scholarly journals Control of adenovirus late promoter expression in two human cell lines.

1985 ◽  
Vol 5 (9) ◽  
pp. 2433-2442 ◽  
Author(s):  
E D Lewis ◽  
J L Manley
Keyword(s):  
Hela Cells ◽  
Transcriptional Start Site ◽  
Regulatory Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Late Promoter ◽  
Enhancer Element ◽  
Cis Acting ◽  
Early Region ◽  
293 Cells

We investigated the nucleotide sequence requirements of the adenovirus 2 late promoter when activated by either a trans-acting regulatory protein or a cis-acting enhancer element. Using deletion mutants in transient expression assays, we determined that the 5' limit of the region required for activation by a trans-acting regulatory protein, the adenovirus early region 1a gene product, and the simian virus 40 enhancer is the same in both 293 and HeLa cells. Surprisingly, the 3' limit of required sequences varied, depending on the mechanism of activation. Activation mediated by the early region 1a protein endogenous in 293 cells or produced after cotransfection of HeLa cells requires the region around the transcriptional start site, whereas activation brought about by an enhancer element in HeLa cells has no requirement for these sequences. Under no conditions tested did the simian virus 40 enhancer activate the late promoter in 293 cells, even when sequences sufficient for enhancer-mediated activation in HeLa cells, but not for early region 1a activation, were present. These results suggest the existence of at least two different mechanisms for positive regulation of promoter activity.

Control of adenovirus late promoter expression in two human cell lines

1985 ◽  
Vol 5 (9) ◽  
pp. 2433-2442
Author(s):  
E D Lewis ◽  
J L Manley
Keyword(s):  
Hela Cells ◽  
Transcriptional Start Site ◽  
Regulatory Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Late Promoter ◽  
Enhancer Element ◽  
Cis Acting ◽  
Early Region ◽  
293 Cells

We investigated the nucleotide sequence requirements of the adenovirus 2 late promoter when activated by either a trans-acting regulatory protein or a cis-acting enhancer element. Using deletion mutants in transient expression assays, we determined that the 5' limit of the region required for activation by a trans-acting regulatory protein, the adenovirus early region 1a gene product, and the simian virus 40 enhancer is the same in both 293 and HeLa cells. Surprisingly, the 3' limit of required sequences varied, depending on the mechanism of activation. Activation mediated by the early region 1a protein endogenous in 293 cells or produced after cotransfection of HeLa cells requires the region around the transcriptional start site, whereas activation brought about by an enhancer element in HeLa cells has no requirement for these sequences. Under no conditions tested did the simian virus 40 enhancer activate the late promoter in 293 cells, even when sequences sufficient for enhancer-mediated activation in HeLa cells, but not for early region 1a activation, were present. These results suggest the existence of at least two different mechanisms for positive regulation of promoter activity.

scholarly journals Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific.

1986 ◽  
Vol 6 (12) ◽  
pp. 4697-4708 ◽  
Author(s):  
R H Costa ◽  
E Lai ◽  
J E Darnell
Keyword(s):  
Hela Cells ◽  
Hepg2 Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Proximal Promoter ◽  
Start Site ◽  
Beta Globin ◽  
Human Hepatoma Cells ◽  
Enhancer Element ◽  
A Cell

The mouse genomic clone for the prealbumin (transthyretin) gene was cloned, and its upstream regulatory regions were analyzed. The 200 nucleotides 5' to the cap site when placed within a recombinant plasmid were sufficient to direct transient expression in HepG2 (human hepatoma) cells, but this DNA region did not support expression in HeLa cells. The sequence of the 200-nucleotide region is highly conserved between mouse and human DNA and can be considered a cell-specific promoter. Deletions of this promoter region identified a crucial element for cell-specific expression between 151 and 110 nucleotides 5' to the RNA start site. A region situated at about 1.6 to 2.15 kilobases upstream of the RNA start site was found to stimulate expression 10-fold in HepG2 cells but not in HeLa cells. This far upstream element was invertible and increased expression from the beta-globin promoter in HepG2 cells. Unlike the simian virus 40 enhancer, the prealbumin enhancer would not stimulate beta-globin synthesis in HeLa cells, and even the simian virus 40 enhancer did not stimulate the prealbumin promoter in HeLa cells. Thus, we identified in the prealbumin gene two DNA elements that respond in a cell-specific manner: a proximal promoter including a crucial sequence between -108 and -151 nucleotides and a distant enhancer element located between 1.6 and 2.15 kilobases upstream.

scholarly journals Two distinct calcium pools in the endoplasmic reticulum of HEK-293T cells

2011 ◽  
Vol 435 (1) ◽  
pp. 227-235 ◽  
Author(s):  
Francisco J. Aulestia ◽  
Pedro C. Redondo ◽  
Arancha Rodríguez-García ◽  
Juan A. Rosado ◽  
Ginés M. Salido ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Simian Virus 40 ◽  
Cyclopiazonic Acid ◽  
Simian Virus ◽  
T Antigen ◽  
Atpase Inhibitor ◽  
Ph Gradients ◽  
293 Cells ◽  
Functional Features

Agonist-sensitive intracellular Ca2+ stores may be heterogeneous and exhibit distinct functional features. We have studied the properties of intracellular Ca2+ stores using targeted aequorins for selective measurements in different subcellular compartments. Both, HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and HeLa cells accumulated Ca2+ into the ER (endoplasmic reticulum) to near millimolar concentrations and the IP3-generating agonists, carbachol and ATP, mobilized this Ca2+ pool. We find in HEK-293T, but not in HeLa cells, a distinct agonist-releasable Ca2+ pool insensitive to the SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) inhibitor TBH [2,5-di-(t-butyl)-benzohydroquinone]. TG (thapsigargin) and CPA (cyclopiazonic acid) completely emptied this pool, whereas lysosomal disruption or manoeuvres collapsing endomembrane pH gradients did not. Our results indicate that SERCA3d is important for filling the TBH-resistant store as: (i) SERCA3d is more abundant in HEK-293T than in HeLa cells; (ii) the SERCA 3 ATPase activity of HEK-293T cells is not fully blocked by TBH; and (iii) the expression of SERCA3d in HeLa cells generated a TBH-resistant agonist-mobilizable compartment in the ER. Therefore the distribution of SERCA isoforms may originate the heterogeneity of the ER Ca2+ stores and this may be the basis for store specialization in diverse functions. This adds to recent evidence indicating that SERCA3 isoforms may subserve important physiological and pathophysiological mechanisms.

Multiple cis- and trans-acting elements involved in regulation of the neu gene

1990 ◽  
Vol 10 (12) ◽  
pp. 6306-6315
Author(s):  
T C Suen ◽  
M C Hung
Keyword(s):  
Transcriptional Start Site ◽  
Receptor Gene ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Chloramphenicol Acetyltransferase ◽  
Start Site ◽  
Sequence Motifs ◽  
S1 Nuclease ◽  
Cis Acting ◽  
Nuclease Mapping

A 2.4-kb rat neu genomic DNA fragment that hybridized to the 5'-most coding sequence of the rat neu cDNA was cloned. S1 nuclease mapping identified multiple transcriptional initiation sites. DNA sequence analysis revealed that this fragment contained 64 bp of the first intron, 81 bp of the first exon, and the upstream noncoding sequence of the neu gene. The sequence immediately upstream of the translation start site was G + C rich (greater than 75%) and contained a consensus CCAAT sequence despite the absence of a TATA box. An Sp1-binding site was found, in addition to various sequence motifs common to the promoters of the human neu gene (erbB2), the epidermal growth factor receptor gene, and the simian virus 40 enhancer. A 2.2-kb EcoRI-Narl fragment containing sequences upstream from the 3'-most transcriptional start site was fused to the bacterial chloramphenicol acetyltransferase reporter gene and shown to promote transcription efficiently. A series of promoter deletion constructs was made, and results from transfection and subsequent chloramphenicol acetyltransferase assays suggested the presence of multiple cis-acting elements that contributed either positively or negatively to the transcription activity. Cotransfection competition experiments using subcloned cis-acting elements confirmed the existence of trans-acting factors interacting with these DNA fragments. In addition, a gel retardation assay was performed to demonstrate the physical binding of nuclear factors to certain fragments. The results complemented those of the deletion studies and led us to conclude that transcriptional regulation of the neu proto-oncogene involves at least one negative and three positive trans-acting factors interacting with different cis-acting elements along the neu gene promoter.

scholarly journals trans activation of type 1 interferon promoters by simian virus 40 T antigen.

1988 ◽  
Vol 8 (8) ◽  
pp. 3397-3405 ◽  
Author(s):  
J Hiscott ◽  
A Wong ◽  
D Alper ◽  
S Xanthoudakis
Keyword(s):  
Hela Cells ◽  
Sendai Virus ◽  
Expression System ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Relative Contribution ◽  
Ifn Alpha ◽  
293 Cells

A human transient expression system was used to measure the influence of simian virus 40 T antigen and adenovirus E1a proteins on the activation of alpha interferon subtype 1 (IFN-alpha 1) and IFN-beta promoters linked to the reporter chloramphenicol acetyltransferase gene. Large T-antigen production, amplified by expression plasmid replication in transfected 293 cells, was able to trans activate the IFN-beta promoter 5- to 10-fold, increasing both the constitutive and Sendai virus-induced levels of expression. Surprisingly, the previously quiescent transfected IFN-alpha 1 promoter in T-antigen-expressing cells displayed a level of inducibility similar to IFN-beta. The endogenous IFN-alpha 1 gene was also inducible to a limited extent in cells expressing T antigen. A truncated IFN-beta promoter deleted to position -37 relative to the CAP site was neither inducible nor trans activated by T antigen, suggesting that sequences required for efficient induction were also needed for trans activation. Since 293 cells express adenoviral E1a proteins, experiments were also performed in HeLa cells to assess the relative contribution of T antigen and E1a proteins to IFN trans activation. In HeLa cells, T-antigen coexpression increased the constitutive level of IFN-beta and IFN-alpha 1 promoter activity without augmenting relative inducibility. Coexpression of T antigen and E1a proteins did not have a cooperative effect on type 1 IFN expression.

scholarly journals Multiple cis- and trans-acting elements involved in regulation of the neu gene.

1990 ◽  
Vol 10 (12) ◽  
pp. 6306-6315 ◽  
Author(s):  
T C Suen ◽  
M C Hung
Keyword(s):  
Transcriptional Start Site ◽  
Receptor Gene ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Chloramphenicol Acetyltransferase ◽  
Start Site ◽  
Sequence Motifs ◽  
S1 Nuclease ◽  
Cis Acting ◽  
Nuclease Mapping

A 2.4-kb rat neu genomic DNA fragment that hybridized to the 5'-most coding sequence of the rat neu cDNA was cloned. S1 nuclease mapping identified multiple transcriptional initiation sites. DNA sequence analysis revealed that this fragment contained 64 bp of the first intron, 81 bp of the first exon, and the upstream noncoding sequence of the neu gene. The sequence immediately upstream of the translation start site was G + C rich (greater than 75%) and contained a consensus CCAAT sequence despite the absence of a TATA box. An Sp1-binding site was found, in addition to various sequence motifs common to the promoters of the human neu gene (erbB2), the epidermal growth factor receptor gene, and the simian virus 40 enhancer. A 2.2-kb EcoRI-Narl fragment containing sequences upstream from the 3'-most transcriptional start site was fused to the bacterial chloramphenicol acetyltransferase reporter gene and shown to promote transcription efficiently. A series of promoter deletion constructs was made, and results from transfection and subsequent chloramphenicol acetyltransferase assays suggested the presence of multiple cis-acting elements that contributed either positively or negatively to the transcription activity. Cotransfection competition experiments using subcloned cis-acting elements confirmed the existence of trans-acting factors interacting with these DNA fragments. In addition, a gel retardation assay was performed to demonstrate the physical binding of nuclear factors to certain fragments. The results complemented those of the deletion studies and led us to conclude that transcriptional regulation of the neu proto-oncogene involves at least one negative and three positive trans-acting factors interacting with different cis-acting elements along the neu gene promoter.

scholarly journals Sequences involved in initiation of simian virus 40 late transcription in the absence of T antigen.

1986 ◽  
Vol 6 (6) ◽  
pp. 1875-1885 ◽  
Author(s):  
F Omilli ◽  
M Ernoult-Lange ◽  
J Borde ◽  
E May
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Late Promoter ◽  
Late Gene Expression ◽  
Rich Region ◽  
Enhancer Element ◽  
Late Initiation ◽  
Late Transcription

We analyzed the sequences involved in vivo in the initiation of simian virus 40 (SV40) late transcription occurring in the absence of both SV40 origin sequences and T antigen. The constituent elements of the SV40 late promoters have already been the subject of extensive studies. In vitro studies have resulted in the description of two putative domains of the late promoters. The first domain consists of an 11-base-pair (bp) sequence, 5'-GGTACCTAACC-3', located 25 nucleotides (nt) upstream of the SV40 major late initiation site (MLIS) (J. Brady, M. Radonovich, M. Vodkin, V. Natarajan, M. Thoren, G. Das, J. Janik, and N. P. Salzman, Cell 31:624-633, 1982). The second domain is located within the G-C-rich region (J. Brady, M. Radonovich, M. Thoren, G. Das, and N. P. Salzman, Mol. Cell. Biol. 4:133-141; U. Hansen and P. A. Sharp, EMBO J. 2:2293-2303). Our previous in vivo studies permitted us to define a domain of the late promoter which extends from nt 332 to nt 113 and includes the 72-bp enhancer sequences. Here, by using transfection of the appropriate chimeric plasmids into HeLa cells in conjunction with quantitative S1 nuclease analysis, we analyzed in more detail the sequences required for the control of SV40 late-gene expression occurring before the onset of viral DNA replication. We showed that the major late promoter element is in fact the 72-bp repeat enhancer element. This element was able to drive efficient late transcription in the absence of T antigen. Under our experimental conditions, removal of the G-C-rich region (21-bp repeats) entailed a significant increase in the level of late-gene expression. Moreover, translocation of this element closer to the MLIS (53 nt upstream of the MLIS) enhanced the level of transcripts initiated at natural late initiation sites. Our results suggest that the G-C-rich regions have to be positioned between the enhancer element and the initiation sites to stimulate transcription from downstream sites. Thus, the relative arrangement of the various promoter elements is a critical factor contributing to the situation in which the early promoter is stronger than late promoters before viral DNA replication.

trans activation of type 1 interferon promoters by simian virus 40 T antigen

1988 ◽  
Vol 8 (8) ◽  
pp. 3397-3405
Author(s):  
J Hiscott ◽  
A Wong ◽  
D Alper ◽  
S Xanthoudakis
Keyword(s):  
Hela Cells ◽  
Sendai Virus ◽  
Expression System ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Relative Contribution ◽  
Ifn Alpha ◽  
293 Cells

A human transient expression system was used to measure the influence of simian virus 40 T antigen and adenovirus E1a proteins on the activation of alpha interferon subtype 1 (IFN-alpha 1) and IFN-beta promoters linked to the reporter chloramphenicol acetyltransferase gene. Large T-antigen production, amplified by expression plasmid replication in transfected 293 cells, was able to trans activate the IFN-beta promoter 5- to 10-fold, increasing both the constitutive and Sendai virus-induced levels of expression. Surprisingly, the previously quiescent transfected IFN-alpha 1 promoter in T-antigen-expressing cells displayed a level of inducibility similar to IFN-beta. The endogenous IFN-alpha 1 gene was also inducible to a limited extent in cells expressing T antigen. A truncated IFN-beta promoter deleted to position -37 relative to the CAP site was neither inducible nor trans activated by T antigen, suggesting that sequences required for efficient induction were also needed for trans activation. Since 293 cells express adenoviral E1a proteins, experiments were also performed in HeLa cells to assess the relative contribution of T antigen and E1a proteins to IFN trans activation. In HeLa cells, T-antigen coexpression increased the constitutive level of IFN-beta and IFN-alpha 1 promoter activity without augmenting relative inducibility. Coexpression of T antigen and E1a proteins did not have a cooperative effect on type 1 IFN expression.

Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific

1986 ◽  
Vol 6 (12) ◽  
pp. 4697-4708
Author(s):  
R H Costa ◽  
E Lai ◽  
J E Darnell
Keyword(s):  
Hela Cells ◽  
Hepg2 Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Proximal Promoter ◽  
Start Site ◽  
Beta Globin ◽  
Human Hepatoma Cells ◽  
Enhancer Element ◽  
A Cell

The mouse genomic clone for the prealbumin (transthyretin) gene was cloned, and its upstream regulatory regions were analyzed. The 200 nucleotides 5' to the cap site when placed within a recombinant plasmid were sufficient to direct transient expression in HepG2 (human hepatoma) cells, but this DNA region did not support expression in HeLa cells. The sequence of the 200-nucleotide region is highly conserved between mouse and human DNA and can be considered a cell-specific promoter. Deletions of this promoter region identified a crucial element for cell-specific expression between 151 and 110 nucleotides 5' to the RNA start site. A region situated at about 1.6 to 2.15 kilobases upstream of the RNA start site was found to stimulate expression 10-fold in HepG2 cells but not in HeLa cells. This far upstream element was invertible and increased expression from the beta-globin promoter in HepG2 cells. Unlike the simian virus 40 enhancer, the prealbumin enhancer would not stimulate beta-globin synthesis in HeLa cells, and even the simian virus 40 enhancer did not stimulate the prealbumin promoter in HeLa cells. Thus, we identified in the prealbumin gene two DNA elements that respond in a cell-specific manner: a proximal promoter including a crucial sequence between -108 and -151 nucleotides and a distant enhancer element located between 1.6 and 2.15 kilobases upstream.

Sign in / Sign up

Export Citation Format

Share Document