA synthesis with M + U in exponentially growing cells, in which one S period usually occurs between two successive divisions, or in heat-shocked cells, when successive S periods are known to occur between divisions, resulted in the complete suppression of the following division. In further experiments in which the division activities were reassociated with the DNA synthetic cycle by premature termination of the heat-shock treatment, it was shown that (a) the completion of one S period during the treatment was sufficient for cell division, (b) the beginning of division events suppressed the initiation of further S periods, and (c) if further S periods were initiated while the heat-shock treatment was continued, division preparations could not begin until the necessary portion of the S period was completed, even though DNA had previously been duplicated. It was concluded that a temporal incompatibility exists between DNA synthesis and division which may reflect a coupling mechanism which insures their coordination during the normal cell cycle.

scholarly journals [6N]METHYL ADENINE IN THE NUCLEAR DNA OF A EUCARYOTE, TETRAHYMENA PYRIFORMIS

1973 ◽  
Vol 56 (3) ◽  
pp. 697-701 ◽  
Author(s):  
Martin A. Gorovsky ◽  
Stanley Hattman ◽  
Gloria Lorick Pleger
Keyword(s):  
Nuclear Dna ◽  
Tetrahymena Pyriformis ◽  
The Other ◽  
Other Hand ◽  
Different Strains

DNA isolated from macronuclei of the ciliate, Tetrahymena pyriformis, has been found to contain [6N]methyl adenine (MeAde); this represents the first clear demonstration of significant amounts of MeAde in the DNA of a eucaryote. The amounts of macronuclear MeAde differed slightly between different strains of Tetrahymena, with approximately 0.65–0.80% of the adenine bases being methylated. The MeAde content of macronuclear DNA did not seem to vary in different physiological states. The level of MeAde in DNA isolated from micronuclei, on the other hand, was quite low (at least tenfold lower than in macronuclear DNA).

Regulation of ribosomal RNA synthesis in Tetrahymena pyriformis

1978 ◽  
Vol 31 (1) ◽  
pp. 13-23
Author(s):  
J. Keiding ◽  
H.A. Andersen
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Tetrahymena Pyriformis ◽  
Rrna Synthesis ◽  
Constant Rate ◽  
Normal Increase ◽  
S Period ◽  
Dosis Effect

Ribosomal RNA is synthesized at constant rate during most of the cell cycle in heat-shock synchronized populations of Tetrahymena pyriformis. Early in each macronuclear S-period the rate of synthesis increases abruptly, concomitant with replication of the genes coding for ribosomal RNA. The increase is prevented by inhibitors of DNA replication, added prior to the S-period. Similarly, in cultures synchronized by starvation/refeeding, inhibition of DNA replication, at the time when the rDNA is replicated, will prevent the normal increase in rate of RNA synthesis which follows refeeding. We conclude that inhibition of rDNA replication interferes with the synthesis of rRNA, and we suggest that with respect to rRNA synthesis a gene dosis effect is operating in fast-growing Tetrahymena cells.

scholarly journals CELL DIVISION AND DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS DEPRIVED OF ESSENTIAL AMINO ACIDS

1964 ◽  
Vol 21 (2) ◽  
pp. 275-281 ◽  
Author(s):  
G. E. Stone ◽  
D. M. Prescott
Keyword(s):  
Amino Acids ◽  
Cell Division ◽  
Amino Acid ◽  
Dna Synthesis ◽  
Tetrahymena Pyriformis ◽  
Essential Amino Acids ◽  
The Other ◽  
Cent Increase ◽  
Amino Acid Requirements

The question of amino acid requirements for DNA synthesis and cell division has been studied in Tetrahymena pyriformis by depriving cells of histidine and tryptophan at defined stages in the interdivision interval. Deprivation any time before DNA synthesis does not prevent the initiation of such synthesis but completely inhibits the following division and limits the increase in DNA, as measured microspectrophotometrically, to 20 per cent. H3-thymidine added to the medium is not incorporated during the 20 per cent increase. Deprivation after DNA synthesis is initiated does not prevent the continuation (to completion) of DNA synthesis, and cell division ensues. H3-thymidine added to the medium under these conditions is incorporated into macronuclear DNA. The data indicate that some amino acid-dependent event occurs, about the time of the beginning of the DNA synthesis period, which is not essential for initiation of DNA synthesis but which is essential for the maintenance of synthesis once it has begun. These results are further discussed in terms of enzymes required to convert thymidine (and possibly the other three deoxyribonucleosides) to the immediate precursor of DNA synthesis.

Inhibition of rRNA synthesis following incorporation of 5-bromodeoxyuridine into DNA of Tetrahymena pyriformis

1975 ◽  
Vol 17 (3) ◽  
pp. 495-502
Author(s):  
A.E. Lykkesfeldt ◽  
H.A. Andersen
Keyword(s):  
Dna Replication ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Nuclear Dna ◽  
Tetrahymena Pyriformis ◽  
Growth Conditions ◽  
Defined Medium ◽  
Rrna Synthesis ◽  
Chemically Defined Medium ◽  
Dividing Cells

Tetrahymena pyriformis was grown on chemically defined medium in the presence of 5-bromodeoxyuridine (BUdR). Under these growth conditions more than 60% of the thymidine sites in DNA were substituted with BUdR. It was found that RNA synthesis was strongly inhibited by the presence of BUdR in DNA. To assure that incorporation of BUdR into DNA was a prerequisite of the effect observed, BUdR was added to synchronously dividing cells. BUdR had no effect on the cells when present outside the period of nuclear DNA replication, whereas RNA synthesis was strongly inhibited as soon as the genes coding for ribosomal RNA had replicated in the presence of BUdR.

scholarly journals THE EFFECT OF 5-BROMODEOXYURIDINE ON DNA REPLICATION AND CELL DIVISION IN TETRAHYMENA PYRIFORMIS

1974 ◽  
Vol 62 (2) ◽  
pp. 316-321 ◽  
Author(s):  
Anne E. Lykkesfeldt ◽  
H. A. Andersen
Keyword(s):  
Cell Division ◽  
Structural Change ◽  
Dna Replication ◽  
First Generation ◽  
Normal Growth ◽  
Tetrahymena Pyriformis ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Normal Hybrid ◽  
Hybrid Dna

Populations of Tetrahymena pyriformis were grown in a chemically defined medium containing the thymidine analogue 5-bromodeoxyuridine (BUdR). About 65% of the thymidine sites in DNA were substituted by BUdR. During the first generation in the presence of BUdR, all DNA became hybrid. After the following cell division, in about 80% of the cells the second DNA replication round was initiated but no further cell division took place. The cells could be rescued by removing BUdR and adding thymidine. New replication took place before the first cell division. However, although the cells contained double heavy as well as hybrid DNA, only the hybrid DNA was replicated. After a full replication of the hybrid DNA, normal growth was restored. Melting profiles of normal, hybrid, and double heavy DNA indicated a structural change of the double heavy DNA.

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