Posttranscriptional control of human gamma interferon gene expression in transfected mouse fibroblasts

1986 ◽  
Vol 6 (6) ◽  
pp. 2253-2256
Author(s):  
H A Young ◽  
L Varesio ◽  
P Hwu
Keyword(s):  
Gene Expression ◽  
Genomic Dna ◽  
Gamma Interferon ◽  
Biologically Active ◽  
Posttranscriptional Control ◽  
Transfected Cells ◽  
3T3 Fibroblasts ◽  
Nih 3T3 ◽  
Interferon Gene ◽  
Calcium Phosphate Precipitation

Human gamma interferon genomic DNA was introduced into NIH 3T3 fibroblasts by calcium phosphate precipitation and was not expressed in these cells at the cytoplasmic mRNA or protein level. Treatment of the transfected cells with cycloheximide (1 microgram/ml) induced the accumulation of cytoplasmic gamma interferon mRNA and biologically active human gamma interferon. Analysis of the nuclear enriched RNA from untreated cells indicated that human gamma interferon mRNA was present, suggesting that cycloheximide may act by inhibiting a specific nuclease or may enhance the processing or transport of the RNA from the nucleus to the cytoplasm.

scholarly journals Posttranscriptional control of human gamma interferon gene expression in transfected mouse fibroblasts.

1986 ◽  
Vol 6 (6) ◽  
pp. 2253-2256 ◽  
Author(s):  
H A Young ◽  
L Varesio ◽  
P Hwu
Keyword(s):  
Gene Expression ◽  
Genomic Dna ◽  
Gamma Interferon ◽  
Biologically Active ◽  
Posttranscriptional Control ◽  
Transfected Cells ◽  
3T3 Fibroblasts ◽  
Nih 3T3 ◽  
Interferon Gene ◽  
Calcium Phosphate Precipitation

Human gamma interferon genomic DNA was introduced into NIH 3T3 fibroblasts by calcium phosphate precipitation and was not expressed in these cells at the cytoplasmic mRNA or protein level. Treatment of the transfected cells with cycloheximide (1 microgram/ml) induced the accumulation of cytoplasmic gamma interferon mRNA and biologically active human gamma interferon. Analysis of the nuclear enriched RNA from untreated cells indicated that human gamma interferon mRNA was present, suggesting that cycloheximide may act by inhibiting a specific nuclease or may enhance the processing or transport of the RNA from the nucleus to the cytoplasm.

GAMMA-INTERFERON GENE EXPRESSION IN HUMAN RENAL ALLOGRAFT FINE-NEEDLE ASPIRATES

1994 ◽  
Vol 57 (4) ◽  
pp. 498-501 ◽  
Author(s):  
CYNTHIA C. NAST ◽  
XIAO-JING ZUO ◽  
JOHN PREHN ◽  
GABRIEL M. DANOVITCH ◽  
ALAN WILKINSON ◽  
...  
Keyword(s):  
Gene Expression ◽  
Renal Allograft ◽  
Gamma Interferon ◽  
Fine Needle ◽  
Fine Needle Aspirates ◽  
Interferon Gene

scholarly journals Regulation of Calreticulin Gene Expression by Calcium

1997 ◽  
Vol 138 (3) ◽  
pp. 547-557 ◽  
Author(s):  
Mathilde Waser ◽  
Nasrin Mesaeli ◽  
Charlotte Spencer ◽  
Marek Michalak
Keyword(s):  
Gene Expression ◽  
Genomic Dna ◽  
Chloramphenicol Acetyltransferase ◽  
Ionophore A23187 ◽  
Mrna Levels ◽  
3T3 Cells ◽  
Transcription Analysis ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We have isolated and characterized a 12-kb mouse genomic DNA fragment containing the entire calreticulin gene and 2.14 kb of the promoter region. The mouse calreticulin gene consists of nine exons and eight introns, and it spans 4.2 kb of genomic DNA. A 1.8-kb fragment of the calreticulin promoter was subcloned into a reporter gene plasmid containing chloramphenicol acetyltransferase. This construct was then used in transient and stable transfection of NIH/ 3T3 cells. Treatment of transfected cells either with the Ca2+ ionophore A23187, or with the ER Ca2+-ATPase inhibitor thapsigargin, resulted in a five- to sevenfold increase of the expression of chloramphenicol acetyltransferase protein. Transactivation of the calreticulin promoter was also increased by fourfold in NIH/3T3 cells treated with bradykinin, a hormone that induces Ca2+ release from the intracellular Ca2+ stores. Analysis of the promoter deletion constructs revealed that A23187- and thapsigargin-responsive regions are confined to two regions (−115 to −260 and −685 to −1,763) in the calreticulin promoter that contain the CCAAT nucleotide sequences. Northern blot analysis of cells treated with A23187, or with thapsigargin, revealed a fivefold increase in calreticulin mRNA levels. Thapsigargin also induced a fourfold increase in calreticulun protein levels. Importantly, we show by nuclear run-on transcription analysis that calreticulin gene transcription is increased in NIH/3T3 cells treated with A23187 and thapsigargin in vivo. This increase in gene expression required over 4 h of continuous incubation with the drugs and was also sensitive to treatment with cycloheximide, suggesting that it is dependent on protein synthesis. Changes in the concentration of extracellular and cytoplasmic Ca2+ did not affect the increased expression of the calreticulin gene. These studies suggest that stress response to the depletion of intracellular Ca2+ stores induces expression of the calreticulin gene in vitro and in vivo.

scholarly journals GAMMA-INTERFERON GENE EXPRESSION IN HUMAN RENAL ALLOGRAFT FINE-NEEDLE ASPIRATES

1994 ◽  
Vol 57 (4) ◽  
pp. 498-501 ◽  
Author(s):  
CYNTHIA C. NAST ◽  
XIAO-JING ZUO ◽  
JOHN PREHN ◽  
GABRIEL M. DANOVITCH ◽  
ALAN WILKINSON ◽  
...  
Keyword(s):  
Gene Expression ◽  
Renal Allograft ◽  
Gamma Interferon ◽  
Fine Needle ◽  
Fine Needle Aspirates ◽  
Interferon Gene

scholarly journals The gene expression profile induced by Wnt 3a in NIH 3T3 fibroblasts

2007 ◽  
Vol 1 (3-4) ◽  
pp. 175-183 ◽  
Author(s):  
Shaoqiong Chen ◽  
Sarah McLean ◽  
David E. Carter ◽  
Andrew Leask
Keyword(s):  
Gene Expression ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
3T3 Fibroblasts ◽  
Nih 3T3

scholarly journals Differential processing of colony-stimulating factor 1 precursors encoded by two human cDNAs.

1988 ◽  
Vol 8 (11) ◽  
pp. 5026-5034 ◽  
Author(s):  
C W Rettenmier ◽  
M F Roussel
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Biologically Active ◽  
Colony Stimulating Factor ◽  
Acid Product ◽  
Human Cdna ◽  
3T3 Fibroblasts ◽  
Nih 3T3 ◽  
Stimulating Factor

The biosynthesis of macrophage colony-stimulating factor 1 (CSF-1) was examined in mouse NIH-3T3 fibroblasts transfected with a retroviral vector expressing the 554-amino-acid product of a human 4-kilobase (kb) CSF-1 cDNA. Similar to results previously obtained with a 1.6-kb human cDNA that codes for a 256-amino-acid CSF-1 precursor, the results of the present study showed that NIH-3T3 cells expressing the product of the 4-kb clone produced biologically active human CSF-1 and were transformed by an autocrine mechanism when cotransfected with a vector containing a human c-fms (CSF-1 receptor) cDNA. The 4-kb CSF-1 cDNA product was synthesized as an integral transmembrane glycoprotein that was assembled into disulfide-linked dimers and rapidly underwent proteolytic cleavage to generate a soluble growth factor. Although the smaller CSF-1 precursor specified by the 1.6-kb human cDNA was stably expressed as a membrane-bound glycoprotein at the cell surface and was slowly cleaved to release the extracellular growth factor, the cell-associated product of the 4-kb clone was efficiently processed to the secreted form and was not detected on the plasma membrane. Digestion with glycosidic enzymes indicated that soluble CSF-1 encoded by the 4-kb cDNA contained both asparagine(N)-linked and O-linked carbohydrate chains, whereas the product of the 1.6-kb clone had only N-linked oligosaccharides. Removal of the carbohydrate indicated that the polypeptide chain of the secreted 4-kb cDNA product was longer than that of the corresponding form encoded by the smaller clone. These differences in posttranslational processing may reflect diverse physiological roles for the products of the two CSF-1 precursors in vivo.

scholarly journals Induction of peroxisome proliferator-activated receptor gamma during the conversion of 3T3 fibroblasts into adipocytes is mediated by C/EBPbeta, C/EBPdelta, and glucocorticoids.

1996 ◽  
Vol 16 (8) ◽  
pp. 4128-4136 ◽  
Author(s):  
Z Wu ◽  
N L Bucher ◽  
S R Farmer
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Ectopic Expression ◽  
Response To Treatment ◽  
Peroxisome Proliferator ◽  
Differentiation Process ◽  
Peroxisome Proliferator Activated Receptor ◽  
Initial Exposure ◽  
3T3 Fibroblasts ◽  
Nih 3T3

The differentiation of 3T3 preadipocytes into adipocytes is accompanied by a transient induction of C/EBPbeta and C/EBPdelta expression in response to treatment of the cells with methylisobutylxanthine (MIX) and dexamethasone (DEX), respectively. In this report, we demonstrate that peroxisome proliferator-activated receptor gamma (PPARgamma) expression in 3T3-L1 preadipocytes is induced by MIX and DEX, suggesting that C/EBPbeta and C/EBPdelta may be involved in this process. Using a tetracycline-responsive expression system, we have recently shown that the conditional ectopic expression of C/EBPbeta in NIH 3T3 fibroblasts (beta2 cells) in the presence of DEX activates the synthesis of peroxisome PPARgamma mRNA. Subsequent exposure of these cells to PPAR activators stimulates their conversion into adipocytes; however, neither the expression of C/EBPbeta nor exposure to DEX alone is capable of inducing PPARgamma expression in the beta2 cell line. We find that unlike the case for 3T3 preadipocytes, C/EBPdelta is not induced by DEX in these 3T3 fibroblasts and therefore is not relaying the effect of this glucocorticoid to the PPARgamma gene. To define the role of glucocorticoids in regulating PPARgamma expression and the possible involvement of C/EBPdelta, we have established an additional set of NIH 3T3 cell lines expressing either C/EBPdelta alone (delta23 cells) or C/EBPdelta and C/EBPbeta together (beta/delta39 cells), using the tetracycline-responsive system. Culture of these cells in tetracycline-deficient medium containing DEX, MIX, insulin, and fetal bovine serum shows that the beta/delta39 cells express PPARgamma and aP2 mRNAs at levels that are almost equivalent to those observed in fully differentiated 3T3-L1 adipocytes. These levels are approximately threefold higher than their levels of expression in the beta2 cells. Despite the fact that these beta/delta39 cells produce abundant amounts of C/EBPbeta and C/EBPdelta (in the absence of tetracycline), they still require glucocorticoids to attain maximum expression of PPARgamma mRNA. Furthermore, the induction of PPARgamma mRNA by exposure of these cells to DEX occurs in the absence of ongoing protein synthesis. The delta23 cells, on the other hand, are not capable of activating PPARgamma gene expression when exposed to the same adipogenic inducers. Finally, attenuation of ectopic C/EBPbeta production at various stages during the differentiation process results in a concomitant inhibition of PPARgamma and the adipogenic program. These data strongly suggest that the induction of PPARgamma gene expression in multipotential mesenchymal stem cells (NIH 3T3 fibroblasts) is dependent on elevated levels of C/EBPbeta throughout the differentiation process, as well as an initial exposure to glucocorticoids. C/EBPdelta may function by synergizing with C/EBPbeta to enhance the level of PPARgamma expression.

scholarly journals Involvement of the MAP kinase cascade in resetting of the mammalian circadian clock

2000 ◽  
Vol 14 (6) ◽  
pp. 645-649 ◽  
Author(s):  
Makoto Akashi ◽  
Eisuke Nishida
Keyword(s):  
Gene Expression ◽  
Circadian Clock ◽  
Mek Inhibitor ◽  
Circadian Oscillation ◽  
Humoral Factors ◽  
3T3 Fibroblasts ◽  
Immortalized Cells ◽  
Kinase Cascade ◽  
Peripheral Cells ◽  
Nih 3T3

Although the suprachiasmatic nucleus (SCN) is the major pacemaker in mammals, the peripheral cells or immortalized cells also contain a circadian clock. The SCN and the periphery may use different entraining signals—light and some humoral factors, respectively. We show that induction of the circadian oscillation of gene expression is triggered by TPA treatment of NIH-3T3 fibroblasts, which is inhibited by a MEK inhibitor, and that prolonged activation of the MAPK cascade is sufficient to trigger circadian gene expression. Therefore, such prolonged activation of MAPK by entraining cues may be involved in the resetting of the circadian clock.

Differential processing of colony-stimulating factor 1 precursors encoded by two human cDNAs

1988 ◽  
Vol 8 (11) ◽  
pp. 5026-5034
Author(s):  
C W Rettenmier ◽  
M F Roussel
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Biologically Active ◽  
Colony Stimulating Factor ◽  
Acid Product ◽  
Human Cdna ◽  
3T3 Fibroblasts ◽  
Nih 3T3 ◽  
Stimulating Factor

The biosynthesis of macrophage colony-stimulating factor 1 (CSF-1) was examined in mouse NIH-3T3 fibroblasts transfected with a retroviral vector expressing the 554-amino-acid product of a human 4-kilobase (kb) CSF-1 cDNA. Similar to results previously obtained with a 1.6-kb human cDNA that codes for a 256-amino-acid CSF-1 precursor, the results of the present study showed that NIH-3T3 cells expressing the product of the 4-kb clone produced biologically active human CSF-1 and were transformed by an autocrine mechanism when cotransfected with a vector containing a human c-fms (CSF-1 receptor) cDNA. The 4-kb CSF-1 cDNA product was synthesized as an integral transmembrane glycoprotein that was assembled into disulfide-linked dimers and rapidly underwent proteolytic cleavage to generate a soluble growth factor. Although the smaller CSF-1 precursor specified by the 1.6-kb human cDNA was stably expressed as a membrane-bound glycoprotein at the cell surface and was slowly cleaved to release the extracellular growth factor, the cell-associated product of the 4-kb clone was efficiently processed to the secreted form and was not detected on the plasma membrane. Digestion with glycosidic enzymes indicated that soluble CSF-1 encoded by the 4-kb cDNA contained both asparagine(N)-linked and O-linked carbohydrate chains, whereas the product of the 1.6-kb clone had only N-linked oligosaccharides. Removal of the carbohydrate indicated that the polypeptide chain of the secreted 4-kb cDNA product was longer than that of the corresponding form encoded by the smaller clone. These differences in posttranslational processing may reflect diverse physiological roles for the products of the two CSF-1 precursors in vivo.

Sign in / Sign up

Export Citation Format

Share Document