scholarly journals DNA REPLICATION: DIRECTION AND RATE OF CHAIN GROWTH IN MAMMALIAN CELLS

1973 ◽  
Vol 58 (2) ◽  
pp. 410-418 ◽  
Author(s):  
Roger Hand ◽  
Igor Tamm
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Thymidine Incorporation ◽  
Specific Activity ◽  
High Specific Activity ◽  
Chain Elongation ◽  
Prolonged Treatment ◽  
Chain Growth ◽  
The Common ◽  
Dna Chain

Using pulse labeling techniques with [3H]thymidine or [3H]cytidine, combined with DNA fiber autoradiography, we have investigated the direction and rate of DNA chain growth in mammalian cells. In general, chain elongation proceeds bidirectionally from the common origin of pairs of adjacent replication sections. This type of replication is noted whether the DNA is labeled first with [3H]thymidine of high specific activity, followed by [3H]thymidine of low specific activity or the sequence is reversed. Approximately one-fifth of the growing points have unique origins and in these replication units, chain growth proceeds in one direction only. Fluorodeoxyuridine and hydroxyurea both inhibit DNA chain propagation. Fluorodeoxyuridine exerts its effect on chain growth within 15–23 min, while the effect of hydroxyurea is evident within 15 min under conditions where the endogenous thymidine pool has been depleted by prior treatment with fluorodeoxyuridine. Puromycin has no effect on chain growth until 60 min after addition of the compound, even though thymidine incorporation is more than 50% reduced within 15 min. After 2 h of treatment with puromycin, the rate of chain growth is reduced by 50%, whereas thymidine incorporation is reduced by 75%. Cycloheximide reduces the rates of DNA chain growth and thymidine incorporation 50% within 15 min, and, on prolonged treatment, the decrease in rate of chain growth generally parallels the reduction in thymidine incorporation.

scholarly journals DNA replication in mammalian cells. Altered patterns of initiation during inhibition of protein synthesis.

1975 ◽  
Vol 67 (3) ◽  
pp. 761-773 ◽  
Author(s):  
R Hand
Keyword(s):  
Protein Synthesis ◽  
Dna Replication ◽  
Antibiotic Treatment ◽  
Mammalian Cells ◽  
Specific Activity ◽  
High Specific Activity ◽  
Normal Pattern ◽  
Inhibition Of Protein Synthesis ◽  
Initiation Of Dna Replication ◽  
Cellular Dna

The effects of inhibition of protein synthesis by the antibiotics cycloheximide and puromycin on the initiation of DNA replication in mouse L cells were studied. Cellular DNA was pulse labeled with [3H]thymidine of high, then of low specific activity and prepared for fiber autoradiography. Autoradiograms containing multiple (up to four) replication units were analyzed. In control cells, the proportion of replication units that initiated during a 10-min, high specific activity pulse was approximately equal to the proportion initiating immediately before the pulse. The addition of cycloheximide or puromycin at the start of the pulse inhibited the frequency of initiation in that there was a decrease by up to one-third of units initiating during the pulse relative to controls. Replication direction was also altered. Addition of the antibiotics 2 h before the pulse reduced the proportion of bidirectional units observed from 0.98 to 0.70. Antibiotic treatment for 2 h also decreased initiation synchrony in that the proportion of multiunit autoradiograms on which neighboring units showed similar replication patterns (indicating temporally coordinated initiation) was reduced by one-half. These observations indicate that inhibition of protein synthesis alters the normal pattern of DNA initiation.

scholarly journals The Human Tim/Tipin Complex Coordinates an Intra-S Checkpoint Response to UV That Slows Replication Fork Displacement

2007 ◽  
Vol 27 (8) ◽  
pp. 3131-3142 ◽  
Author(s):  
Keziban Ünsal-Kaçmaz ◽  
Paul D. Chastain ◽  
Ping-Ping Qu ◽  
Parviz Minoo ◽  
Marila Cordeiro-Stone ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Protein A ◽  
Chain Elongation ◽  
Replication Forks ◽  
Interacting Protein ◽  
Checkpoint Response ◽  
Replication Fork Progression ◽  
Dna Chain ◽  
Interfering Rna

ABSTRACT UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m2 UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.

THE CHARACTERISTICS AND GENETIC MAP LOCATION OF A TEMPERATURE SENSITIVE DNA MUTANT OF E. COLI K12

Genetics ◽  
1972 ◽  
Vol 72 (4) ◽  
pp. 569-593
Author(s):  
Beverly Wolf
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Chain Elongation ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Bacterial Dna ◽  
E Coli ◽  
Dna Chain ◽  
Inhibition Of Dna Synthesis ◽  
Dna Mutant

ABSTRACT A temperature sensitive strain of E. coli K12 has been isolated in which residual DNA synthesis occurs at the 40°C restrictive temperature; syntheses of RNA, protein and DNA precursors are not directly affected. The mutation has been designated dna-325 and is located at 89 min on the E. coli map in the same region where the dnaC locus is found. dnaC mutants are considered to be defective in DNA initiation. Some of the data are consistent with the view that the dna-325 mutation is temperature sensitive in the process of DNA initiation rather than DNA chain elongation: (1) more than two cell divisions occur after a shift to 40°C; (2) upon a shift down to 30°C, cell division occurs again only after the DNA content of the cells has doubled; (3) 80% more DNA is made at 30°C in the presence of chloramphenicol after prior inhibition of DNA synthesis at 40°C. These three observations indicate that rounds of DNA replication were completed at 40°C. Also (4) infective λ particles can be made at 40°C long after bacterial DNA replication has ceased. It appears however that some DNA initiation can occur at 40°C since (1) a limited amount of DNA synthesis does occur at 40°C after prior alignment of the chromosomes by amino acid starvation at 30°C, and (2) after incubation in bromouracil at the restrictive temperature, heavy DNA is found with both strands containing bromouracil.

Role for Megakaryocyte Phosphatidylinositol Transfer Proteins-Alpha and -Beta in TGF Beta-Mediated Regulation of Hematopoietic Homeostasis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 780-780
Author(s):  
Hal E. Broxmeyer ◽  
Maegan L. Capitano ◽  
Liang Zhao ◽  
Scott Cooper ◽  
Charles S. Abrams
Keyword(s):  
Colony Formation ◽  
Mammalian Cells ◽  
Conflicts Of Interest ◽  
Specific Activity ◽  
High Specific Activity ◽  
Homeostatic Regulation ◽  
Hematopoietic Stem ◽  
Phosphatidylinositol Transfer ◽  
Phosphatidylinositol Transfer Proteins

Abstract There are still unknowns regarding homeostatic regulation of hematopoietic stem (HSC) and progenitor (HPC) cells. Deciphering these processes are important for understanding and treating hematopoietic diseases. Phosphatidylinositol is a rare membrane structure lipid, but is critical for cellular signaling upon phosphorylation by lipid kinases to generate phosphoinositide. While phosphoinositide pathways contribute to events linked to the cytoskeleton, little is known of these pathways in regulating hematopoiesis. Critical to this pathway are phosphatidylinositol transfer proteins (PITPs) that in vitro enhance transfer of aqueous insoluble phosphatidylinositol from one membrane to another. Class I PITP proteins PITP α and β are highly conserved, small, and ubiquitously expressed in mammalian cells. To test the hypothesis that phosphatidylinositol signaling contributes to hematopoiesis, we generated conditional knock out mice that lack either PITPα single isoform (PITPαfl/fl PF4Cre+) or both PITPα and PITPβ (PITPαfl/fl βfl/fl PF4Cre+) specifically in their platelets and megakaryocytes, and observed a bone marrow (BM) HSC/HPC phenotype. BM from these mice and their littermate controls were evaluated for absolute numbers of nucleated cells, HSC, and HPC. Cells were analyzed by rigorous phenotyping for long-term (LT)-HSC, short-term (ST)-HSC, multipotential (MPP), common myeloid (CMP), and granulocyte macrophage (GMP) progenitors. They were also assessed for functional HPC by colony assays in vitro for multi-cytokine (Epo, GM-CSF, IL-3, SCF, hemin) stimulated granulocyte macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitors, and for their cycling status using a high specific activity tritiated thymidine kill assay. PITPα-/-, and to a greater extent PITPα/β-/-, progenitor cells demonstrated significant decreases in LT-HSC and ST-HSC per femur. While there were no significant changes in numbers of MPP, CMP, and GMP in the PITPα and PITPα/β-/- BM compared to controls, there were significant decreases of approximately 50% in numbers of CFU-GM, BFU-E, and CFU-GEMM per femur. PITC-/- HPC were in a slow or non-cycling state compared to the rapid cell cycle (40-57% in S-phase) of control HPC. Thus PITPα-/- and PITPα/β -/- BM cells were associated with decreased HSC and functional HPC numbers. To evaluate mechanisms for this phenotype, we focused on BM megakaryocytes, as they have been implicated in microenvironmental regulation of hematopoiesis, and PITPα and PITPα/β activities are associated with megakaryocyte/platelet function. BM derived TPO-culture expanded megakaryocytes were allowed to condition medium for 48 hours, and conditioned medium (CM) from PITPα-/-, PITPα/β-/-, and control BM megakaryocytes were assayed for effects on colony formation by multicytokine stimulated BM cells derived from normal mice. CM from PITPα-/- and PITPα/β-/- megakaryocytes, but not from control mice, significantly suppressed colony formation by CFU-GM, BFU-E and CFU-GEMM (by ~50%). Limiting dilution analysis of the CM demonstrated that PITPα/β-/- cells had more potent suppressor activity than PITPα-/- cells. Bioplex analysis of the CM from PITPα -/- and PITPα/β -/- megakaryocytes demonstrated significantly higher levels of cytokines/chemokines with known myelosuppressive activities (including: TNF-α, VEGF, LIF, IP-10, ENA-78, MDC, MIG, and MIP-1α). However, ELISA analysis of TGF-β1, demonstrated minimal protein in BM flushes from control mice, but large amounts of TGF-β (>350 pg/ml) in BM flushes from the PITPα/β -/- mice. CM from PITPα and α/β-/- megakaryocytes also contained highly elevated TGF-β protein. Thus, we hypothesized that the effect of PITP -/- on the suppression of HPC colony formation was mediated by TGF-β. The myelosuppressive CM derived from PITPα and PITPα/β -/- megakaryocytes was completely neutralized by a monoclonal TGF-β antibody. This demonstrates that PITPα and PITP α/β-/- megakaryocytes produce elevated TGF-β that at least in part, and possibly in synergy with other myelosuppressive cytokines/chemokines, decreases numbers of HSC and functional HPC. Our studies demonstrate a link between PITPα and α/β and TGF-β levels with significant effects on HSCs and HPCs, thus demonstrating involvement of the phosphoinositide pathway in homeostatic regulation of hematopoiesis. Disclosures No relevant conflicts of interest to declare.

The Relevance of Intra-Cellular Damage to Effects in the Complex Organism

1968 ◽  
Vol 70 (2) ◽  
pp. 152-162
Author(s):  
R. H. Mole
Keyword(s):  
Cell Division ◽  
Mammalian Cells ◽  
Radiation Effects ◽  
Specific Activity ◽  
A Priori ◽  
High Specific Activity ◽  
Cellular Damage ◽  
Complex Organism ◽  
Interesting Approach

As the three previous papers in this Symposium have shown (Ord and Stocken 1968; Evans 1968; Court-Brown 1968) the exploration of radiation effects within the cell has its own intrinsic fascination quite apart from its relevance to an understanding of the phenomena which may be observed after irradiation of the complex organism, vertebrate or invertebrate. Whitmore (cf. Whitmore Gulyas and Botond 1965) has been one of the pioneer workers on a previously unsuspected phenomenon, the quantitatively substantial recovery of the ability to divide which occurs within the first hour or two after exposure to low LET radiation. In cultures of mammalian cells in vitro this kind of recovery was shown to have nothing to do with cell division, to occur at all stages of the cell cycle, and to be little, if at all, affected by a reduction in temperature which would be expected a priori to modify profoundly the rate of chemical reactions. Whitmore's technique for synchronising asynchronously dividing cell cultures by the selective suicide of DNA synthesising cells following the incorporation of radioactively labelled DNA precursors with high specific activity has a wide application (Whitmore and Gulyas 1966). Workers from his laboratory have proposed an interesting approach to the real meaning of “ability to divide” and “recovery” based on the idea that there is a certain probability of failure at each cell division and that this probability may be permanently increased by exposure to radiation (Till, McCulloch and Siminovitch 1964). Thus radiation damage may be expressed by failure of cell division not only at the first or second divisions after exposure but also at any subsequent division in the distant future.

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