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.

Continuous Nucleolar DNA Synthesis in Late-Interphase Nuclei of Physarum Polycephalum after Transplantation into Postmitotic Plasmodia

1974 ◽  
Vol 15 (1) ◽  
pp. 131-143
Author(s):  
E. GUTTES
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Physarum Polycephalum ◽  
Nuclear Dna ◽  
S Phase ◽  
Interphase Nuclei ◽  
G2 Phase

In the myxomycete, Physarum polycephalum, nuclear DNA synthesis commences immediately upon completion of mitosis. While the synthesis of extranucleolar DNA is completed within a few hours, nucleolar DNA synthesis occurs during most of the S-phase and the entire G2 phase of the intermitotic period. When large (polyploid), late-interphase nuclei were allowed to bypass mitosis by transplantation into recipient plasmodia which were at early interphase and which belonged to a strain having smaller nuclei, the nucleolar DNA of the transplanted nuclei continued to be labelled (autoradiographs) after incubation of the host plasmodia with [3H]thymidine until they entered prophase along with the nuclei of the host plasmodium, approximately one intermitotic period later. This labelling was DNase-sensitive and RNase-resistant. When late-interphase nuclei were labelled with [3H]thymidine just prior to transplantation, there was no decrease of label after transplantation during the additional intermitotic period. We conclude from these experiments that there is no obligatory alternation between nucleolar DNA duplication and mitosis in Physarum polycephalum and that nucleolar DNA replication might exhibit amplification during an experimentally prolonged intermitotic period.

scholarly journals REGULATION OF DNA REPLICATION IN THE NUCLEI OF THE SLIME MOLD PHYSARUM POLYCEPHALUM

1968 ◽  
Vol 37 (3) ◽  
pp. 761-772 ◽  
Author(s):  
Sophie Guttes ◽  
Edmund Guttes
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Physarum Polycephalum ◽  
Slime Mold ◽  
S Phase ◽  
G2 Phase

Nuclei in G2 phase of the slime mold Physarum polycephalum, when transplanted, by plasmodial coalescence, into an S-phase plasmodium, failed to start another round of DNA synthesis. In the reciprocal combination, S-phase nuclei in a G2-phase host continued DNA synthesis for several hours without appreciable decrease in rate. It is suggested that the beginning of DNA replication is determined by an event, either during or shortly after mitosis, which renders the chromosomes structurally competent for DNA replication.

scholarly journals Microbubbles in replicating nuclear deoxyribonucleic acid from Physarum polycephalum

1979 ◽  
Vol 183 (2) ◽  
pp. 477-480 ◽  
Author(s):  
D A F Gillespie ◽  
N Hardman
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Dna Synthesis ◽  
Physarum Polycephalum ◽  
Deoxyribonucleic Acid ◽  
Nuclear Dna ◽  
S Phase ◽  
Dna Structures ◽  
Nuclear Deoxyribonucleic Acid

Clusters of microbubbles, represent probable sites of newly initiated DNA synthesis, were identified in nuclear DNA from Physarum polycephalum by using the electron microscope. Their presence is associated specifically with S-phase. Each microbubble corresponds in size to a replicating segment of DNA about 100-5000 nucleotide residues in length. The DNA structures containing microbubbles are metastable, and revert to native DNA in the presence of moderate concentrations of formamide used to prepare samples for electron microscopy. It is suggested that each cluster of microbubbles may correspond to a unit of replication (a replicon) in Physarum DNA.

scholarly journals THE EFFECT OF ACTIDIONE ON MITOSIS IN THE SLIME MOLD PHYSARUM POLYCEPHALUM

1965 ◽  
Vol 27 (2) ◽  
pp. 337-341 ◽  
Author(s):  
J. E. Cummins ◽  
E. N. Brewer ◽  
H. P. Rusch
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Nucleic Acid ◽  
Physarum Polycephalum ◽  
Specific Inhibitor ◽  
Slime Mold ◽  
Amino Acid Incorporation ◽  
Late Prophase ◽  
Acid Incorporation ◽  
Inhibitor Of Protein Synthesis

Actidione, reportedly a specific inhibitor of protein synthesis, was found to reduce the incorporation of labeled amino acids into proteins of the slime mold Physarum polycephalum without drastically inhibiting the incorporation of nucleic acid precursors into RNA. This inhibitor was found to completely block the ensuing mitosis if it was added at any time between telophase and late prophase. Plasmodia given Actidione in late prophase (about the time of nucleolar dissolution) went on through telophase to reconstruction even though nuclear amino acid incorporation was drastically reduced during that period.

The Inifiation, Maintenance and Termination DNA Synthesis: A Study of Nuclear DNA Replication Using Amoeba Proteus As a Cell Model

1971 ◽  
Vol 9 (1) ◽  
pp. 1-21
Author(s):  
M. J. ORD
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Nuclear Dna ◽  
Thymidine Incorporation ◽  
Cell Model ◽  
Amoeba Proteus ◽  
Early Prophase ◽  
Precursor Pool ◽  
Endogenous Precursor ◽  
The Individual

By means of the nuclear transfer technique for amoeba, combinations of nuclei and cytoplasma from all parts of the cell cycle were available for examining the individual roles of the nucleus and cytoplasm in nuclear DNA replication. Neither S-phase nor division sphere cytoplasm proved capable of initiating a new round of nuclear DNA synthesis in the G2 nucleus. There was some indication that G2 nuclei which were transferred into early prophase cells, i.e. before the formation of a regular division sphere, did incorporate more [3H]thymidine than control G2 nuclei. Positive proof of the induction of DNA synthesis in ‘immature’ nuclei was observed in only two cases. When young G2 nuclei were transplanted into late G2 amoebae, the addition of the donor nucleus generally resulted in the older nucleus being held in a late G2 phase until the younger nucleus passed through its G2. Division of 90% of heterophasic homokaryons was synchronous, with a subsequent synchrony of DNA synthesis. A study of variance in [3H]thymidine incorporation by S nuclei sharing the same cytoplasm - using binucleate, trinucleate and multinucleate homokaryons - showed that nuclei through the peak-S period synthesized DNA at approximately similar rates. The large differences in [3H]thymidine incorporation by nuclei of amoebae of equal age appear due to differences in endogenous precursor pools. These would vary both with differences in food intake and with the draining of remote precursor pools for simultaneous cellular activities, particularly RNA synthesis. When sharing the same cytoplasm nuclei in peak S incorporated similar amounts of [3H]thymidine. Though cytoplasm did not influence the progress of DNA replication by a nucleus, it did influence the use of exogenous [3H]thymidine by the cell, and in so doing caused much of the variation observed in the labelling of nuclei during S. Nuclei sharing the same cytoplasm, and so subject to the same precursor pool changes, incorporated similar amounts of exogenous thymidine. Once DNA synthesis had been initiated it continued to completion regardless of the cytoplasm which surrounded it. Thus neither the maintenance nor termination of DNA synthesis required a special cytoplasmic state.

scholarly journals DNA replication in Physarum polycephalum: electron microscopic analysis of patterns of DNA replication in the presence of cycloheximide.

1982 ◽  
Vol 95 (1) ◽  
pp. 323-331 ◽  
Author(s):  
F Haugli ◽  
R Andreassen ◽  
S Funderud
Keyword(s):  
Protein Synthesis ◽  
Dna Replication ◽  
Physarum Polycephalum ◽  
Replication Fork ◽  
Microscopic Analysis ◽  
S Phase ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Electron Microscopic ◽  
Replication Fork Progression

DNA from synchronously replicating nuclei of Physarum polycephalum was studied electron microscopically after 15, 30, 60, and 90 or 120 min of replication in the presence or absence of the protein synthesis inhibitor cycloheximide. The replication-loop size-distribution showed that replication fork progression is severely retarded in the presence of cycloheximide. Analysis of replication-loop frequency showed a similar pattern in control and cyclo-heximide-treated samples, with an increase from 15 to 30 and 60 min. This suggests, surprisingly, that initiations of new replicons either may not be inhibited by cycloheximide or, alternatively, that all initiations have already taken place at the very start of S-phase. The latter conclusion is favored in the light of previous results in our laboratory, discussed here.

scholarly journals MACROPHAGE-MELANOMA CELL HETEROKARYONS

1971 ◽  
Vol 134 (4) ◽  
pp. 935-946 ◽  
Author(s):  
Saimon Gordon ◽  
Zanvil Cohn
Keyword(s):  
Protein Synthesis ◽  
Dna Synthesis ◽  
Melanoma Cell ◽  
Melanoma Cells ◽  
Irreversible Inhibitor ◽  
Cell Pretreatment ◽  
Lag Period ◽  
Inhibitor Of Protein Synthesis ◽  
Physical Changes ◽  
The Relationship

Dormant macrophage nuclei initiate DNA synthesis 2–3 hr after fusion of macrophages with exponentially growing melanoma cells. Cycloheximide treatment (1–5 µg/ml) of heterokaryons during the preceding lag period inhibits the initiation of macrophage DNA synthesis, in a reversible fashion. Each type of cell was also treated with streptovitacin A, an irreversible inhibitor of protein synthesis. Pretreatment of the melanoma cells (0.5–2 µg/ml), 1 hr before fusion, inhibited the induction of macrophage DNA synthesis in heterokaryons, whereas pretreatment of macrophages (1–20 µg/ml) had no effect. Melanoma cell pretreatment reduced the incorporation of leucine-3H into the cytoplasm and nuclei of heterokaryons, whereas macrophage pretreatment had no effect. These experiments suggested that melanoma proteins played an important role in the initiation of macrophage DNA synthesis. The relationship between the melanoma cell cycle and macrophage DNA synthesis was studied with synchronous melanoma cells. If the melanoma cells were in S phase at the time of fusion, macrophage DNA synthesis occurred 2 hr later. However, the fusion of melanoma cells in G1 delayed macrophage DNA synthesis until the melanoma nuclei had entered S. Experiments with actinomycin and cycloheximide showed that RNA and protein, essential to achieve DNA synthesis in the macrophage nucleus, were made during late G1 as well as S. Melanoma cells and macrophages differ in their radiolabeled acid-soluble products after incubation in thymidine-3H. Thymidine taken up by the macrophage remained unphosphorylated, whereas it was recovered mainly as thymidine triphosphate from melanoma cells. These findings, as well as those reported previously, suggest that the melanoma cell provides the RNA, protein, and precursors which initiate macrophage DNA synthesis. In the absence of a requirement for new macrophage RNA and protein synthesis, other changes must be responsible for the 2 hr delay in DNA synthesis. These may involve physical changes in DNA, associated with swelling, as well as the transport of melanoma products into the macrophage nucleus.

scholarly journals Human Cyclin a Is Required for Mitosis until Mid Prophase

1999 ◽  
Vol 147 (2) ◽  
pp. 295-306 ◽  
Author(s):  
Nobuaki Furuno ◽  
Nicole den Elzen ◽  
Jonathon Pines
Keyword(s):  
Cyclin A ◽  
Time Lapse ◽  
S Phase ◽  
Early Prophase ◽  
Cyclin Dependent Kinase ◽  
Late Prophase ◽  
Daughter Cells ◽  
G2 Phase ◽  
Rate Limiting

We have used microinjection and time-lapse video microscopy to study the role of cyclin A in mitosis. We have injected purified, active cyclin A/cyclin-dependent kinase 2 (CDK2) into synchronized cells at specific points in the cell cycle and assayed its effect on cell division. We find that cyclin A/CDK2 will drive G2 phase cells into mitosis within 30 min of microinjection, up to 4 h before control cells enter mitosis. Often this premature mitosis is abnormal; the chromosomes do not completely condense and daughter cells fuse. Remarkably, microinjecting cyclin A/CDK2 into S phase cells has no effect on progress through the following G2 phase or mitosis. In complementary experiments we have microinjected the amino terminus of p21Cip1/Waf1/Sdi1 (p21N) into cells to inhibit cyclin A/CDK2 activity. We find that p21N will prevent S phase or G2 phase cells from entering mitosis, and will cause early prophase cells to return to interphase. These results suggest that cyclin A/CDK2 is a rate-limiting component required for entry into mitosis, and for progress through mitosis until late prophase. They also suggest that cyclin A/CDK2 may be the target of the recently described prophase checkpoint.

scholarly journals MAMMALIAN CHROMOSOMES IN VITRO

1964 ◽  
Vol 23 (1) ◽  
pp. 53-62 ◽  
Author(s):  
T. C. Hsu
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Sex Chromosomes ◽  
Chinese Hamster ◽  
Distal Portion ◽  
S Phase ◽  
Chromosome 1 ◽  
Chromosome 6 ◽  
Chromosome 2

The complete DNA replication sequence of the entire complement of chromosomes in the Chinese hamster may be studied by using the method of continuous H3-thymidine labeling and the method of 5-fluorodeoxyuridine block with H3-thymidine pulse labeling as relief. Many chromosomes start DNA synthesis simultaneously at multiple sites, but the sex chromosomes (the Y and the long arm of the X) begin DNA replication approximately 4.5 hours later and are the last members of the complement to finish replication. Generally, chromosomes or segments of chromosomes that begin replication early complete it early, and those which begin late, complete it late. Many chromosomes bear characteristically late replicating regions. During the last hour of the S phase, the entire Y, the long arm of the X, and chromosomes 10 and 11 are heavily labeled. The short arm of chromosome 1, long arm of chromosome 2, distal portion of chromosome 6, and short arms of chromosomes 7, 8, and 9 are moderately labeled. The long arm of chromosome 1 and the short arm of chromosome 2 also have late replicating zones or bands. The centromeres of chromosomes 4 and 5, and occasionally a band on the short arm of the X are lightly labeled.

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