Preferential mitochondrial and plastid DNA synthesis before multiple cell divisions in Nicotiana tabacum

1992 ◽  
Vol 103 (3) ◽  
pp. 831-837 ◽  
Author(s):  
T. Suzuki ◽  
S. Kawano ◽  
A. Sakai ◽  
M. Fujie ◽  
H. Kuroiwa ◽  
...  
Keyword(s):  
Nicotiana Tabacum ◽  
Stationary Phase ◽  
Dna Synthesis ◽  
Cell Line ◽  
Nuclear Dna ◽  
Root Meristem ◽  
Low Frequency ◽  
Plastid Dna ◽  
Lag Phase ◽  
Quiescent Center

Organelle DNA synthesis in root meristem and cultured cell line BY-2, both derived from Nicotiana tabacum cv. Bright Yellow 2, was examined by immunofluorescence microscopy of Technovit sections with antibody against 5- bromodeoxyuridine (BrdU) and co-fluorescent staining with 4′,6-diamidino-2-phenylindole (DAPI) and quantitative Southern hybridization. In the root meristem, the mitochondrial DNAs (mtDNAs) were synthesized in a specific region near to the quiescent center, where a low frequency of DNA synthesis of cell nuclei was observed. The mitochondrial nuclei (nucleoids) changed morphologically from long ellipsoids with a high frequency of DNA synthesis, in the region just above the quiescent center, to granules with a low frequency of DNA synthesis, as cell distance from the quiescent center increased. Similar patterns were observed in the cultured tobacco cell line (BY-2), in which large amounts of preferential synthesis of DNA of both mitochondria and plastids occurred prior to cell nuclear DNA synthesis just after stationary phase cells were transferred to fresh medium. Granular mitochondria which vigorously synthesized mtDNA were observed in both lag phase and logarithmic growth phase cells. However, long, ellipsoidal mitochondria which showed a low frequency of mtDNA synthesis were observed in stationary phase cells. Morphological changes of plastids were more conspicuous than those of mitochondria. After the medium was renewed, spherical plastids became extremely elongated and string-like, for 24 h, but were divided into small pieces after the third day. Vigorous synthesis of plastid DNA (ptDNA) occurred during this period of plastids elongation.

scholarly journals Proplastid Dna Synthesis at Pachytene in Pollen Mother Cells of Lilium Henryi

1982 ◽  
Vol 56 (1) ◽  
pp. 293-302
Author(s):  
D. R. SMYTH
Keyword(s):  
Dna Synthesis ◽  
Dna Sequences ◽  
Nuclear Dna ◽  
Buoyant Density ◽  
Plastid Dna ◽  
Electron Microscopic ◽  
Pollen Mother Cells ◽  
Specific Hybridization ◽  
Chloroplast Dna Sequences ◽  
Mother Cells

About three-quarters of the DNA synthesis occurring in pachytene pollen mother cells of Lilium henryi takes place in proplastids. Only around 15% can be attributed to mitochondrial labelling and 10% to nuclear DNA synthesis. Label was identified in the proplastid genome by its location in electron microscopic autoradiographs, by its buoyant density (1.698 g/ml), and by its specific hybridization to chloroplast DNA sequences from spinach. Proplastids, while apparently not dividing at pachytene, may be replicating their DNA in readiness for subsequent proliferation in developing microspores. The annealing properties of plastid DNA closely parallel those of labelled pachytene DNA sequences implicated in meiotic exchange events.

Studies on the Behavior of Mitochondrial DNA

1992 ◽  
Vol 101 (3) ◽  
pp. 483-493
Author(s):  
TSUNEYOSHI KUROIWA ◽  
MAKOTO FUJIE ◽  
HARUKO KUROIWA
Keyword(s):  
Mitochondrial Dna ◽  
Mitotic Cycle ◽  
Nuclear Dna ◽  
Root Meristem ◽  
Photon Counting ◽  
S Phase ◽  
Quiescent Center ◽  
Elongation Zone ◽  
Pelargonium Zonale ◽  
Thin Sections

The fate of mitochondrial nuclei (known as nucleoids or mt-nuclei), which contain extremely small amounts of DNA, was followed in thin sections of the root meristem of Pelargonium zonale by embedding of samples in Technovit 7100 resin and double staining with 4′-6-diamidino-2-phenylindole (DAPI) and acridine orange, in combination with light-microscopic autoradiography and microphotometry. The synthesis of cell-nuclear DNA and cell division occurs actively in the root meristem, between 150 μm and 700 μm from the tip of the root. For simplicity, cells in S phase in the cortex were selected for main analysis as the model system for examination of cell proliferation. It is estimated, on the basis of the length of the cells in longitudinal median sections, that the cells in the cortex, which are generated in the area just above the quiescent center (QC) about 150 μm from the tip, enter the elongation zone after at least five divisions. In the entire cortex, individual cells in S phase have approximately 230 mitochondria that each contain one mt-nucleus. The observation suggests that individual mitochondria divide once per mitotic cycle in the entire region of the meristem. By contrast, on the basis of incorporation of [3H]thymidine into mt-nuclei, the synthesis of mitochondrial DNA (mtDNA) occurs independently of the mitotic cycle in a restricted region just above the QC. Fluorimetry, using a video-intensified microscope photon-counting system (VIMPICS), revealed that the mtDNA content per mt-nucleus in the cells just above QC, where the synthesis of mtDNA is active, corresponds to approximately 3000 kilobase pairs (kbp) but, in the meristematic cells just below the elongation zone of the root it falls to less than 170 kbp. These findings strongly suggest that the amount of mtDNA per mitochondrion which has been synthesized in the region just above the QC is reduced stepwise as a result of continuous divisions of mitochondria in the absence of the synthesis of mtDNA. This phenomenon would explain why differentiated cells with a large vacuole in the elongation zone have mitochondria that contain only extremely small amounts of mtDNA.

scholarly journals Involvement of deoxyribonucleic acid polymerase β in nuclear deoxyribonucleic acid synthesis

1978 ◽  
Vol 173 (1) ◽  
pp. 309-314 ◽  
Author(s):  
T R Butt ◽  
W M Wood ◽  
E L McKay ◽  
R L P Adams
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Nuclear Dna ◽  
Dna Polymerase Beta ◽  
Cell Nuclei ◽  
High Molecular Weight Dna ◽  
Dna Polymerase Alpha ◽  
Polymerase Beta

The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.

Radiation effects on DNA synthesis in a defined chromosomal replicon

1994 ◽  
Vol 14 (3) ◽  
pp. 1901-1908
Author(s):  
J M Larner ◽  
H Lee ◽  
J L Hamlin
Keyword(s):  
Dna Synthesis ◽  
Cell Line ◽  
Radiation Effects ◽  
Signal Transduction Pathway ◽  
Mapping Method ◽  
G1 Arrest ◽  
Tumor Suppressor P53 ◽  
Damage Sensing ◽  
Direct Demonstration ◽  
S Period

It has recently been shown that the tumor suppressor p53 mediates a signal transduction pathway that responds to DNA damage by arresting cells in the late G1 period of the cell cycle. However, the operation of this pathway alone cannot explain the 50% reduction in the rate of DNA synthesis that occurs within 30 min of irradiation of an asynchronous cell population. We are using the amplified dihydrofolate reductase (DHFR) domain in the methotrexate-resistant CHO cell line, CHOC 400, as a model replicon in which to study this acute radiation effect. We first show that the CHOC 400 cell line retains the classical acute-phase response but does not display the late G1 arrest that characterizes the p53-mediated checkpoint. Using a two-dimensional gel replicon-mapping method, we then show that when asynchronous cultures are irradiated with 900 cGy, initiation in the DHFR locus is completely inhibited within 30 min and does not resume for 3 to 4 h. Since initiation in this locus occurs throughout the first 2 h of the S period, this result implies the existence of a p53-independent S-phase damage-sensing pathway that functions at the level of individual origins. Results obtained with the replication inhibitor mimosine define a position near the G1/S boundary beyond which cells are unable to prevent initiation at early-firing origins in response to irradiation. This is the first direct demonstration at a defined chromosomal origin that radiation quantitatively down-regulates initiation.

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.

Continuous DNA replication in the nucleus of the dinoflagellate Prorocentrum micans (Ehrenberg)

1977 ◽  
Vol 27 (1) ◽  
pp. 81-90
Author(s):  
S.A. Filfilan ◽  
D.C. Sigee
Keyword(s):  
Electron Microscope ◽  
Dna Synthesis ◽  
Nuclear Dna ◽  
Continuous Process ◽  
Prorocentrum Micans ◽  
Interphase Nuclei ◽  
Electron Microscope Autoradiography ◽  
Dividing Cells ◽  
High Degree ◽  
Very High

The uptake of tritiated thymine into cells of a heterogeneous population of Prorocentrum micans was investigated using light-microscope and electron-microscope autoradiography. Specificity of thymine uptake into DNA was demonstrated by the specific removal of label from wax-embedded material using DNase and by the high degree of localization of nuclear label to chromosomes in the electron-microscope autoradiographs. All nuclei, including both dividing and non-dividing cells, showed a substantial uptake of label, indicating that nuclear DNA synthesis in Prorocentrum micans is a continuous process. The level of DNA synthesis does show considerable variation, however, with very high levels in some interphase nuclei. The continuous replication of nuclear DNA provides further evidence of dinoflagellate affinity to the prokaryotes, and indicates that Prorocentrum micans is a very primitive eukaryote cell.

scholarly journals Meiotic DNA synthesis during mouse spermatogenesis.

1975 ◽  
Vol 64 (1) ◽  
pp. 211-222 ◽  
Author(s):  
M L Meistrich ◽  
B O Reid ◽  
W J Barcellona
Keyword(s):  
Dna Synthesis ◽  
Nuclear Dna ◽  
Sodium Dodecyl ◽  
Phenol Extraction ◽  
Sperm Dna ◽  
Sperm Nuclei ◽  
Cauda Epididymidis ◽  
Contamination Levels ◽  
Triton X ◽  
Mechanical Shearing

The incorporation of radioactivity into various cells in the sequence of spermatogenesis was measured by preparing highly purified spermatozoan nuclei from the cauda epididymidis of mice at daily intervals after injection of (3H)thymidine. The stages of differentiation of these sperm at the time of thymidine administration were calculated from the kinetics of spermatogenesis. The procedure for purification of sperm nuclei included sonication, mechanical shearing, and treatment with trypsin, DNase, Triton X-100, 2M NaC1, and sodium dodecyl sulfate. DNA was isolated from these nuclei by treatment with dithiothreitol and pronase, followed by phenol extraction and ethanol precipitation. The levels of radioactivity in the epididymal sperm head preparations were low (less than 13 dpm/mouse) for 27 days after injection, and then rose dramatically to over 4 times 104 dpm/mouse. Further experiments demonstrated that the 11 dpm of 3H radioactivity contained in sperm heads at 21 or 26 days after injection of (3H)TdR was significantly above background and contamination levels from other cells or other sources. Most of the radioactivity was in the sperm DNA and represented incorporation of tritium from (3H)TdR into the nuclear DNA of meiotic cells at 0.002 percent of the rate of incorporation into S-phase cells. Little, if any, (3H)TdR was incorporation into the DNA of spermatids. The levels of DNA synthesis during the meiotic prophase in the mouse appear to be much lower than those reported for other organisms.

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