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  • Life and Medical Sciences  (5)
  • 1975-1979  (5)
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  • 1
    Electronic Resource
    Electronic Resource
    The relative amounts of the cytoplasmic RNA species in normal, transformed and senescent cultured cell lines (1977)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 90 (1977), S. 465-470 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have examined the relative quantities of 18S and 28S rRNA, 4S RNA and poly (A) + mRNA in the following cultured cells: the mouse fibroblast lines 3T3 and 3T6 in the resting (contact inhibited) and growing (sparse) states, 3T3 clones transformed with SV40 (SV3T3) and with both SV40 and polyoma (SV-Py 3T3), hamster lung fibroblasts (V79), human cervical carcinoma cells (HeLa), and human diploid fibroblasts at early and late passage. The relative quantities of the RNA species were determined by labeling the cells to equilibrium with 32PO4 and measuring the amount of label in each RNA species.The ratio of mRNA to rRNA varied from 1.1% to 2.7% in the different cell lines, the more rapidly growing cell lines usually giving a higher ratio. In cells experiencing growth limitation either by contact inhibition or due to senescence, the ratio of mRNA to rRNA was about 30% lower than in the corresponding cells in the growing state. In most cell lines the ratio of 4S RNA to 18S rRNA was between 0.8 and 1.2, but in senescent fibroblasts, this ratio increased to greater than 1.7. Senescent fibroblasts also contained much more total RNA per unit of DNA than the same cells at early passage or than 3T6 or 3T3 cells.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1040900310
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  • 2
    Electronic Resource
    Electronic Resource
    Regulation of dihydrofolate reductase gene expression in mouse fibroblasts during the transition from the resting to growing state (1978)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 97 (1978), S. 397-406 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The rate of accumulation of dihydrofolate reductase (DHFR) was studied in resting, growing and serum stimulated mouse 3T6 fibroblasts by first exposing the cells briefly to 10-6 M methotrexate (MTX) to inactivate specifically and irreversibly the pre-existing enzyme, then determining the rate of recovery of reductase activity after removal of MTX. DHFR activity was quantitated by measuring the ability of a cell extract to reduce 3H-folic acid or to bind 3H-MTX. In all cases, recovery of enzyme activity was inhibited by cyclo-heximide, indicating that the recovery was due to de novo synthesis of reductase.We found that the rate of accumulation of DHFR was high in exponentially growing cells, as expected, but about 40-fold lower in resting (G0) 3T6 cells. When resting 3T6 cells were induced to re-enter the cell cycle following serum stimulation, we found that the rate of accumulation of DHFR increased sharply about ten hours after serum stimulation. DNA replication also began at this time. When resting cells were serum stimulated in the presence of inhibitors of DNA synthesis (hydroxyurea or cytosine arabinoside), the increase in DHFR synthesis was the same as in control stimulated cells. This indicates a lack of tight coupling between DNA synthesis and reductase gene expression. The increase in DHFR accumulation was inhibited by Actinomycin D (5 μg/ml) if the drug was added 7.5 hours after stimulation, but was not inhibited if the drug was added 15 hours after stimulation. This is consistent with the idea that DHFR gene expression is regulated at the level of transcription, and that reductase mRNA is transcribed only between 7.5 and 15 hours following stimulation.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1040970314
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  • 3
    Electronic Resource
    Electronic Resource
    Increasing content of poly A(+) mRNA of serum-stimulated cells in the absence of ribosome synthesisThese investigations were aided by grants from the National Cancer Institute and the National Science Foundation. (1976)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 87 (1976), S. 141-146 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When 3T6 cells undergo a serum-induced transition from resting to growing state, the number of ribosomes and the amounts of mRNA and tRNA increase as the cells prepare for DNA synthesis. We have examined the effect of preventing ribosome synthesis during this transition. When resting cells are stimulated to grow in the presence of 5-fluorouridine, mRNA accumulates normally during the first eight hours, though new ribosome formation is completely blocked by the drug. At later times, mRNA continues to accumulate, but at a reduced rate. The ratio of poly A(+) mRNA to rRNA increases from the value characteristic of resting 3T6 (1.8%) to that of growing 3T6 (2.7%) by five hours, and continues to increase to abnormally high values after this time.Although labelling of tRNA is not affected after brief exposure of cells to fluorouridine, the drug prevents the later accumulation of tRNA that ordinarily occurs following serum stimulation of resting cells. This failure of accumulation is not the result of increased lability of fluorinated tRNA, but is probably due to failure of the transcription rate of pre-tRNA to increase. It is possible that this effect might be due to a regulatory system coupling tRNA content to ribosome content.In cultures stimulated with serum in the presence of fluorouridine the rate of protein synthesis increases with poly A(+) mRNA content during the first eight hours; it then fails to increase further, possibly because ribosomes become rate-limiting.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1040870202
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  • 4
    Electronic Resource
    Electronic Resource
    Rapid increase in poly(A) (+) mRNA content following inhibition of protein synthesis (1977)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 92 (1977), S. 57-64 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When resting 3T6 cells undergo a serum-induced transition to the growing state, the cytoplasmic content of ribosomal, transfer and messenger RNA increase as the cells prepare for DNA synthesis. The normal linear increase in mRNA content occurs even when the production of ribosomes is blocked. In this paper we determine the effect of inhibiting protein synthesis on the increase in poly(A) (+) mRNA content. Resting cells were serum stimulated in the presence of cycloheximide or puromycin at levels which inhibit protein synthesis by greater than 95%. Cytoplasmic poly(A) (+) mRNA content was determined at various times thereafter. We found that mRNA content increased five to ten times more rapidly in drug treated cells than in control cells stimulated in the absence of inhibitors. mRNA content increased 50-70% by one hour, and 60-90% by two hours following stimulation in the presence of inhibitor, and remained more or less constant thereafter. In contrast, mRNA content increased linearly in control stimulated cultures and did not double until about 15 hours after stimulation. The rapid increase in mRNA content is most likely the result of inhibition of protein synthesis rather than a secondary effect of the drug since the same observations were made in growth stimulated cells if protein synthesis was blocked with either puromycin or cycloheximide.A similar effect was also observed with resting 3T6, exponentially growing 3T6 and growing HeLa cells following exposure to cycloheximide, although the magnitude of the increase was less than that observed with growth stimulated cells. Puromycin had negligible effect on mRNA content in resting or exponentially growing cells.The rapid increase in cytoplasmic poly(A) (+) mRNA content was not due to rapid unbalanced export of nuclear poly(A) (+) RNA into the cytoplasm since there was no decrease in nuclear poly(A) content following serum stimulation in the presence of cycloheximide.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1040920108
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  • 5
    Electronic Resource
    Electronic Resource
    Rapid changes in poly (A)(+) mRNA content in growth stimulated fibroblasts following perturbations in protein synthesis (1979)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 100 (1979), S. 531-538 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have previously shown that when resting 3T6 cells are serum stimulated in the presence of inhibitors of protein synthesis, poly(A)(+) mRNA content increases extremely rapidly relative to cells stimulated in the absence of drug. Poly(A)(+) mRNA content nearly doubles within two hours, but then remains constant for at least ten hours (Johnson and Meister, '77). In this report we show that continuous exposure to both serum and cycloheximide are required to maintain this elevated mRNA level. Removal of either leads to an equally rapid decrease in poly(A)(+) mRNA content. If cycloheximide is withdrawn at either two or ten hours following serum stimulation in the presence of the drug, allowing the rapid (〈 30 minutes) restoration of the rate of protein synthesis, we observe that poly(A)(+) mRNA content decreases within two hours to a level nearly equal to that found in resting cells prior to stimulation. If the drug is withdrawn but the serum stimulus is not, the rapid decrease in poly(A)(+) mRNA content is followed by an increase which is parallel to that which occurs in cultures stimulated in the absence of drug, but displaced from the latter by an interval approximately equal to the length of exposure of the drug. These results show that the mammalian cell is able to decrease as well as increase its content of poly(A)(+) mRNA in response to drug induced perturbations in the rate of protein synthesis. The changes in poly(A)(+) mRNA content occur extremely rapidly and may represent an attempt by the cell to correct the perturbation.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041000315
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