Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells

1988 ◽  
Vol 8 (11) ◽  
pp. 4799-4807
Author(s):  
L J Brunet ◽  
A J Berk
Keyword(s):  
Adenovirus Type ◽  
In Vitro Transcription ◽  
Mrna Levels ◽  
Adenovirus E1a ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Tumor Virus ◽  
E1a Gene ◽  
High Level

The adenovirus E1A proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level E1A expression in the absence of viral infection would facilitate biochemical studies of E1A-mediated transactivation. Toward this end, we introduced the adenovirus type 2 E1A gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable E1A mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of E1A expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying E1A concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same E1A concentration and that transcription from each promoter increased as the E1A concentration increased. These results indicate that E1A-mediated transactivation is proportional to the concentration of E1A protein. E1A-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the E1A proteins was sufficient to increase the transcriptional activity of nuclear extracts.

scholarly journals Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells.

1988 ◽  
Vol 8 (11) ◽  
pp. 4799-4807 ◽  
Author(s):  
L J Brunet ◽  
A J Berk
Keyword(s):  
Adenovirus Type ◽  
In Vitro Transcription ◽  
Mrna Levels ◽  
Adenovirus E1a ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Tumor Virus ◽  
E1a Gene ◽  
High Level

The adenovirus E1A proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level E1A expression in the absence of viral infection would facilitate biochemical studies of E1A-mediated transactivation. Toward this end, we introduced the adenovirus type 2 E1A gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable E1A mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of E1A expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying E1A concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same E1A concentration and that transcription from each promoter increased as the E1A concentration increased. These results indicate that E1A-mediated transactivation is proportional to the concentration of E1A protein. E1A-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the E1A proteins was sufficient to increase the transcriptional activity of nuclear extracts.

scholarly journals Induction of cell cycle progression by adenovirus E1A gene 13S- and 12S-mRNA products in quiescent rat cells.

1987 ◽  
Vol 7 (10) ◽  
pp. 3846-3852 ◽  
Author(s):  
T Nakajima ◽  
M Masuda-Murata ◽  
E Hara ◽  
K Oda
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Adenovirus Type ◽  
Inducible Promoter ◽  
Similar Rate ◽  
Cycle Progression ◽  
Adenovirus E1a ◽  
Tumor Virus ◽  
E1a Gene

Rat 3Y1 cell lines that express either adenovirus type 12 E1A 13S mRNA or 12S mRNA in response to dexamethasone treatment were established by introduction of recombinant vector DNA containing the E1A 13S- or 12S-mRNA cDNA placed downstream of the hormone-inducible promoter of mouse mammary tumor virus. These cell lines were growth arrested, and the induction of cell cycle progression was analyzed by flow cytometry after switch on of the cDNA by the addition of dexamethasone. The results indicate that the 13S- or 12S-mRNA product alone has the ability to cause progression of the cell cycle at a similar rate. The simultaneous addition of epidermal growth factor accelerated the rate of cell cycle progression in the transition from the G0/G1 phase to the S phase.

scholarly journals Tumorigenicity of fibroblast lines expressing the adenovirus E1a, cellular p53, or normal c-myc genes.

1986 ◽  
Vol 6 (1) ◽  
pp. 7-14 ◽  
Author(s):  
A Kelekar ◽  
M D Cole
Keyword(s):  
Simian Virus 40 ◽  
Adenovirus Type ◽  
Simian Virus ◽  
Primary Cells ◽  
Adenovirus E1a ◽  
Immortalized Cells ◽  
Transforming Activity ◽  
E1a Gene

Cellular and viral oncogenes have been linked to the transformation of established cell lines in vitro, to the induction of tumors in vivo, and to the partial transformation or immortalization of primary cells. Based on the ability to cooperate with mutated ras oncogenes in the transformation of primary cells, the adenovirus E1a and cellular p53 genes have been assigned an immortalizing activity. It is demonstrated in this paper that the adenovirus type 5 E1a gene and simian virus 40 promoter-linked p53 cDNA are able to transform previously immortalized cells to a tumorigenic phenotype without a significant change in cell morphology. It is also shown that, when linked to a constitutive promoter, the normal mouse and human c-myc genes have the same transforming activity. Cells transformed by each of these oncogenes have an increased capacity to grow in the absence of growth factors and a limited anchorage-independent growth capability.

scholarly journals Molecular cloning and sequence of a cholesterol-repressible enzyme related to prenyltransferase in the isoprene biosynthetic pathway.

1987 ◽  
Vol 7 (9) ◽  
pp. 3138-3146 ◽  
Author(s):  
C F Clarke ◽  
R D Tanaka ◽  
K Svenson ◽  
M Wamsley ◽  
A M Fogelman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Molecular Cloning ◽  
In Vitro Transcription ◽  
In Vitro Translation ◽  
Mrna Levels ◽  
Hmg Coa Reductase ◽  
Transcription System ◽  
Coa Reductase

Differential hybridization and molecular cloning have been used to isolate CR39, a cDNA which hybridizes to a 1.2-kilobase (kb) mRNA in rat liver. The level of CR39 mRNA was increased seven- to ninefold over normal levels by dietary cholestyramine and mevinolin and decreased about fourfold compared with normal levels by cholesterol feeding or administration of mevalonate. Similar changes in the mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase were observed under the various conditions. In vitro translation of either CR39 hybrid selected RNA or 1.2-kb CR39 RNA generated by an SP6 in vitro transcription system produced a polypeptide of 39,000 daltons. As deduced from the nucleotide sequence of a full-length CR39 cDNA, the rat CR39 polypeptide contained 344 amino acids and had a molecular weight of 39,615. The predicted amino acid composition and submit molecular weight of the rat CR39 were very similar to those of prenyltransferases isolated from chicken, pig, and human. The sequence of amino acid residues 173 through 203 in the rat CR39 polypeptide showed that 17 out of 30 matched an active-site peptide of avian liver prenyltransferase. Thus, alterations in the rate of cholesterogenesis resulted in the coordinate regulation of three mRNAs encoding HMG-CoA reductase, HMG-CoA synthase, and CR39, the latter being tentatively identified as prenyltransferase.

scholarly journals In vitro transcription and delimitation of promoter elements of the murine dihydrofolate reductase gene.

1986 ◽  
Vol 6 (7) ◽  
pp. 2392-2401 ◽  
Author(s):  
P J Farnham ◽  
R T Schimke
Keyword(s):  
Dihydrofolate Reductase ◽  
In Vitro Transcription ◽  
S Phase ◽  
Specific Factor ◽  
Or Efficiency ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Reductase Gene

We have developed an in vitro transcription system for the murine dihydrofolate reductase gene. Although transcription in vitro from a linearized template was initiated at the same start sites as in vivo, the correct ratios were more closely approximated when a supercoiled template was used. In addition, whereas the dihydrofolate reductase promoter functions bidirectionally in vivo, the initiation signals directed unidirectional transcription in this in vitro system. The dihydrofolate reductase gene does not have a typical TATA box, but has four GGGCGG hexanucleotides within 300 base pairs 5' of the AUG codon. Deletion analysis suggested that, although sequences surrounding each of the GC boxes could specify initiation approximately 40 to 50 nucleotides downstream, three of the four GC boxes could be removed without changing the accuracy or efficiency of initiation at the major in vivo site. The dihydrofolate reductase promoter initiated transcription very rapidly in vitro, with transcripts visible by 1 min and almost maximal by 2 min at 30 degrees C with no preincubation. Nuclear extracts prepared from cells blocked in the S phase by aphidicolin or from adenovirus-infected cells at 16 h postinfection had enhanced dihydrofolate reductase transcriptional activity. This increased in vitro transcription mimicked the increase in dihydrofolate reductase mRNA seen in S-phase cells and suggested the presence of a cell-cycle-specific factor(s) which stimulated transcription from the dihydrofolate reductase gene.

Molecular cloning and sequence of a cholesterol-repressible enzyme related to prenyltransferase in the isoprene biosynthetic pathway

1987 ◽  
Vol 7 (9) ◽  
pp. 3138-3146
Author(s):  
C F Clarke ◽  
R D Tanaka ◽  
K Svenson ◽  
M Wamsley ◽  
A M Fogelman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Molecular Cloning ◽  
In Vitro Transcription ◽  
In Vitro Translation ◽  
Mrna Levels ◽  
Hmg Coa Reductase ◽  
Transcription System ◽  
Coa Reductase

Differential hybridization and molecular cloning have been used to isolate CR39, a cDNA which hybridizes to a 1.2-kilobase (kb) mRNA in rat liver. The level of CR39 mRNA was increased seven- to ninefold over normal levels by dietary cholestyramine and mevinolin and decreased about fourfold compared with normal levels by cholesterol feeding or administration of mevalonate. Similar changes in the mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase were observed under the various conditions. In vitro translation of either CR39 hybrid selected RNA or 1.2-kb CR39 RNA generated by an SP6 in vitro transcription system produced a polypeptide of 39,000 daltons. As deduced from the nucleotide sequence of a full-length CR39 cDNA, the rat CR39 polypeptide contained 344 amino acids and had a molecular weight of 39,615. The predicted amino acid composition and submit molecular weight of the rat CR39 were very similar to those of prenyltransferases isolated from chicken, pig, and human. The sequence of amino acid residues 173 through 203 in the rat CR39 polypeptide showed that 17 out of 30 matched an active-site peptide of avian liver prenyltransferase. Thus, alterations in the rate of cholesterogenesis resulted in the coordinate regulation of three mRNAs encoding HMG-CoA reductase, HMG-CoA synthase, and CR39, the latter being tentatively identified as prenyltransferase.

scholarly journals Transcriptional and posttranscriptional regulation of the rat prolactin gene by calcium.

1990 ◽  
Vol 10 (2) ◽  
pp. 442-448 ◽  
Author(s):  
G M Preston ◽  
W M Billis ◽  
B A White
Keyword(s):  
Posttranscriptional Regulation ◽  
In Vitro Transcription ◽  
Gh3 Cells ◽  
Mrna Levels ◽  
Nuclear Extracts ◽  
Nuclear Site ◽  
Gene Treatment ◽  
Prolactin Gene ◽  
Prolactin Mrna

The rat prolactin gene is expressed at a high basal level in the pituitary tumor GH3 cell line. Culturing GH3 cells in a low-Ca2+, serum-free medium (SFM) depresses prolactin mRNA levels, and subsequent addition of Ca2+ to the SFM results in a specific, gradual, and sustained increase in prolactin mRNA levels. We have now examined whether the observed increase in prolactin mRNA levels can be attributed solely to an increase in the transcriptional rate of the prolactin gene. Treatment of GH3 cells in SFM with 0.4 mM CaCl2 for 24 to 48 h increased cytoplasmic prolactin mRNA levels by 5- to 10-fold, whereas the transcriptional rate of the prolactin gene was increased by less than twofold over values for SFM controls. Prolactin mRNA levels increased progressively during the 24-h period after Ca2+ addition, whereas prolactin gene transcription never exceeded a twofold increase over values for SFM controls. The activities of nuclear extracts from control and Ca2(+)-induced cells were examined in an in vitro transcription assay. The two extracts directed transcription from the prolactin promoter and the adenovirus major late promoter equally well. Cycloheximide had no effect on the ability of Ca2+ to increase or maintain prolactin mRNA levels. In dactinomycin mRNA clearance experiments, prolactin mRNA was cleared at the same rate in the absence and presence of Ca2+. These results demonstrate that although Ca2+ has a small effect on the transcriptional rate of the prolactin gene, Ca2+ produces a significant increase in prolactin mRNA levels by acting at a posttranscriptional site(s). Furthermore, Ca(2+) appears to increase prolactin mRNA levels by posttranslational modification of a stable protein, probably at a nuclear site.

scholarly journals In Vitro Transcription of pe38/Polyhedrin Hybrid Promoters Reveals Sequences Essential for Recognition by the Baculovirus-Induced RNA Polymerase and for the Strength of Very Late Viral Promoters

1998 ◽  
Vol 72 (4) ◽  
pp. 2991-2998 ◽  
Author(s):  
Ruud M. W. Mans ◽  
Dagmar Knebel-Mörsdorf
Keyword(s):  
Rna Polymerase ◽  
In Vitro Transcription ◽  
Sequence Motif ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Polyhedrin Promoter ◽  
Viral Promoters ◽  
Nuclear Polyhedrosis ◽  
Hybrid Promoters

ABSTRACT In vitro transcription was used to analyze the promoter specificity of the α-amanitin-resistant RNA polymerase that is induced late during infection of Autographa californica multicapsid nuclear polyhedrosis virus. By modifying the preparation of crude nuclear extracts, we have established an assay that permits differentiation between weak late and strong very late viral promoters. The virus-induced RNA polymerase initiates at a TAAG sequence motif in both late and very late promoters. Based on the sensitivity of our in vitro transcription system, we have investigated the sequences responsible for a functional TAAG motif and their putative role with respect to the strength of very late promoters. By constructing hybrid promoters between the early pe38 and the very late polyhedrin promoters, we demonstrated that the replacement of 7 nucleotides upstream of the nonfunctional TAAG sequences in the pe38 promoter with the corresponding sequences of the polyhedrin promoter was sufficient for recognition by the virus-induced RNA polymerase. The strength of the very late polyhedrin promoter was established after replacing the 5′ untranslated sequences of the pe38 promoter by those of the polyhedrin promoter in addition to the 7 nucleotides upstream of the TAAG motif.

Induction of cell cycle progression by adenovirus E1A gene 13S- and 12S-mRNA products in quiescent rat cells

1987 ◽  
Vol 7 (10) ◽  
pp. 3846-3852
Author(s):  
T Nakajima ◽  
M Masuda-Murata ◽  
E Hara ◽  
K Oda
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Adenovirus Type ◽  
Inducible Promoter ◽  
Similar Rate ◽  
Cycle Progression ◽  
Adenovirus E1a ◽  
Tumor Virus ◽  
E1a Gene

Rat 3Y1 cell lines that express either adenovirus type 12 E1A 13S mRNA or 12S mRNA in response to dexamethasone treatment were established by introduction of recombinant vector DNA containing the E1A 13S- or 12S-mRNA cDNA placed downstream of the hormone-inducible promoter of mouse mammary tumor virus. These cell lines were growth arrested, and the induction of cell cycle progression was analyzed by flow cytometry after switch on of the cDNA by the addition of dexamethasone. The results indicate that the 13S- or 12S-mRNA product alone has the ability to cause progression of the cell cycle at a similar rate. The simultaneous addition of epidermal growth factor accelerated the rate of cell cycle progression in the transition from the G0/G1 phase to the S phase.

Tumorigenicity of fibroblast lines expressing the adenovirus E1a, cellular p53, or normal c-myc genes

1986 ◽  
Vol 6 (1) ◽  
pp. 7-14
Author(s):  
A Kelekar ◽  
M D Cole
Keyword(s):  
Simian Virus 40 ◽  
Adenovirus Type ◽  
Simian Virus ◽  
Primary Cells ◽  
Adenovirus E1a ◽  
Immortalized Cells ◽  
Transforming Activity ◽  
E1a Gene

Cellular and viral oncogenes have been linked to the transformation of established cell lines in vitro, to the induction of tumors in vivo, and to the partial transformation or immortalization of primary cells. Based on the ability to cooperate with mutated ras oncogenes in the transformation of primary cells, the adenovirus E1a and cellular p53 genes have been assigned an immortalizing activity. It is demonstrated in this paper that the adenovirus type 5 E1a gene and simian virus 40 promoter-linked p53 cDNA are able to transform previously immortalized cells to a tumorigenic phenotype without a significant change in cell morphology. It is also shown that, when linked to a constitutive promoter, the normal mouse and human c-myc genes have the same transforming activity. Cells transformed by each of these oncogenes have an increased capacity to grow in the absence of growth factors and a limited anchorage-independent growth capability.

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