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 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 Lethal Action of Quinolones against a Temperature-Sensitive dnaB Replication Mutant of Escherichia coli

2006 ◽  
Vol 50 (1) ◽  
pp. 362-364 ◽  
Author(s):  
Xilin Zhao ◽  
Muhammad Malik ◽  
Nymph Chan ◽  
Alex Drlica-Wagner ◽  
Jian-Ying Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Dna Replication ◽  
Nalidixic Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Lethal Action ◽  
Inhibition Of Protein Synthesis

ABSTRACT Inhibition of DNA replication in an Escherichia coli dnaB-22 mutant failed to block quinolone-mediated lethality. Inhibition of protein synthesis by chloramphenicol inhibited nalidixic acid lethality and, to a lesser extent, ciprofloxacin lethality in both dnaB-22 and wild-type cells. Thus, major features of quinolone-mediated lethality do not depend on ongoing replication.

A murine replication protein accumulates temporarily in the heterochromatic regions of nuclei prior to initiation of DNA replication

1995 ◽  
Vol 108 (3) ◽  
pp. 927-934 ◽  
Author(s):  
M. Starborg ◽  
E. Brundell ◽  
K. Gell ◽  
C. Larsson ◽  
I. White ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Mammalian Cells ◽  
Mitotic Cell ◽  
Yeast Cells ◽  
Metaphase Chromosomes ◽  
Licensing Factor ◽  
Mammalian Homologue ◽  
Initiation Of Dna Replication

We have analyzed the expression of the murine P1 gene, the mammalian homologue of the yeast MCM3 protein, during the mitotic cell cycle. The MCM3 protein has previously been shown to be of importance for initiation of DNA replication in Saccharomyces cerevisiae. We found that the murine P1 protein was present in the nuclei of mammalian cells throughout interphase of the cell cycle. This is in contrast to the MCM3 protein, which is located in the nuclei of yeast cells only between the M and the S phase of the cell cycle. Detailed analysis of the intranuclear localization of the P1 protein during the cell cycle revealed that it accumulates transiently in the heterochromatic regions towards the end of G1. The accumulation of the P1 protein in the heterochromatic regions prior to activation of DNA replication suggests that the mammalian P1 protein is also of importance for initiation of DNA replication. The MCM2-3.5 proteins have been suggested to represent yeast equivalents of a hypothetical replication licensing factor initially described in Xenopus. Our data support this model and indicate that the murine P1 protein could function as replication licensing factor. The chromosomal localization of the P1 gene was determined by fluorescence in situ hybridization to region 6p12 in human metaphase chromosomes.

Temporal order of DNA replication in the H-2 major histocompatibility complex of the mouse

1992 ◽  
Vol 12 (11) ◽  
pp. 5174-5188
Author(s):  
E G Spack ◽  
E D Lewis ◽  
B Paradowski ◽  
R T Schimke ◽  
P P Jones
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Temporal Order ◽  
Chromosomal Region ◽  
S Phase ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
T Lymphoma Cells ◽  
Initiation Of Dna Replication ◽  
T Lymphoma

As an approach to mapping replicons in an extended chromosomal region, the temporal order of DNA replication was analyzed in the murine major histocompatibility gene complex (MHC). Replicating DNA from T-lymphoma and myelomonocyte cell lines was density labeled with bromodeoxyuridine and extracted from cells which had been fractionated into different stages of S phase by centrifugal elutriation. The replicating DNA from each fraction of S phase was separated from nonreplicating DNA on density gradients, blotted, and hybridized with 34 specific MHC probes. The earliest replication occurred in the vicinity of transcribed genes K, HAM1 and HAM2, RD, B144, D, L, T18, and T3. The temporal order of replication of groups of DNA segments suggests the location of five or six replicons within the H-2 complex, some of which appear to be either unidirectional or markedly asymmetric. The rates of replication through each of these apparent replicons appear to be similar. The TL region of the S49.1 T-lymphoma cells, which contains at least three transcribed genes, replicates earlier than the inactive TL region of WEHI-3 myelomonocytic cells. These results provide further evidence of a relationship between transcription and the initiation of DNA replication in mammalian cells. The mouse MHC examined in this study is the largest chromosomal region (> 2,000 kb) measured for timing of replication to date.

scholarly journals PR48, a Novel Regulatory Subunit of Protein Phosphatase 2A, Interacts with Cdc6 and Modulates DNA Replication in Human Cells

2000 ◽  
Vol 20 (3) ◽  
pp. 1021-1029 ◽  
Author(s):  
Zhen Yan ◽  
Sergei A. Fedorov ◽  
Marc C. Mumby ◽  
R. Sanders Williams
Keyword(s):  
Dna Replication ◽  
Protein Phosphatase ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Protein Phosphatase 2A ◽  
Specific Interaction ◽  
Regulatory Subunit ◽  
Amino Terminal ◽  
Phosphatase 2A ◽  
Initiation Of Dna Replication

ABSTRACT Initiation of DNA replication in eukaryotes is dependent on the activity of protein phosphatase 2A (PP2A), but specific phosphoprotein substrates pertinent to this requirement have not been identified. A novel regulatory subunit of PP2A, termed PR48, was identified by a yeast two-hybrid screen of a human placental cDNA library, using human Cdc6, an essential component of prereplicative complexes, as bait. PR48 binds specifically to an amino-terminal segment of Cdc6 and forms functional holoenzyme complexes with A and C subunits of PP2A. PR48 localizes to the nucleus of mammalian cells, and its forced overexpression perturbs cell cycle progression, causing a G1 arrest. These results suggest that dephosphorylation of Cdc6 by PP2A, mediated by a specific interaction with PR48, is a regulatory event controlling initiation of DNA replication in mammalian cells.

scholarly journals Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts.

1993 ◽  
Vol 123 (6) ◽  
pp. 1321-1331 ◽  
Author(s):  
Y Kubota ◽  
H Takisawa
Keyword(s):  
Dna Replication ◽  
Specific Activity ◽  
S Phase ◽  
Egg Cell ◽  
Sperm Dna ◽  
Before And After ◽  
M Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Initiation Of Dna Replication

Xenopus egg extracts prepared before and after egg activation retain M- and S-phase specific activity, respectively. Staurosporine, a potent inhibitor of protein kinase, converted M-phase extracts into interphase-like extracts that were capable of forming nuclei upon the addition of sperm DNA. The nuclei formed in the staurosporine treated M-phase extract were incapable of replicating DNA, and they were unable to initiate replication upon the addition of S-phase extracts. Furthermore, replication was inhibited when the staurosporine-treated M-phase extract was added in excess to the staurosporine-treated S-phase extract before the addition of DNA. The membrane-depleted S-phase extract supported neither nuclear formation nor replication; however, preincubation of sperm DNA with these extracts allowed them to form replication-competent nuclei upon the addition of excess staurosporine-treated M-phase extract. These results demonstrate that positive factors in the S-phase extracts determined the initiation of DNA replication before nuclear formation, although these factors were unable to initiate replication after nuclear formation.

scholarly journals Inhibition of protein synthesis and early protein processing by thapsigargin in cultured cells

1993 ◽  
Vol 289 (1) ◽  
pp. 71-79 ◽  
Author(s):  
W L Wong ◽  
M A Brostrom ◽  
G Kuznetsov ◽  
D Gmitter-Yellen ◽  
C O Brostrom
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Extracellular Fluid ◽  
Mrna Translation ◽  
Protein Processing ◽  
Pituitary Cells ◽  
Translational Initiation ◽  
Early Protein ◽  
Inhibition Of Protein Synthesis

Thapsigargin, a tumour-promoting sesquiterpene lactone, selectively inhibits the Ca(2+)-ATPase responsible for Ca2+ accumulation by the endoplasmic reticulum (ER). Mobilization of ER-sequestered Ca2+ to the cytosol and to the extracellular fluid subsequently ensues, with concomitant alteration of cellular functions. Thapsigargin was found to serve as a rapid, potent and efficacious inhibitor of amino acid incorporation in cultured mammalian cells. At concentrations mobilizing cell-associated Ca2+ to the extracellular fluid, thapsigargin provoked extensive inhibition of protein synthesis within 10 min. The inhibition in GH3 pituitary cells involved the synthesis of almost all polypeptides, was not associated with increased cytosolic free Ca2+ concentration ([Ca2+]i), and was not reversed at high extracellular Ca2+. The transient rise in [Ca2+]i triggered by ionomycin was diminished by thapsigargin. Polysomes failed to accumulate in the presence of the drug, indicative of impaired translational initiation. With longer (1-3 h) exposures to thapsigargin, recovery of translational activity was observed accompanied by increased synthesis of the ER protein glucose-regulated stress protein 78 or immunoglobulin heavy-chain binding protein (‘GRP78/BiP’) and its mRNA. Such inductions were comparable with those observed previously with Ca2+ ionophores which mobilize the cation from all intracellular sequestered sites. Actin mRNA concentrations declined significantly during such treatments. In HepG2 cells processing and secretion of the glycoprotein alpha 1-antitrypsin were rapidly suppressed by thapsigargin. Ca2+ sequestered specifically by the ER is concluded to be essential for optimal protein synthesis and processing. These rapid effects of thapsigargin on mRNA translation, protein processing and gene expression should be considered when evaluating potential mechanisms by which this tumour promoter influences cellular events.

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