Multiple mechanisms are responsible for altered expression of ornithine decarboxylase in overproducing variant cells

1986 ◽  
Vol 6 (8) ◽  
pp. 2865-2871
Author(s):  
L McConlogue ◽  
S L Dana ◽  
P Coffino
Keyword(s):  
Steady State ◽  
Gene Amplification ◽  
Ornithine Decarboxylase ◽  
Mrna Levels ◽  
Wild Type ◽  
Altered Expression ◽  
Cells Of Origin ◽  
Multistep Process ◽  
Steady State Levels ◽  
Synthesis And Degradation

We selected and characterized a series of mouse S49 cell variants that overproduce ornithine decarboxylase (ODC). Previously, we described variants that have an amplified ODC gene and produce about 500-fold more ODC than the wild-type cells of origin (L. McConlogue and P. Coffino, J. Biol. Chem. 258:12083-12086, 1983). We examined a series of independent variants that overproduce ODC to a lesser degree and found that a number of mechanisms other than gene amplification are responsible for the increased ODC activity. Variants were selected for resistance to 0.1 mM difluoromethylornithine, an inhibitor of ODC, by either a single or a multistep process. All showed increased ODC activity and increased ODC mRNA steady-state levels. The half-life of the enzyme was not increased in any of the variants. In one class of variant the increase of ODC mRNA was sufficient to account for ODC overproduction. In a second class, the rate of synthesis of ODC polypeptide per ODC mRNA was at least four- to eightfold higher than that in wild-type cells. Therefore, these variants were altered in the translatability of ODC mRNA. Southern analysis showed that gene amplification does not account for the increased ODC mRNA levels in any of the variants. In both variant and wild-type cells, ODC activity was responsive to changes in polyamine pools; activity was reduced following augmentation of pool size. This change in activity was associated with modification of the rate of synthesis and degradation of ODC but no change in the level of ODC mRNA.

scholarly journals Multiple mechanisms are responsible for altered expression of ornithine decarboxylase in overproducing variant cells.

1986 ◽  
Vol 6 (8) ◽  
pp. 2865-2871 ◽  
Author(s):  
L McConlogue ◽  
S L Dana ◽  
P Coffino
Keyword(s):  
Steady State ◽  
Gene Amplification ◽  
Ornithine Decarboxylase ◽  
Mrna Levels ◽  
Wild Type ◽  
Altered Expression ◽  
Cells Of Origin ◽  
Multistep Process ◽  
Steady State Levels ◽  
Synthesis And Degradation

We selected and characterized a series of mouse S49 cell variants that overproduce ornithine decarboxylase (ODC). Previously, we described variants that have an amplified ODC gene and produce about 500-fold more ODC than the wild-type cells of origin (L. McConlogue and P. Coffino, J. Biol. Chem. 258:12083-12086, 1983). We examined a series of independent variants that overproduce ODC to a lesser degree and found that a number of mechanisms other than gene amplification are responsible for the increased ODC activity. Variants were selected for resistance to 0.1 mM difluoromethylornithine, an inhibitor of ODC, by either a single or a multistep process. All showed increased ODC activity and increased ODC mRNA steady-state levels. The half-life of the enzyme was not increased in any of the variants. In one class of variant the increase of ODC mRNA was sufficient to account for ODC overproduction. In a second class, the rate of synthesis of ODC polypeptide per ODC mRNA was at least four- to eightfold higher than that in wild-type cells. Therefore, these variants were altered in the translatability of ODC mRNA. Southern analysis showed that gene amplification does not account for the increased ODC mRNA levels in any of the variants. In both variant and wild-type cells, ODC activity was responsive to changes in polyamine pools; activity was reduced following augmentation of pool size. This change in activity was associated with modification of the rate of synthesis and degradation of ODC but no change in the level of ODC mRNA.

scholarly journals Effects of nonsense mutations on nuclear and cytoplasmic adenine phosphoribosyltransferase RNA.

1996 ◽  
Vol 16 (8) ◽  
pp. 4426-4435 ◽  
Author(s):  
O Kessler ◽  
L A Chasin
Keyword(s):  
Steady State ◽  
Actinomycin D ◽  
Chinese Hamster ◽  
Nuclear Pore ◽  
Mrna Levels ◽  
Wild Type ◽  
Adenine Phosphoribosyltransferase ◽  
Aprt Gene ◽  
Steady State Levels ◽  
Nonsense Codons

We have analyzed Chinese hamster ovary (CHO) cell mutants bearing nonsense codons in four of the five exons of the adenine phosphoribosyltransferase (aprt) gene and have found a pattern of mRNA reduction similar to that seen in systems studied previously: a decrease in steady-state mRNA levels of 5- to 10-fold for mutations in exons 1, 2, and 4 but little effect for mutations in the 3'-most exon (exon 5). Nuclear aprt mRNA levels showed a similar decrease. Nonsense-containing aprt mRNA decayed at the same rate as wild-type mRNA in these cell lines after inhibition of transcription with actinomycin D. Nonsense-containing aprt mRNA is associated with polysomes, ruling out a model in which stable residual mRNA escapes degradation by avoiding translation initiation. A tetracycline-responsive form of the aprt gene was used to compare the stability of nonsense-containing and wild-type aprt mRNAs without globally inhibiting transcription. In contrast to measurements made in the presence of actinomycin D, after inhibition of aprt transcription with tetracycline, a nonsense-mediated destabilization of aprt mRNA was indeed demonstrable. The increased rate of decay of cytoplasmic aprt mRNA seen here could account for the nonsense-mediated reduction in steady-state levels of aprt mRNA. However, the low levels of nonsense-bearing aprt mRNA in the nucleus suggest a sensibility of mRNA to translation or translatability before it exits that compartment. Quantitation of the steady-state levels of transcripts containing introns revealed no accumulation of partially spliced aprt RNA and hence no indication of nonsense-mediated aberrancies in splicing. Our results are consistent with a model in which translation facilitates the export of mRNA through a nuclear pore. However, the mechanism of this intriguing nucleocytoplasmic communication remains to be determined.

scholarly journals Mouse Adenovirus Type 1 Early Region 1A Is Dispensable for Growth in Cultured Fibroblasts

1998 ◽  
Vol 72 (8) ◽  
pp. 6325-6331 ◽  
Author(s):  
Baoling Ying ◽  
Kimberley Smith ◽  
Katherine R. Spindler
Keyword(s):  
Steady State ◽  
Adenovirus Type ◽  
Wild Type Virus ◽  
Northern Analysis ◽  
Mrna Levels ◽  
Wild Type ◽  
Cultured Fibroblasts ◽  
Early Region ◽  
Steady State Levels

ABSTRACT Mouse adenovirus type 1 (MAV-1) mutants with deletions of conserved regions of early region 1A (E1A) or with point mutations that eliminate translation of E1A were used to determine the role of E1A in MAV-1 replication. MAV-1 E1A mutants expressing no E1A protein grew to titers comparable to wild-type MAV-1 titers on mouse fibroblasts (3T6 fibroblasts and fibroblasts derived from Rb+/+,Rb+/−, and Rb−/− transgenic embryos). To test the hypothesis that E1A could induce a quiescent cell to reenter the cell cycle, fibroblasts were serum starved to stop DNA replication and cellular replication and then infected with the E1A mutant and wild-type viruses. All grew to equivalent titers. Steady-state levels of MAV-1 early mRNAs (E1A, E1B, E2, E3, and E4) from 3T6 cells infected with wild-type or E1A mutant virus were examined by Northern analysis. Steady-state levels of mRNAs from the mutant-infected cells were comparable to or greater than the levels found in wild-type virus infections for most of the early regions and for two late genes. The E2 mRNA levels were slightly reduced in all mutant infections relative to wild-type infections. E1A mRNA was not detected from infections with the MAV-1 E1A null mutant, pmE109, or from infections with similar MAV-1 E1A null mutants, pmE112 andpmE113. The implications for the lack of a requirement of E1A in cell culture are discussed.

scholarly journals The critical role of T cells in glucocorticoid-induced osteoporosis

2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Lin Song ◽  
Lijuan Cao ◽  
Rui Liu ◽  
Hui Ma ◽  
Yanan Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Steady State ◽  
Side Effects ◽  
Critical Role ◽  
Wild Type ◽  
Receptor Activator ◽  
Steady State Levels ◽  
Induced Apoptosis

AbstractGlucocorticoids (GC) are widely used clinically, despite the presence of significant side effects, including glucocorticoid-induced osteoporosis (GIOP). While GC are believed to act directly on osteoblasts and osteoclasts to promote osteoporosis, the detailed underlying molecular mechanism of GC-induced osteoporosis is still not fully elucidated. Here, we show that lymphocytes play a pivotal role in regulating GC-induced osteoporosis. We show that GIOP could not be induced in SCID mice that lack T cells, but it could be re-established by adoptive transfer of splenic T cells from wild-type mice. As expected, T cells in the periphery are greatly reduced by GC; instead, they accumulate in the bone marrow where they are protected from GC-induced apoptosis. These bone marrow T cells in GC-treated mice express high steady-state levels of NF-κB receptor activator ligand (RANKL), which promotes the formation and maturation of osteoclasts and induces osteoporosis. Taken together, these findings reveal a critical role for T cells in GIOP.

Quantitative assessment of ground squirrel mRNA levels in multiple stages of hibernation

2002 ◽  
Vol 10 (2) ◽  
pp. 93-102 ◽  
Author(s):  
L. Elaine Epperson ◽  
Sandra L. Martin
Keyword(s):  
Steady State ◽  
Core Body Temperature ◽  
Ground Squirrels ◽  
Apolipoprotein A1 ◽  
Mrna Levels ◽  
Cdna Subtraction ◽  
Global Issues ◽  
Α2 Macroglobulin ◽  
Steady State Levels ◽  
Molecular Components

Hibernators in torpor dramatically reduce their metabolic, respiratory, and heart rates and core body temperature. These extreme physiological conditions are frequently and rapidly reversed during the winter hibernation season via endogenous mechanisms. This phenotype must derive from regulated expression of the hibernator’s genome; to identify its molecular components, a cDNA subtraction was used to enrich for seasonally upregulated mRNAs in liver of golden-mantled ground squirrels. The relative steady-state levels for seven mRNAs identified by this screen, plus five others, were measured and analyzed for seasonal and stage-specific differences using kinetic RT-PCR. Four mRNAs show seasonal upregulation in which all five winter stages differ significantly from and are higher than summer (α2-macroglobulin, apolipoprotein A1, cathepsin H, and thyroxine-binding globulin). One of these mRNAs, α2-macroglobulin, varies during the winter stages with significantly lower levels at late torpor. None of the 12 mRNAs increased during torpor. The implications for these newly recognized upregulated mRNAs for hibernation as well as more global issues of maintaining steady-state levels of mRNA during torpor are discussed.

Posttranscriptional regulation of cytochrome c expression during the developmental cycle of Trypanosoma brucei

1988 ◽  
Vol 8 (11) ◽  
pp. 4625-4633
Author(s):  
A F Torri ◽  
S L Hajduk
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Trypanosoma Brucei ◽  
Posttranscriptional Regulation ◽  
Mitochondrial Protein ◽  
Developmental Cycle ◽  
Mrna Levels ◽  
Developmentally Regulated ◽  
Partial Cdna ◽  
Steady State Levels

We examined the expression of a nucleus-encoded mitochondrial protein, cytochrome c, during the life cycle of Trypanosoma brucei. The bloodstream forms of T. brucei, the long slender and short stumpy trypanosomes, have inactive mitochondria with no detectable cytochrome-mediated respiration. The insect form of T. brucei, the procyclic trypanosomes, has fully functional mitochondria. Cytochrome c is spectrally undetectable in the bloodstream forms of trypanosomes, but during differentiation to the procyclic form, spectrally detected holo-cytochrome c accumulates rapidly. We have purified T. brucei cytochrome c and raised antibodies that react to both holo- and apo-cytochrome c. In addition, we isolated a partial cDNA to trypanosome cytochrome c. An examination of protein expression and steady-state mRNA levels in T. brucei indicated that bloodstream trypanosomes did not express cytochrome c but maintained significant steady-state levels of cytochrome c mRNA. The results suggest that in T. brucei, cytochrome c is developmentally regulated by a posttranscriptional mechanism which prevents either translation or accumulation of cytochrome c in the bloodstream trypanosomes.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

scholarly journals Modulation of Fibronectin Adhesins and Other Virulence Factors in a Teicoplanin-Resistant Derivative of Methicillin-Resistant Staphylococcus aureus

2004 ◽  
Vol 48 (8) ◽  
pp. 2958-2965 ◽  
Author(s):  
Adriana Renzoni ◽  
Patrice Francois ◽  
Dongmei Li ◽  
William L. Kelley ◽  
Daniel P. Lew ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Steady State ◽  
Resistant Strain ◽  
Mrna Levels ◽  
Glycopeptide Resistance ◽  
Methicillin Resistant ◽  
Qrt Pcr ◽  
Sigma B ◽  
Steady State Levels ◽  
The Impact

ABSTRACT The impact of glycopeptide resistance on the molecular regulation of Staphylococcus aureus virulence and attachment to host tissues is poorly documented. We compared stable teicoplanin-resistant methicillin-resistant S. aureus (MRSA) strain 14-4 with its teicoplanin-susceptible MRSA parent, strain MRGR3, which exhibits a high degree of virulence in a rat model of chronic foreign body MRSA infection. The levels of fibronectin-mediated adhesion and surface display of fibronectin-binding proteins were higher in teicoplanin-resistant strain 14-4 than in its teicoplanin-susceptible parent or a teicoplanin-susceptible revertant (strain 14-4rev) that spontaneously emerged during tissue cage infection. Quantitative reverse transcription-PCR (qRT-PCR) showed four- and twofold higher steady-state levels of fnbA and fnbB transcripts, respectively, in strain 14-4 than in its teicoplanin-susceptible counterparts. Analysis of global regulatory activities by qRT-PCR revealed a strong reduction in the steady-state levels of RNAIII and RNAII in the teicoplanin-resistant strain compared to in its teicoplanin-susceptible counterparts. In contrast, sarA mRNA levels were more than fivefold higher in strain 14-4 than in MRGR3 and 14-4rev. Furthermore, the alternative transcription factor sigma B had a higher level of functional activity in the teicoplanin-resistant strain than in its teicoplanin-susceptible counterparts, as evidenced by significant increases in both the sigma B-dependent asp23 mRNA levels and the sarA P3 promoter-derived transcript levels, as assayed by qRT-PCR and Northern blotting, respectively. These data provide further evidence that the emergence of glycopeptide resistance is linked by still poorly understood molecular pathways with significant pleiotropic changes in the expression and regulation of some major virulence genes. These molecular and phenotypic changes may have a profound impact on the bacterial adhesion and colonization properties of such multiresistant organisms.

Modulation of human DNA methyltransferase activity and mRNA levels in the monoblast cell line U937 induced to differentiate with dibutyryl cyclic AMP and phorbol ester

1993 ◽  
Vol 11 (2) ◽  
pp. 191-200 ◽  
Author(s):  
P Soultanas ◽  
P D Andrews ◽  
D R Burton ◽  
D P Hornby
Keyword(s):  
Steady State ◽  
Cyclic Amp ◽  
Gene Transcription ◽  
Phorbol Ester ◽  
Specific Activity ◽  
Mrna Levels ◽  
Dibutyryl Cyclic Amp ◽  
Nuclear Extracts ◽  
Steady State Levels ◽  
Stimulation Of

ABSTRACT The regulation of DNA (cytosine-5) methyltransferase (DNA MeTase) enzyme activity and gene expression was examined in the monoblastoid U937 cell line induced to differentiate with either dibutyryl cyclic AMP (dbcAMP) or phorbol ester. dbcAMP treatment was found to cause the rapid (<4 h) suppression of DNA MeTase specific activity, with no DNA MeTase activity detectable after 10 h. Equally, no DNA MeTase activity was detectable in nuclear extracts of fresh peripheral blood monocytes. Using both a U937 DNA MeTase cDNA and a mouse DNA MeTase cDNA as probes, steady-state levels of DNA MeTase mRNA were found to decline sharply between 4 and 15 h after dbcAMP treatment. No DNA MeTase mRNA was detectable after 20 h of dbcAMP treatment. Nuclear run-on analysis showed there to be only a small (40%) suppression of DNA MeTase gene transcription in cells treated with dbcAMP for 24 h, implying a role for post-transcriptional processes in the regulation of DNA MeTase mRNA levels. The observed decline in DNA MeTase activity/mRNA levels appeared to precede the dbcAMP-induced arrest in DNA replication, as judged by the incorporation of tritiated thymidine into DNA. In contrast to the effect of dbcAMP, treatment of U937 cells with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) led to an overall stimulation of DNA MeTase specific activity. The TPA response was found to be complex and broadly consisted of an early (0–15 h) burst of DNA MeTase activity followed by a more gradual sustained increase in DNA MeTase activity after prolonged (16–40 h) TPA treatment. The early phase of high DNA MeTase activity was not mirrored by an increase in steady-state levels of DNA MeTase mRNA, as judged by Northern blot analysis. However, a substantial induction of DNA MeTase mRNA levels was observed after 20–24 h of TPA treatment. Nuclear run-on analysis showed this not to be due to any significant increase in DNA MeTase gene transcription. The observed increases in DNA MeTase activity/mRNA levels were observed whilst cells were undergoing deproliferation. Interestingly, the addition of TPA and more physiological protein kinase C (PKC) activators, such as diacylglycerol and phosphatidylserine, to DNA MeTase-enriched nuclear extracts generated a 4·5-fold and a 1·5-fold increase in DNA MeTase specific activity respectively. The TPA-induced stimulation of DNA MeTase activity could be inhibited by the PKC inhibitor H-9, implicating a role for PKC in the regulation of DNA MeTase activity in vivo.

scholarly journals Glucocorticoid regulation of human pulmonary surfactant protein-B (SP-B) mRNA stability is independent of activated glucocorticoid receptor

2011 ◽  
Vol 300 (6) ◽  
pp. L940-L950 ◽  
Author(s):  
Ceá C. Tillis ◽  
Helen W. Huang ◽  
Weizhen Bi ◽  
Su Pan ◽  
Shirley R. Bruce ◽  
...  
Keyword(s):  
Steady State ◽  
Glucocorticoid Receptor ◽  
Mrna Stability ◽  
Pulmonary Surfactant ◽  
Surfactant Protein ◽  
Airway Epithelial Cells ◽  
Mrna Levels ◽  
Airway Epithelial ◽  
Surfactant Protein B ◽  
Steady State Levels

Adequate expression of surfactant protein-B (SP-B) is critical in the function of pulmonary surfactant to reduce alveolar surface tension. Expression of SP-B mRNA is restricted to specific lung-airway epithelial cells, and human SP-B mRNA stability is increased in the presence of the synthetic glucocorticoid dexamethasone (DEX). Although the mechanism of SP-B mRNA stabilization by DEX is unknown, studies suggest involvement of the glucocorticoid receptor (GR). We developed a dual-cistronic plasmid-based expression assay in which steady-state levels of SP-B mRNA, determined by Northern analysis, reproducibly reflect changes in SP-B mRNA stability. Using this assay, we found that steady-state levels of SP-B mRNA increased greater than twofold in transfected human-airway epithelial cells (A549) incubated with DEX (10−7 M). DEX-mediated changes in SP-B mRNA levels required the presence of the SP-B mRNA 3′-untranslated region but did not require ongoing protein synthesis. The effect of DEX on SP-B mRNA levels was dose dependent, with maximal effect at 10−7 M. DEX increased levels of SP-B mRNA in cells lacking GR, and the presence of the GR antagonist RU486 did not interfere with the effect of DEX. Surprisingly, other steroid hormones (progesterone, estradiol, and vitamin D; 10−7 M) significantly increased SP-B mRNA levels, suggesting a common pathway of steroid hormone action on SP-B mRNA stability. These results indicate that the effect of DEX to increase SP-B mRNA stability is independent of activated GR and suggests that the mechanism is mediated by posttranscriptional or nongenomic effects of glucocorticoids.

Sign in / Sign up

Export Citation Format

Share Document