scholarly journals The replication activation potential of selected RNA polymerase II promoter elements at the simian virus 40 origin.

1992 ◽  
Vol 12 (7) ◽  
pp. 3087-3093 ◽  
Author(s):  
A T Hoang ◽  
W Wang ◽  
J D Gralla
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cos Cells ◽  
Activation Potential ◽  
Multiple Copies ◽  
Cellular Transcription ◽  
Transcriptional Stimulation

Binding sites for cellular transcription factors were placed near the simian virus 40 origin of replication, and their effect on replication and TATA-dependent transcription was measured in COS cells. The hierarchy of transcriptional stimulation changed when the plasmids replicated. Only one of seven inserted sequences, a moderately weak transcription element, stimulated replication detectably. However, when two nonstimulatory sites were present in multiple copies they did activate replication. Multiple sites for the chimeric activator GAL4-VP16 did not stimulate replication even though transcription was stimulated strongly. The results indicate that the ability of a binding site to stimulate replication from the simian virus 40 ori is not based on its transcriptional activation potential but is instead related to a separate replication activation potential that can be increased by having multiple sites.

The replication activation potential of selected RNA polymerase II promoter elements at the simian virus 40 origin

1992 ◽  
Vol 12 (7) ◽  
pp. 3087-3093
Author(s):  
A T Hoang ◽  
W Wang ◽  
J D Gralla
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cos Cells ◽  
Activation Potential ◽  
Multiple Copies ◽  
Cellular Transcription ◽  
Transcriptional Stimulation

Binding sites for cellular transcription factors were placed near the simian virus 40 origin of replication, and their effect on replication and TATA-dependent transcription was measured in COS cells. The hierarchy of transcriptional stimulation changed when the plasmids replicated. Only one of seven inserted sequences, a moderately weak transcription element, stimulated replication detectably. However, when two nonstimulatory sites were present in multiple copies they did activate replication. Multiple sites for the chimeric activator GAL4-VP16 did not stimulate replication even though transcription was stimulated strongly. The results indicate that the ability of a binding site to stimulate replication from the simian virus 40 ori is not based on its transcriptional activation potential but is instead related to a separate replication activation potential that can be increased by having multiple sites.

scholarly journals Differential ability of proximal and remote element pairs to cooperate in activating RNA polymerase II transcription.

1991 ◽  
Vol 11 (9) ◽  
pp. 4561-4571 ◽  
Author(s):  
W D Wang ◽  
J D Gralla
Keyword(s):  
Rna Polymerase Ii ◽  
Binding Sites ◽  
Tandem Repeats ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cooperative Interaction ◽  
Promoter Strength ◽  
Synthetic Promoters ◽  
Major Late Promoter ◽  
Test Gene

To investigate the synergism or cooperative interaction between transcription elements, we have designed and constructed a series of synthetic polymerase II promoters with different combinations of elements. These include three different CCAAT boxes, which correspond to the binding sites for CP1, CP2, and NFI, a GC box, a CACCC box, and an ATF/CREB-binding site. The synthetic promoters containing these elements in proximal positions were linked to a test gene (CAT). Tandem repeats of AP1- and AP2-binding sites, the simian virus 40 enhancer, and DNA-binding sites for GAL-estrogen receptor were cloned downstream of the test gene. The strength of these promoters was then tested in transient-expression assays in HeLa TK- cells. In the context of the adenovirus major late promoter TATA box, the promoters containing only certain combinations of elements are active in this assay. Some elements appear to cooperate nearly universally, but others exhibit strong selectivity. These results indicate strongly selective synergistic interactions between elements and suggest that levels of promoter strength may be determined by the extent of compatibility between factors bound to proximal and enhancer sites.

Replication from a proximal simian virus 40 origin is severely inhibited by multiple reiterations of the 72-base-pair repeat enhancer sequence

1988 ◽  
Vol 8 (4) ◽  
pp. 1509-1517
Author(s):  
R Kumar ◽  
K P Yoon ◽  
K N Subramanian
Keyword(s):  
Transcriptional Activation ◽  
Base Pair ◽  
Copy Number ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Origin Of Replication ◽  
Replication Efficiency ◽  
Sv40 Origin ◽  
Multiple Copies ◽  
In Cis

In a previous study in our laboratory, the effect of the reiteration frequency of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer on transcription from the proximal SV40 early promoter was investigated (R. Kumar, T. A. Firak, C. T. Schroll, and K. N. Subramanian, Proc. Natl. Acad. Sci. USA 83:3199-3203, 1986). Increasing the enhancer copy number to four increased transcription proportionately; further increments in enhancer copy number reversed this effect, resulting in a decrease in the transcriptional activation. In the present study, the effect of enhancer reiteration on the replication efficiency of plasmids containing the SV40 origin of replication was investigated in transient replication assays in vivo in COS-1 monkey kidney cells producing the SV40 large tumor antigen required for replication. A plasmid containing the SV40 core origin and three copies of the replication-activating, G+C-rich 21-bp repeat promoter element replicated efficiently. Plasmids containing multiple copies of the 72-bp repeat enhancer cloned in head-to-tail linkage adjacent to the 21-bp repeat and the core origin replicated less efficiently; the decrease in replication efficiency could be correlated with the number of copies of the 72-bp repeat; replication was severely curtailed when 10 or more copies of the 72-bp repeat were present. Replication was not significantly inhibited by an increase in the number of copies of the 21-bp repeat to 15 or by the presence of three copies of a 360-bp pBR322 sequence in the immediate vicinity. Multiple copies of the 72-bp enhancer in cis were unable to inhibit replication from a second SV40 origin of replication situated 2 kilobase pairs away from the enhancer reiteration. Replication of four different test plasmids was not inhibited in trans by cotransfection of an excess of a potential competitor plasmid containing a 24-copy reiteration of the 72-bp enhancer. These results indicate that multiple tandem reiterations of the 72-bp enhancer inhibit replication only when they are present in cis adjacent to the origin of replication. Possible explanations for this inhibitory effect, such as an unfavorable local chromatin structure induced by the multimeric enhancer region or reduced or improper communications between factors bound to the multimeric region and the adjacent replication origin, are discussed.

scholarly journals Replication from a proximal simian virus 40 origin is severely inhibited by multiple reiterations of the 72-base-pair repeat enhancer sequence.

1988 ◽  
Vol 8 (4) ◽  
pp. 1509-1517 ◽  
Author(s):  
R Kumar ◽  
K P Yoon ◽  
K N Subramanian
Keyword(s):  
Transcriptional Activation ◽  
Base Pair ◽  
Copy Number ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Origin Of Replication ◽  
Replication Efficiency ◽  
Sv40 Origin ◽  
Multiple Copies ◽  
In Cis

In a previous study in our laboratory, the effect of the reiteration frequency of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer on transcription from the proximal SV40 early promoter was investigated (R. Kumar, T. A. Firak, C. T. Schroll, and K. N. Subramanian, Proc. Natl. Acad. Sci. USA 83:3199-3203, 1986). Increasing the enhancer copy number to four increased transcription proportionately; further increments in enhancer copy number reversed this effect, resulting in a decrease in the transcriptional activation. In the present study, the effect of enhancer reiteration on the replication efficiency of plasmids containing the SV40 origin of replication was investigated in transient replication assays in vivo in COS-1 monkey kidney cells producing the SV40 large tumor antigen required for replication. A plasmid containing the SV40 core origin and three copies of the replication-activating, G+C-rich 21-bp repeat promoter element replicated efficiently. Plasmids containing multiple copies of the 72-bp repeat enhancer cloned in head-to-tail linkage adjacent to the 21-bp repeat and the core origin replicated less efficiently; the decrease in replication efficiency could be correlated with the number of copies of the 72-bp repeat; replication was severely curtailed when 10 or more copies of the 72-bp repeat were present. Replication was not significantly inhibited by an increase in the number of copies of the 21-bp repeat to 15 or by the presence of three copies of a 360-bp pBR322 sequence in the immediate vicinity. Multiple copies of the 72-bp enhancer in cis were unable to inhibit replication from a second SV40 origin of replication situated 2 kilobase pairs away from the enhancer reiteration. Replication of four different test plasmids was not inhibited in trans by cotransfection of an excess of a potential competitor plasmid containing a 24-copy reiteration of the 72-bp enhancer. These results indicate that multiple tandem reiterations of the 72-bp enhancer inhibit replication only when they are present in cis adjacent to the origin of replication. Possible explanations for this inhibitory effect, such as an unfavorable local chromatin structure induced by the multimeric enhancer region or reduced or improper communications between factors bound to the multimeric region and the adjacent replication origin, are discussed.

Multiple positive and negative cis-acting elements mediate induced arginase (CAR1) gene expression in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (10) ◽  
pp. 5087-5097
Author(s):  
L Kovari ◽  
R Sumrada ◽  
I Kovari ◽  
T G Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cis Acting ◽  
The Third ◽  
Significant Homology ◽  
Mammalian Genes ◽  
Car1 Gene

Expression of the arginase (CAR1) gene in Saccharomyces cerevisiae is induced by arginine or its analog homoarginine. Induction has been previously shown to require a negatively acting upstream repression sequence, which maintains expression of the gene at a low level in the absence of inducer. The objective of this work was to identify the cis-acting elements responsible for CAR1 transcriptional activation and response to inducer. We identified three upstream activation sequences (UASs) that support transcriptional activation in a heterologous expression vector. Two of these UAS elements function in the absence of inducer, whereas the third functions only when inducer is present. One of the inducer-independent UAS elements exhibits significant homology to the Sp1 factor-binding sites identified in simian virus 40 and various mammalian genes.

Differential ability of proximal and remote element pairs to cooperate in activating RNA polymerase II transcription

1991 ◽  
Vol 11 (9) ◽  
pp. 4561-4571
Author(s):  
W D Wang ◽  
J D Gralla
Keyword(s):  
Rna Polymerase Ii ◽  
Binding Sites ◽  
Tandem Repeats ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cooperative Interaction ◽  
Promoter Strength ◽  
Synthetic Promoters ◽  
Major Late Promoter ◽  
Test Gene

To investigate the synergism or cooperative interaction between transcription elements, we have designed and constructed a series of synthetic polymerase II promoters with different combinations of elements. These include three different CCAAT boxes, which correspond to the binding sites for CP1, CP2, and NFI, a GC box, a CACCC box, and an ATF/CREB-binding site. The synthetic promoters containing these elements in proximal positions were linked to a test gene (CAT). Tandem repeats of AP1- and AP2-binding sites, the simian virus 40 enhancer, and DNA-binding sites for GAL-estrogen receptor were cloned downstream of the test gene. The strength of these promoters was then tested in transient-expression assays in HeLa TK- cells. In the context of the adenovirus major late promoter TATA box, the promoters containing only certain combinations of elements are active in this assay. Some elements appear to cooperate nearly universally, but others exhibit strong selectivity. These results indicate strongly selective synergistic interactions between elements and suggest that levels of promoter strength may be determined by the extent of compatibility between factors bound to proximal and enhancer sites.

scholarly journals Multiple positive and negative cis-acting elements mediate induced arginase (CAR1) gene expression in Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (10) ◽  
pp. 5087-5097 ◽  
Author(s):  
L Kovari ◽  
R Sumrada ◽  
I Kovari ◽  
T G Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cis Acting ◽  
The Third ◽  
Significant Homology ◽  
Mammalian Genes ◽  
Car1 Gene

Expression of the arginase (CAR1) gene in Saccharomyces cerevisiae is induced by arginine or its analog homoarginine. Induction has been previously shown to require a negatively acting upstream repression sequence, which maintains expression of the gene at a low level in the absence of inducer. The objective of this work was to identify the cis-acting elements responsible for CAR1 transcriptional activation and response to inducer. We identified three upstream activation sequences (UASs) that support transcriptional activation in a heterologous expression vector. Two of these UAS elements function in the absence of inducer, whereas the third functions only when inducer is present. One of the inducer-independent UAS elements exhibits significant homology to the Sp1 factor-binding sites identified in simian virus 40 and various mammalian genes.

scholarly journals Core Promoter Elements and TAFs Contribute to the Diversity of Transcriptional Activation in Vertebrates

2003 ◽  
Vol 23 (20) ◽  
pp. 7350-7362 ◽  
Author(s):  
Zheng Chen ◽  
James L. Manley
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Core Promoter ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Promoter Strength ◽  
Promoter Elements ◽  
The Core ◽  
Activation Mechanisms ◽  
Rnap Ii

ABSTRACT Gene-specific transcriptional activation is a multistep process that requires numerous protein factors and DNA elements, including enhancers and the core promoter. To investigate the roles of core promoter elements in transcriptional activation in vertebrates, we examined expression and factor occupancy on representative promoters in chicken DT40 cells containing a conditional TATA binding protein (TBP)-associated factor 9 allele (TAF9). Characterized core elements, including TATA box-flanking regions and the downstream promoter element, were found to play significant roles in determining promoter strength, response to activators, and factor occupancy and recruitment. The requirement for TAF9 was found to be highly promoter specific, and TAF9 dependence and promoter occupancy were not always correlated. We also describe contrasting examples of factor recruitment and activation mechanisms at different promoters, highlighted by the nearly opposite mechanisms utilized by the simian virus 40 enhancer and p53. With the core promoters analyzed, the former functions by facilitating RNA polymerase II (RNAP II) recruitment to a preassembled TBP/TFIIB-containing scaffold and p53 strongly recruits TBP and TFIIB while RNAP II levels remain modest. Taken together, our results illustrate both the important roles of core promoter elements and the remarkable diversity that characterizes transcriptional activation mechanisms in vertebrates.

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