Some evidence for replication-transcription coupling in Physarum polycephalum

1975 ◽  
Vol 18 (1) ◽  
pp. 27-39
Author(s):  
H. Fouquet ◽  
R. Bohme ◽  
R. Wick ◽  
H.W. Sauer ◽  
K. Scheller
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Dna Sequences ◽  
Physarum Polycephalum ◽  
Rna Synthesis ◽  
Selective Inhibition ◽  
S Phase ◽  
Dna Molecules ◽  
Uridine Incorporation ◽  
G2 Phase

Hydroxyurea, at concentrations of 40–60 mM, selectively and effectively blocked incorporation of thymidine into DNA. Inhibition occurred within 5–10 min of application of the agent when DNA synthesis was in progress, while the onset of replication at the beginning of S-phase and DNA synthesis in G2 phase were not affected. Uridine incorporation into TCA-precipitable material, in the presence of hydroxyurea, was significantly (up to 70%) inhibited in early S-phase of the cell cycle. Selective inhibition of RNA synthesis was confirmed for RNA separated into rRNA-rich and poly(A)-rich RNA fractions and analysed by the 2 kinds of DNA-RNA hybridization reactions. Uridine incorporation into poly (A) RNA was also inhibited under conditions where cycloheximide prevented maturation of nascent DNA molecules in early S-phase. We assume that chromatin which is replicating early DNA sequences may be a more competent template for transcription.

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.

Control of histone gene expression in Physarum polycephalum. I. Protein synthesis during the cell cycle

1982 ◽  
Vol 57 (1) ◽  
pp. 139-150
Author(s):  
P.N. Schofield ◽  
I.O. Walker
Keyword(s):  
Cell Cycle ◽  
Physarum Polycephalum ◽  
Specific Activity ◽  
S Phase ◽  
Histone Gene ◽  
Synchronous Cultures ◽  
Histone Gene Expression ◽  
Core Histones ◽  
G2 Phase ◽  
Histone Synthesis

Synchronous cultures of Physarum polycephalum were pulsed with [3H]lysine hydrochloride in S and G2 phases of the cell cycle. Plasmodial extracts were separated into nuclear, ribosomal and acid-soluble post-ribosomal cytoplasmic fractions. Core histones could be detected by staining in the nuclear fractions of both S and G2 phases, but were not detected by staining in the cytoplasmic fractions. Newly synthesized histone was present in S-phase nuclei but not in S-phase cytoplasm. The specific activity of newly synthesized histone in G2-phase nuclei decreased by at least 95% compared to S phase and no newly synthesized histone was observed in G2-phase cytoplasmic fractions. Thus histone synthesis is restricted to S phase. There are no free pools of histone in the cytoplasm of Physarum in either S or G2 phases of the cell cycle.

scholarly journals Distinct replication-independent and -dependent phases of histone gene expression during the Physarum cell cycle.

1987 ◽  
Vol 7 (5) ◽  
pp. 1933-1937 ◽  
Author(s):  
J J Carrino ◽  
V Kueng ◽  
R Braun ◽  
T G Laffler
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
S Phase ◽  
Histone Gene ◽  
Histone Mrna ◽  
Histone Gene Expression ◽  
Tightly Coupled ◽  
G2 Phase

During the S phase of the cell cycle, histone gene expression and DNA replication are tightly coupled. In mitotically synchronous plasmodia of the myxomycete Physarum polycephalum, which has no G1 phase, histone mRNA synthesis begins in mid-G2 phase. Although histone gene transcription is activated in the absence of significant DNA synthesis, our data demonstrate that histone gene expression became tightly coupled to DNA replication once the S phase began. There was a transition from the replication-independent phase to the replication-dependent phase of histone gene expression. During the first phase, histone mRNA synthesis appears to be under direct cell cycle control; it was not coupled to DNA replication. This allowed a pool of histone mRNA to accumulate in late G2 phase, in anticipation of future demand. The second phase began at the end of mitosis, when the S phase began, and expression became homeostatically coupled to DNA replication. This homeostatic control required continuing protein synthesis, since cycloheximide uncoupled transcription from DNA synthesis. Nuclear run-on assays suggest that in P. polycephalum this coupling occurs at the level of transcription. While histone gene transcription appears to be directly switched on in mid-G2 phase and off at the end of the S phase by cell cycle regulators, only during the S phase was the level of transcription balanced with the rate of DNA synthesis.

Distinct replication-independent and -dependent phases of histone gene expression during the Physarum cell cycle

1987 ◽  
Vol 7 (5) ◽  
pp. 1933-1937
Author(s):  
J J Carrino ◽  
V Kueng ◽  
R Braun ◽  
T G Laffler
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
S Phase ◽  
Histone Gene ◽  
Histone Mrna ◽  
Histone Gene Expression ◽  
Tightly Coupled ◽  
G2 Phase

During the S phase of the cell cycle, histone gene expression and DNA replication are tightly coupled. In mitotically synchronous plasmodia of the myxomycete Physarum polycephalum, which has no G1 phase, histone mRNA synthesis begins in mid-G2 phase. Although histone gene transcription is activated in the absence of significant DNA synthesis, our data demonstrate that histone gene expression became tightly coupled to DNA replication once the S phase began. There was a transition from the replication-independent phase to the replication-dependent phase of histone gene expression. During the first phase, histone mRNA synthesis appears to be under direct cell cycle control; it was not coupled to DNA replication. This allowed a pool of histone mRNA to accumulate in late G2 phase, in anticipation of future demand. The second phase began at the end of mitosis, when the S phase began, and expression became homeostatically coupled to DNA replication. This homeostatic control required continuing protein synthesis, since cycloheximide uncoupled transcription from DNA synthesis. Nuclear run-on assays suggest that in P. polycephalum this coupling occurs at the level of transcription. While histone gene transcription appears to be directly switched on in mid-G2 phase and off at the end of the S phase by cell cycle regulators, only during the S phase was the level of transcription balanced with the rate of DNA synthesis.

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.

SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase

1993 ◽  
Vol 13 (9) ◽  
pp. 5829-5842
Author(s):  
P Zheng ◽  
D S Fay ◽  
J Burton ◽  
H Xiao ◽  
J L Pinkham ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Synthesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Low Frequency ◽  
S Phase ◽  
Upstream Region ◽  
Specific Gene

SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein. Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L). Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated. These colonies were greatly enriched for budded cells, especially those with large buds. Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency. Spk1 protein was identified by immunoprecipitation and immunoblotting. It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays. Spk1 was localized to the nucleus by immunofluorescence. The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements. These elements confer S-phase-specific transcription to many genes involved in DNA synthesis. Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes. The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage. spk1 strains were hypersensitive to UV irradiation. Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability. The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis.

The Schizosaccharomyces pombe hus5 gene encodes a ubiquitin conjugating enzyme required for normal mitosis

1995 ◽  
Vol 108 (2) ◽  
pp. 475-486 ◽  
Author(s):  
F. al-Khodairy ◽  
T. Enoch ◽  
I.M. Hagan ◽  
A.M. Carr
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Cell Cycle Control ◽  
S Phase ◽  
Temperature Sensitive ◽  
Checkpoint Control ◽  
Ubiquitin Conjugating Enzyme ◽  
Efficient Recovery ◽  
Mediate Cell

Normal eukaryotic cells do not enter mitosis unless DNA is fully replicated and repaired. Controls called ‘checkpoints’, mediate cell cycle arrest in response to unreplicated or damaged DNA. Two independent Schizosaccharomyces pombe mutant screens, both of which aimed to isolate new elements involved in checkpoint controls, have identified alleles of the hus5+ gene that are abnormally sensitive to both inhibitors of DNA synthesis and to ionizing radiation. We have cloned and sequenced the hus5+ gene. It is a novel member of the E2 family of ubiquitin conjugating enzymes (UBCs). To understand the role of hus5+ in cell cycle control we have characterized the phenotypes of the hus5 mutants and the hus5 gene disruption. We find that, whilst the mutants are sensitive to inhibitors of DNA synthesis and to irradiation, this is not due to an inability to undergo mitotic arrest. Thus, the hus5+ gene product is not directly involved in checkpoint control. However, in common with a large class of previously characterized checkpoint genes, it is required for efficient recovery from DNA damage or S-phase arrest and manifests a rapid death phenotype in combination with a temperature sensitive S phase and late S/G2 phase cdc mutants. In addition, hus5 deletion mutants are severely impaired in growth and exhibit high levels of abortive mitoses, suggesting a role for hus5+ in chromosome segregation. We conclude that this novel UBC enzyme plays multiple roles and is virtually essential for cell proliferation.

Biochemical and morphological characterization of the nuclear matrix during the synchronous cell cycle of Physarum polycephalum

1993 ◽  
Vol 105 (4) ◽  
pp. 1121-1130 ◽  
Author(s):  
S. Lang ◽  
T. Decristoforo ◽  
W. Waitz ◽  
P. Loidl
Keyword(s):  
Cell Cycle ◽  
Nuclear Matrix ◽  
Physarum Polycephalum ◽  
Nuclear Periphery ◽  
S Phase ◽  
Cycle Phase ◽  
Electron Microscopic ◽  
Nuclear Lamins ◽  
Synchronous Cell ◽  
Matrix Bound

We have investigated biochemical and ultrastructural aspects of the nuclear matrix during the naturally synchronous cell cycle of Physarum polycephalum. The morphology of the in situ nuclear matrix exhibited significant cell cycle changes as revealed by electron microscopic examination, especially during the progression of nuclei through mitosis and S-phase. In mitosis the interchromatin matrix was found to be retracted to the nuclear periphery; during S-phase this interchromatin matrix gradually resembled, concomitant with the reconstruction of a nucleolar remnant structure. During the G2-period no significant changes in matrix morphology were observed. The pattern of nuclear matrix proteins was invariant during the cell cycle; no cycle phase-specific proteins could be detected. In vivo labelling of plasmodia with [35S]methionine/cysteine showed that only a few proteins are synthesized and assembled into nuclear matrix structures in a cell cycle-dependent way; the majority of proteins were synthesized almost continuously. This was also shown for nuclear lamins homologues. In contrast to bulk nuclear histones, those histones that remain tightly bound to the nuclear matrix were synthesized and assembled into nuclear structures in the very first hour of S-phase; assembly was terminated in mid-S-phase, indicating that nuclear matrix-bound chromatin is replicated early in S-phase. Comparison of the acetylation pattern of matrix-bound histone H4 with bulk nuclear H4 revealed a largely elevated acetate content of matrix H4. The percentage of acetylated subspecies was entirely different from that in bulk nuclear H4, indicating that matrix-associated histones represent a subpopulation of nuclear histones with distinct properties, reflecting specific structural requirements of matrix-attached chromatin.

Excision and replication of extrachromosomal DNA of pea (Pisum sativum)

1983 ◽  
Vol 3 (2) ◽  
pp. 172-181
Author(s):  
J Van't Hof ◽  
C A Bjerknes ◽  
N C Delihas
Keyword(s):  
Cell Cycle ◽  
Pisum Sativum ◽  
S Phase ◽  
Velocity Sedimentation ◽  
Extrachromosomal Dna ◽  
Chromosomal Dna ◽  
Root Tips ◽  
Dividing Cells ◽  
G2 Phase ◽  
Pea Roots

Experiments with cultured pea roots were conducted to determine (i) whether extrachromosomal DNA was produced by cells in the late S phase or in the G2 phase of the cell cycle, (ii) whether the maturation of nascent DNA replicated by these cells achieved chromosomal size, (iii) when extrachromosomal DNA was removed from the chromosomal duplex, and (iv) the replication of nascent chains by the extrachromosomal DNA after its release from the chromosomal duplex. Autoradiography and cytophotometry of cells of carbohydrate-starved root tips revealed that extrachromosomal DNA was produced by a small fraction of cells accumulated in the late S phase after they had replicated about 80% of their DNA. Velocity sedimentation of nascent chromosomal DNA in alkaline sucrose gradients indicated that the DNA of cells in the late S phase failed to achieve chromosomal size. After reaching sizes of 70 X 10(6) to 140 X 10(6) daltons, some of the nascent chromosomal molecules were broken, presumably releasing extrachromosomal DNA several hours later. Sedimentation of selectively extracted extrachromosomal DNA either from dividing cells or from those in the late S phase showed that it replicated two nascent chains, one of 3 X 10(6) daltons and another of 7 X 10(6) daltons. Larger molecules of extrachromosomal DNA were detectable after cells were labeled for 24 h. These two observations were compatible with the idea that the extrachromosomal DNA was first replicated as an integral part of the chromosomal duplex, was cut from the duplex, and then, once free of the chromosome, replicated two smaller chains of 3 X 10(6) and 7 X 10(6) daltons.

scholarly journals Enhancement of DNA-mediated gene transfer by high-Mr carrier DNA in synchronized CV-1 cells

1985 ◽  
Vol 225 (2) ◽  
pp. 529-533 ◽  
Author(s):  
A J Strain ◽  
W A H Wallace ◽  
A H Wyllie
Keyword(s):  
Cell Cycle ◽  
Gene Transfer ◽  
Transfection Efficiency ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
S Phase ◽  
Sv40 Virus ◽  
G2 Phase ◽  
Cycle Dependent ◽  
Sv40 Dna

Synchronized CV-1 cells were transfected with SV40 (simian virus 40) DNA-calcium phosphate co-precipitates. In the presence of carrier DNA, the transfection efficiency of SV40 DNA was decreased 5-fold in S-phase cells and was increased 4-fold in preparations of mitotically enriched cells as compared with asynchronous controls. No difference was observed when carrier DNA was omitted, when cells had progressed through S-phase and into G2-phase, or when the infectivity of cells to intact SV40 virus was tested. These results highlight the importance of cell-cycle-dependent factors on DNA-mediated gene transfer.

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