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.

scholarly journals Domain structure of the simian virus 40 core origin of replication.

1986 ◽  
Vol 6 (5) ◽  
pp. 1663-1670 ◽  
Author(s):  
S Deb ◽  
A L DeLucia ◽  
C P Baur ◽  
A Koff ◽  
P Tegtmeyer
Keyword(s):  
Base Pair ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Dna Conformation ◽  
Sequence Specificity ◽  
Origin Of Replication ◽  
Base Pairs ◽  
Interaction Sites ◽  
Sequence Requirements

The simian virus 40 core origin of replication consists of nucleotides 5211 through 31. These 64 base pairs contain three functional domains with strict sequence requirements and two spacer regions with relaxed sequence specificity but precise positional constraints. The early domain extends for 10 contiguous base pairs between nucleotides 5211 and 5220. A 9-base pair spacer from sequences 5221 through 5229 separates the early domain from the 23-base pair central palindrome that directs the binding of T antigen. The late end of the core between nucleotides 12 and 31 also contains spacer and sequence-specific functions that are not yet completely mapped. We propose that the sequence-specific domains are interaction sites for viral and cellular proteins, determinants of DNA conformation, or both. The spacers would position these signals at required distances and rotations relative to one another.

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 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.

Domain structure of the simian virus 40 core origin of replication

1986 ◽  
Vol 6 (5) ◽  
pp. 1663-1670 ◽  
Author(s):  
S Deb ◽  
A L DeLucia ◽  
C P Baur ◽  
A Koff ◽  
P Tegtmeyer
Keyword(s):  
Base Pair ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Dna Conformation ◽  
Sequence Specificity ◽  
Origin Of Replication ◽  
Base Pairs ◽  
Interaction Sites ◽  
Sequence Requirements

The simian virus 40 core origin of replication consists of nucleotides 5211 through 31. These 64 base pairs contain three functional domains with strict sequence requirements and two spacer regions with relaxed sequence specificity but precise positional constraints. The early domain extends for 10 contiguous base pairs between nucleotides 5211 and 5220. A 9-base pair spacer from sequences 5221 through 5229 separates the early domain from the 23-base pair central palindrome that directs the binding of T antigen. The late end of the core between nucleotides 12 and 31 also contains spacer and sequence-specific functions that are not yet completely mapped. We propose that the sequence-specific domains are interaction sites for viral and cellular proteins, determinants of DNA conformation, or both. The spacers would position these signals at required distances and rotations relative to one another.

scholarly journals Analysis of an activatable promoter: sequences in the simian virus 40 late promoter required for T-antigen-mediated trans activation.

1985 ◽  
Vol 5 (8) ◽  
pp. 1859-1869 ◽  
Author(s):  
J M Keller ◽  
J C Alwine
Keyword(s):  
Base Pair ◽  
Promoter Activity ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Origin Of Replication ◽  
Wild Type ◽  
Late Promoter ◽  
Promoter Sequences ◽  
In Trans

The late promoter of simian virus 40 (SV40) is activated in trans by the viral early gene product, T antigen. We inserted the wild-type late-promoter region, and deletion mutants of it, into chloramphenicol acetyltransferase transient expression vectors to identify promoter sequences which are active in the presence of T antigen. We defined two promoter activities. One activity was mediated by a promoter element within simian virus 40 nucleotides 200 to 270. The activity of this element was detectable only in the presence of an intact, functioning origin of replication and accounted for 25 to 35% of the wild-type late-promoter activity in the presence of T antigen. The other activity was mediated by an element located within a 33-base-pair sequence (simian virus nucleotides 168 to 200) which spans the junction of the 72-base-pair repeats. This element functioned in the absence of both the origin of replication and the T-antigen-binding sites and appeared to be responsible for trans-activated gene expression. When inserted into an essentially promoterless plasmid, the 33-base-pair element functioned in an orientation-dependent manner. Under wild-type conditions in the presence of T antigen, the activity of this element accounted for 65 to 75% of the late-promoter activity. The roles of the 33-base-pair element and T antigen in trans-activation are discussed.

Analysis of an activatable promoter: sequences in the simian virus 40 late promoter required for T-antigen-mediated trans activation

1985 ◽  
Vol 5 (8) ◽  
pp. 1859-1869
Author(s):  
J M Keller ◽  
J C Alwine
Keyword(s):  
Base Pair ◽  
Promoter Activity ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Origin Of Replication ◽  
Wild Type ◽  
Late Promoter ◽  
Promoter Sequences ◽  
In Trans

The late promoter of simian virus 40 (SV40) is activated in trans by the viral early gene product, T antigen. We inserted the wild-type late-promoter region, and deletion mutants of it, into chloramphenicol acetyltransferase transient expression vectors to identify promoter sequences which are active in the presence of T antigen. We defined two promoter activities. One activity was mediated by a promoter element within simian virus 40 nucleotides 200 to 270. The activity of this element was detectable only in the presence of an intact, functioning origin of replication and accounted for 25 to 35% of the wild-type late-promoter activity in the presence of T antigen. The other activity was mediated by an element located within a 33-base-pair sequence (simian virus nucleotides 168 to 200) which spans the junction of the 72-base-pair repeats. This element functioned in the absence of both the origin of replication and the T-antigen-binding sites and appeared to be responsible for trans-activated gene expression. When inserted into an essentially promoterless plasmid, the 33-base-pair element functioned in an orientation-dependent manner. Under wild-type conditions in the presence of T antigen, the activity of this element accounted for 65 to 75% of the late-promoter activity. The roles of the 33-base-pair element and T antigen in trans-activation are discussed.

scholarly journals Role for DNA replication in beta-globin gene activation.

1988 ◽  
Vol 8 (3) ◽  
pp. 1301-1308 ◽  
Author(s):  
T Enver ◽  
A C Brewer ◽  
R K Patient
Keyword(s):  
Dna Replication ◽  
Transcriptional Activation ◽  
Globin Gene ◽  
Gene Activation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Covalent Modification ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Template Copy

Transcriptional activation of the Xenopus laevis beta-globin gene requires the synergistic action of the simian virus 40 enhancer and DNA replication in DEAE-dextran-mediated HeLa cell transfections. Replication does not act through covalent modification of the template, since its requirement was not obviated by the prior replication of the transfected DNA in eucaryotic cells. Transfection of DNA over a 100-fold range demonstrates that replication does not contribute to gene activation simply increasing template copy number. Furthermore, in cotransfections of replicating and nonreplicating constructs, only replicating templates were transcribed. Replication is not simply a requirement of chromatin assembly, since even unreplicated templates generated nucleosomal ladders. Stimulation of beta-globin transcription by DNA replication, though less marked, was also observed in calcium phosphate transfections. We interpret these results as revealing a dynamic role for replication in gene activation.

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