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  • 1
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Thymidylate synthase (TS) mRNA content increases about 20-fold when growth-stimulated mouse cells progress from the GO/G1 phase into the S phase of the cell cycle. Previous studies, using a cell line in which the TS gene is amplified (LU3-7), indicated that transcriptional initiation as well as polyadenylation of the mRNA occur at several locations in unsynchronized cells. In the present study, we have used S1 nuclease protection assays to analyze the possible significance of the multiple transcriptional initiation and polyadenylation sites. We found that the same pattern of 5′ and 3′ termini were detected with RNA isolated from the overproducing cells as with RNA isolated from the parental mouse 3T6 cell line, demonstrating that the heterogeneous termini are not a consequence of gene amplification. There was no change in the pattern of 5′ or 3′ termini with either cell line during the progression from G1 phase through S phase in serum-stimulated cells. Therefore, the increase in TS mRNA content is not the result of differential utilization of the various transcriptional initiation or polyadenylation sites. Analyses of poly(A)- deficient cytoplasmic TS RNA showed that the 5′ termini were the same as those found in poly(A)+ mRNA. However, the 3′ termini were extremely heterogeneous in length. Although some of the poly(A)- deficient RNA extended beyond the normal site of polyadenylation, most of it was shorter than full-length TS mRNA.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 54 (1994), S. 387-392 
    ISSN: 0730-2312
    Keywords: growth regulation ; cell cycle ; RNA processing ; intron ; splicing ; translational control ; autogenous control ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Thymidylate synthase (TS) is an essential enzyme that catalyzes the formation of thymidylic acid in the de novo biosynthetic pathway and is the target enzyme for a variety of chemotherapeutic agents. The TS gene is expressed at a much higher level in proliferating cells than in quiescent cells. Control is primarily exerted at the posttranscriptional level. Studies with chimeric TS minigenes have shown that regulation of TS mRNA content in growth-stimulated mouse fibroblasts requires the presence of sequences located upstream of the essential promoter elements. In addition, an efficiently spliced intron must be present within the transcript. Neither sequence by itself is sufficient for proper regulation, suggesting that the upstream and downstream sequences may communicate to effect regulation. A possible mechanism by which the upstream sequences influence the efficiency of splicing of TS transcripts in a cell cycle specific manner is described.Expression of the human TS gene is also controlled at the translational level. The TS enzyme is able to block the translation of its own mRNA by binding to the message in the vicinity of the AUG start codon. The translational block is relieved in the presence of substrates or inhibitors of the enzyme. The autogenous translational regulation of TS mRNA is likely to be responsible for the rapid increase in TS enzyme level that occurs when cells are exposed to certain TS inhibitors. Elucidation of the mechanism by which the translational control is exerted may lead to the design of more effective TS inhibitors.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 104-116 
    ISSN: 0730-2312
    Keywords: mRNA export ; cell cycle ; gene transfection ; cultured mammalian cells ; hnRNP L ; nuclear transport ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The pre-mRNA processing enhancer (PPE) element is an RNA sequence element derived from the intronless HSV-TK gene. Insertion of the element into the highly intron-dependent human β-globin gene leads to efficient expression in the absence of splicing. We have analyzed the effect of the PPE element on the expression of mouse thymidylate synthase (TS) minigenes. We have previously shown that the expression of intronless TS minigenes is moderately (up to 20-fold) stimulated by the inclusion of introns. Furthermore, S phase-specific expression of TS minigenes in growth-stimulated cells depends on the presence of a spliceable intron as well as the TS promoter. The goal of our study was to determine if the PPE element would overcome the dependence on introns for efficient expression and for S phase-specific expression of transfected TS minigenes. We found that insertion of the PPE element into an intronless TS minigene partially overcame intron dependence. However, the increase in expression was much less than that observed for the intronless β-globin gene. We also found that intronless TS or HSV-TK genes that contained the PPE element and that were driven by the TS promoter were expressed at a constant level in serum-stimulated cells. However, when an intron was included in these genes, they were expressed in an S phase-specific manner. Thus the PPE element was not able to overcome the dependence on introns for S phase-specific expression of TS minigenes. J. Cell. Biochem. 69:104-116, 1998. © 1998 Wiley-Liss, Inc.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 90 (1977), S. 465-470 
    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.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 97 (1978), S. 397-406 
    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.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 118 (1984), S. 79-86 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have studied the rate of transcription of the gene for dihydrofolate reductase (DHFR) in mouse 3T6 fibroblasts during serum-induced transitions between the resting (G0) and growing states. As a model system, we have used a methotrexate-resistant 3T6 cell line that overproduces DHFR and its mRNA about 300-fold, yet regulates the expression of the DHFR gene in the same manner as normal 3T6 cells. In previous studies, we showed that the rate of production of cytoplasmic DHFR mRNA relative to total mRNA is about 4 times lower in resting than in exponentially growing cells. The rate increases to the growing value by about 15 hr following serum stimulation of the resting cells. This increase appeared to be controlled by regulating the rate of synthesis of DHFR hnRNA. In this study, we analyze the transcription of the DHFR gene in more detail. We use a variety of labeling times and RNA extraction procedures to measure the rate of synthesis of DHFR hnRNA relative to total hnRNA in pulse-labeled cells or in nuclei isolated from cells at various times following serum stimulation. The amount of labeled DHFR RNA is determined by DNA-excess filter hybridization. In all cases, the relative rate of synthesis of DHFR hnRNA increases at the same time, and to the same extent, as the rate of production of DHFR mRNA, suggesting that the increase in DHFR mRNA production is due to a corresponding increase in the rate of transcription of the DHFR gene. The increase in DHFR gene transcription is not blocked by cytosine arabinoside, showing that the increase does not depend on gene duplication. In isolated nuclei, DHFR RNA synthesis is inhibited by α-amanitin (1 μg/ml), indicating that the DHFR gene is transcribed by RNA polymerase II. Others have shown that when stationary phase cells are stimulated to proliferate, the increase in DHFR mRNA content is controlled primarily at the post-transcriptional level. Therefore, it appears that the rate of production of DHFR mRNA is controlled by different biochemical mechanisms when cells are in different physiological states.
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  • 7
    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.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 92 (1977), S. 57-64 
    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.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 100 (1979), S. 531-538 
    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.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 110 (1982), S. 183-189 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have used a methotrexate-resistant mouse 3T6 cell line (M50L3) that overproduces dihydrofolate reductase (DHFR) and its mRNA by a factor of about 300 to study the regulation of DHFR hnRNA synthesis. We have previously shown that when resting (G0) M50L3 cells are serum stimuled to reenter the cell cycle, the amount and rate of synthesis of DHER and the content of DHER mRNA all begin to increase as the cells enter the S phase of the cell cycle. The increase in DHFR mRNA content is due to an increase in the rate of mRNA production. In the present study, we have used the technique of DNA-excess filter hybridization to determine the rate of synthesis of DHFR hnRNA relative to total hnRNA at various times following serum stimulation. We found that the relative rate of DHFR hnRNA synthesis began to increase at about the same time (6 hours), and increased to about the same extent (three to fourfold by 15 hours following stimulation) as we observed previously for DHFR mRNA production. This suggests that the increase in DHFR mRNA production (and consequently DHFR gene expression) is controlled primarily, if not exclusively, at the level of transcription. We also studied the effect of addition of high concentrations of dibutyryl cAMP and theophyllne on DHFR gene transciption. We found that addition of these drugs at the time of stimulation completely blocked the increase in DHFR hnRNA synthesis as well as entry into S phase. Addition of the drugs at either 13 or 20 hours following stimulation led to a rapid decrease in DHFR hnRNA synthesis. The drugs were found to have little effect on the ability of the cells to complete S phase when they were added at 13 hours following stimulation. Our results suggest that high intracellular concentrations of cAMP may effect DHFR gene expression not only by preventing the progession of cells through the G1 phase of the cell cycle but also by affecting the rate of DHFR gene transcription in a more direct manner.
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