scholarly journals DNA REPLICATION PATTERNS AND CHROMOSOMAL PROTEIN SYNTHESIS IN OPOSSUM LYMPHOCYTES IN VITRO

1967 ◽  
Vol 33 (3) ◽  
pp. 497-509 ◽  
Author(s):  
Lawrence K. Schneider ◽  
William O. Rieke
Keyword(s):  
Protein Synthesis ◽  
Dna Replication ◽  
Dna Synthesis ◽  
X Chromosome ◽  
Sex Chromosomes ◽  
Didelphis Virginiana ◽  
Chromosomal Protein ◽  
Large Size ◽  
Mitotic Figures

DNA replication patterns were determined in the autosomes and sex chromosomes of phytohemagglutinin-stimulated lymphocytes from the opossum (Didelphis virginiana) by employing thymidine-3H labeling and high-resolution radioautography. Opossum chromosomes are desirable experimental material due to their large size, low number (2n = 22), and morphologically distinct sex chromosomes. The autosomes in both sexes began DNA synthesis synchronously and terminated replication asynchronously. One female X chromosome synthesized DNA throughout most of the S phase. Its homologue, however, began replication approximately 3.5 hr later. The two X's terminated DNA synthesis synchronously, slightly later than the autosomes. This form of late replication, in which one X chromosome begins DNA synthesis later than its homologue but completes replication at the same time as its homologue, is apparently unique in the opossum. The male X synthesized DNA throughout S while the Y chromosome exhibited late-replicating characteristics. The two sex chromosomes completed synthesis synchronously, slightly later than the autosomes. Grain counts were performed on all chromosomes to analyze trends in labeling intensity at hourly intervals of S. By analyzing the percent of labeled mitotic figures on radioautographs at various intervals after introduction of arginine-3H, chromosomal protein synthesis was found not to be restricted to any portion of interphase but to increase throughout S and into G2.

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.

Replication of cloned DNA containing the Alu family sequence during cell extract-promoting simian virus 40 DNA synthesis

1984 ◽  
Vol 4 (8) ◽  
pp. 1476-1482
Author(s):  
H Ariga
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Cell Extract ◽  
Simian Virus ◽  
T Antigen ◽  
Sv40 T Antigen ◽  
Sv40 Dna

The replicating activity of several cloned DNAs containing putative origin sequences was examined in a cell-free extract that absolutely depends on simian virus 40 (SV40) T antigen promoting initiation of SV40 DNA replication in vitro. Of the three DNAs containing the human Alu family sequence (BLUR8), the origin of (Saccharomyces cerevisiae plasmid 2 micron DNA (pJD29), and the yeast autonomous replicating sequence (YRp7), only BLUR8 was active as a template. Replication in a reaction mixture with BLUR8 as a template was semiconservative and not primed by a putative RNA polymerase III transcript synthesized on the Alu family sequence in vitro. Pulse-chase experiments showed that the small-sized DNA produced in a short-term incubation was converted to full-length closed circular and open circular DNAs in alkaline sucrose gradients. DNA synthesis in extracts began in a region of the Alu family sequence and was inhibited 80% by the addition of anti-T serum. Furthermore, partially purified T antigen bound the Alu family sequence in BLUR8 by the DNA-binding immunoassay. These results suggest that SV40 T antigen recognizes the Alu family sequence, similar to the origin sequence of SV40 DNA, and initiates semiconservative DNA replication in vitro.

CHROMOSOMES AND DNA OF MICROTUS II. CONFIRMATION OF DELETION OF CONSTITUTIVE HETEROCHROMATIN IN M. AGRESTIS CELLS IN VITRO

1977 ◽  
Vol 19 (3) ◽  
pp. 537-541 ◽  
Author(s):  
J. E. K. Cooper
Keyword(s):  
Somatic Cell ◽  
Cell Line ◽  
Cell Lines ◽  
X Chromosome ◽  
Sex Chromosomes ◽  
Constitutive Heterochromatin ◽  
The Other ◽  
Microtus Agrestis ◽  
C Banding

The distribution of constitutive heterochromatin has been examined by C-banding in two somatic cell lines, grown in vitro, from a female Microtus agrestis. One line retains one intact X chromosome together with the short arm of the other X chromosome, while the other cell line retains only the short arm of one X chromosome. Thus, each cell line has lost substantial amounts of heterochromatin from the sex chromosomes, but this material has been deleted from the cells, and not translocated to other chromosomes. Nonetheless, both cell lines continue to propagate well in vitro.

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.

scholarly journals Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

1986 ◽  
Vol 6 (11) ◽  
pp. 3815-3825 ◽  
Author(s):  
R S Decker ◽  
M Yamaguchi ◽  
R Possenti ◽  
M L DePamphilis
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Early Gene ◽  
Initial Direction ◽  
Dna Polymerase Alpha

Aphidicolin, a specific inhibitor of DNA polymerase alpha, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (ori) and continuation of replication beyond ori. In the presence of sufficient aphidicolin to inhibit total DNA synthesis by 50%, initiation of DNA replication in SV40 chromosomes or ori-containing plasmids continued in vitro, whereas DNA synthesis in the bulk of SV40 replicative intermediate DNA (RI) that had initiated replication in vivo was rapidly inhibited. This resulted in accumulation of early RI in which most nascent DNA was localized within a 600- to 700-base-pair region centered at ori. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 ori-dependent, T-antigen-dependent DNA replication and only when aphidicolin was added to the in vitro system. Increasing aphidicolin concentrations revealed that DNA synthesis in the ori region was not completely resistant to aphidicolin but simply less sensitive than DNA synthesis at forks that were farther away. Since DNA synthesized in the presence of aphidicolin was concentrated in the 300 base pairs on the early gene side of ori, we conclude that the initial direction of DNA synthesis was the same as that of early mRNA synthesis, consistent with the model proposed by Hay and DePamphilis (Cell 28:767-779, 1982). The data were also consistent with initiation of the first DNA chains in ori by CV-1 cell DNA primase-DNA polymerase alpha. Synthesis of pppA/G(pN)6-8(pdN)21-23 chains on a single-stranded DNA template by a purified preparation of this enzyme was completely resistant to aphidicolin, and further incorporation of deoxynucleotide monophosphates was inhibited. Therefore, in the presence of aphidicolin, this enzyme could initiate RNA-primed DNA synthesis at ori first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended distance.

Stimulation of cell division and DNA replication inPrototheca richardsiby passage through larval amphibian guts

Parasitology ◽  
1993 ◽  
Vol 107 (2) ◽  
pp. 119-124 ◽  
Author(s):  
T. J. C. Beebee ◽  
A. L.-C. Wong
Keyword(s):  
Cell Division ◽  
Dna Replication ◽  
Growth Inhibition ◽  
Dna Synthesis ◽  
Free Living ◽  
Leucine Uptake ◽  
Amphibian Larvae ◽  
Larval Amphibian ◽  
Stimulation Of

SUMMARYPrototheca richardsi, an unpigmented heterotrophic alga, causes growth inhibition in amphibian larvae and has proved refractory to culturein Vitro.P. richardsireplication is dependent on regular passaging through tadpole digestive systems; uptake of thymidine by free-livingProtothecacells and incorporation into DNA are very low by comparison with leucine uptake and incorporation into protein, but DNA synthesis is detectable in cells isolated from tadpole intestines. DNA replication was elicited 6–8 h after ingestion in protothecans fed to tadpoles and subsequently re-isolated from them, providing that the tadpoles were fed subsequent to the ingestion. It appears that passaging through tadpole intestines provides an essential stimulus to maintaining an active cell division cycle inP. richardsi.

scholarly journals Insulin and TSH promote growth in size of PC Cl3 rat thyroid cells, possibly via a pathway different from DNA synthesis: comparison with FRTL-5 cells

1999 ◽  
pp. 94-103 ◽  
Author(s):  
T Kimura ◽  
JE Dumont ◽  
A Fusco ◽  
J Golstein
Keyword(s):  
Protein Synthesis ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Insulin Action ◽  
Cell Mass ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Rat Thyroid ◽  
Stimulation Of

In the rat thyroid cell lines PC Cl3, FRTL- 5 and WRT, proliferation is mainly regulated by insulin or IGF, and TSH. However, the mechanism regulating cell mass doubling prior to division is still unknown. Our laboratory has shown that in dog thyroid cells insulin promotes growth in size while TSH in the presence of insulin triggers DNA replication. In the absence of insulin, TSH has no effect on cell growth. In this report we investigated insulin action on both cell mass and DNA synthesis and its modulation by TSH and insulin in PC Cl3 and FRTL-5 cells. In PC Cl3 cells, insulin activated not only DNA synthesis but also protein synthesis and accumulation. Although TSH potentiated the stimulation of DNA synthesis induced by insulin, enhancement of protein synthesis by both agents was additive. All TSH effects were reproduced by forskolin. Similar effects were also obtained in FRTL-5 cells. This suggests that insulin and TSH, via cAMP, modulate both growth in size and DNA replication in these cell lines. Lovastatin, which blocks 3-hydroxy-3-methylglutaryl coenzyme A reductase, decreased the induction of DNA synthesis, but not of protein synthesis induced by insulin or TSH in PC Cl3 cells. In FRTL-5 cells, lovastatin reduced protein and DNA synthesis stimulated by insulin but not TSH-induced protein synthesis. Taking these data together, we propose that insulin and/or TSH both modulate cell mass doubling and DNA synthesis in these cell lines, presumably via different pathways, and that there are at least two pathways which regulate growth in size in FRTL-5 thyroid cells: one triggered by insulin, which is lovastatin sensitive, and the other activated by TSH, which is not sensitive to lovastatin.

scholarly journals The effect of insulin and glucocorticoids on the synthesis and degradation of phosphoenolpyruvate carboxykinase (GTP) in rat adipose tissue cultured in vitro

1976 ◽  
Vol 158 (1) ◽  
pp. 9-16 ◽  
Author(s):  
O Meyuhas ◽  
L Reshef ◽  
F J Ballard ◽  
R W Hanson
Keyword(s):  
Protein Synthesis ◽  
Adipose Tissue ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Epididymal Adipose Tissue ◽  
Large Size ◽  
A Value ◽  
Rat Adipose Tissue ◽  
Synthesis And Degradation

1. Epididymal adipose tissue from the rat was maintained in culture for periods of up to 96h. 2. After an initial decrease in protein synthesis during the first 24h of culture, the adipose tissue recovered its capacity to synthesize and accumulate proteins of a relatively large size. 3. The activity of phosphoenolpyruvate carboxykinase decreased in a parallel manner, but increased again after 24h of incubation of the tissue in culture, to a value twice that noted in the tissue in vivo. This increase in enzyme activity was due to an increase in its rate of synthesis. 4. Both insulin and dexamethasone (9alpha-fluoro-16alpha-methyl-11beta,17,-21-trihydroxypregna-1,4-diene-3,20-dione) inhibited phosphoenolpyruvate carboxykinase synthesis, but dexamethasone also decreased total protein synthesis. 5. The half-life of phosphoenolpyruvate carboxykinase in adipose tissue cultured in vitro was 5-7h and was not altered by insulin or dexamethasone. 6. It is concluded that both insulin and glucocroticoids lower the activity of phosphoenolpyruvate carboxykinase in rat adipose tissue by decreasing its rate of synthesis.

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