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.

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.

scholarly journals Sequence Requirements for the Assembly of Simian Virus 40 T Antigen and the T-Antigen Origin Binding Domain on the Viral Core Origin of Replication

1999 ◽  
Vol 73 (9) ◽  
pp. 7543-7555 ◽  
Author(s):  
Henry Y. Kim ◽  
Brett A. Barbaro ◽  
Woo S. Joo ◽  
Andrea E. Prack ◽  
K. R. Sreekumar ◽  
...  
Keyword(s):  
Central Region ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Binding Domain ◽  
Origin Of Replication ◽  
Viral Core ◽  
The Core ◽  
Flanking Sequences ◽  
Sequence Requirements

ABSTRACT The regions of the simian virus 40 (SV40) core origin that are required for stable assembly of virally encoded T antigen (T-ag) and the T-ag origin binding domain (T-ag-obd131–260) have been determined. Binding of the purified T-ag-obd131–260 is mediated by interactions with the central region of the core origin, site II. In contrast, T-ag binding and hexamer assembly requires a larger region of the core origin that includes both site II and an additional fragment of DNA that may be positioned on either side of site II. These studies indicate that in the context of T-ag, the origin binding domain can engage the pentanucleotides in site II only if a second region of T-ag interacts with one of the flanking sequences. The requirements for T-ag double-hexamer assembly are complex; the nucleotide cofactor present in the reaction modulates the sequence requirements for oligomerization. Nevertheless, these experiments provide additional evidence that only a subset of the SV40 core origin is required for assembly of T-ag double hexamers.

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 Quantitative Analysis of the Binding of Simian Virus 40 Large T Antigen to DNA

2007 ◽  
Vol 81 (17) ◽  
pp. 9162-9174 ◽  
Author(s):  
Amélie Fradet-Turcotte ◽  
Caroline Vincent ◽  
Simon Joubert ◽  
Peter A. Bullock ◽  
Jacques Archambault
Keyword(s):  
Specific Binding ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Sequence Specificity ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Viral Origin ◽  
Single Stranded Dna ◽  
Terminal Domain

ABSTRACT SV40 large T antigen (T-ag) is a multifunctional protein that successively binds to 5′-GAGGC-3′ sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork, and interacts with host DNA replication factors to promote replication of the simian virus 40 genome. The transition of T-ag from a sequence-specific binding protein to a nonspecific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for nonspecific double- and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild-type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5′-GAGGC-3′ sequence and of the residue downstream of it, which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with an ∼10-fold higher affinity than to nonspecific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer.

Regulation of transcription of the adenovirus EII promoter by EIa gene products: absence of sequence specificity

1984 ◽  
Vol 4 (10) ◽  
pp. 1970-1977
Author(s):  
R E Kingston ◽  
R J Kaufman ◽  
P A Sharp
Keyword(s):  
Cell Lines ◽  
Simian Virus 40 ◽  
Initiation Site ◽  
Simian Virus ◽  
Regulation Of Transcription ◽  
Sequence Specificity ◽  
Base Pairs ◽  
In Trans ◽  
In Cis ◽  
Specific Sequences

During adenovirus infection, the EII promoter is positively regulated by products of the EIa region. We have studied this regulation by fusing a DNA segment containing the adenovirus EII promoter to a dihydrofolate reductase cDNA segment. Expression of this hybrid gene is stimulated in trans when cell lines containing an integrated copy are either transfected with plasmids carrying the EIa region or infected with adenovirus. This suggests that EIa activity regulates transcription of the EII promoter in the absence of other viral proteins and that this stimulation can occur when the EII promoter is organized in cellular chromatin. Transcription from the EII promoter is initiated at two sites in cell lines lacking EIa activity. Introduction of the EIa region preferentially stimulated transcription from one of these two sites. A sensitive, stable cotransfection assay was used to test for specific EII sequences required for stimulation. EIa activity stimulates all mutant promoters; the most extensive deletion retained only 18 base pairs of sequences upstream of the initiation site. We suggest that regulation of a promoter by the EIa region does not depend on the presence of a set of specific sequences, but instead reflects a characteristic of promoters that have been exogenously introduced into cells. Insertion of the 72-base-pair repeat of simian-virus 40 in cis enhances transcription from the EII promoter. The stimulatory effects of EIa activity and of the simian virus 40 sequence are additive and appear to differ mechanistically.

Stimulation of the adenovirus E2 promoter by simian virus 40 T antigen or E1A occurs by different mechanisms

1986 ◽  
Vol 6 (6) ◽  
pp. 2020-2026
Author(s):  
M R Loeken ◽  
G Khoury ◽  
J Brady
Keyword(s):  
Simian Virus 40 ◽  
Promoter Sequence ◽  
Simian Virus ◽  
Chloramphenicol Acetyltransferase ◽  
T Antigen ◽  
Base Pairs ◽  
Promoter Sequences ◽  
Adenovirus E1a ◽  
E1a Gene ◽  
Stimulation Of

We have examined the ability of simian virus 40 T antigen to stimulate transcription from the adenovirus E2 promoter. T antigen, produced from a cotransfected plasmid, stimulated chloramphenicol acetyltransferase enzyme and mRNA production from an E2 promoter-chloramphenicol acetyltransferase fusion plasmid (pEC113) in monkey kidney CV-1 cells. The level of stimulation of E2 transcription by simian virus 40 T antigen was equal to that observed in cotransfections of pEC113 and the adenovirus E1A gene product. Deletion mutations from the 5' end of the E2 promoter were examined for their ability to express basal, T-antigen, or E1A trans-activated promoter activity. In each case, deletion of upstream promoter sequences to -70 base pairs reduced chloramphenicol acetyltransferase expression to approximately 30% of the level observed with the intact E2 promoter. Deletion to -59 base pairs resulted in chloramphenicol acetyltransferase expression that was 3 to 5% of that observed with the intact E2 promoter. At saturating levels of the stimulatory proteins, the chloramphenicol acetyltransferase levels obtained in response to T antigen and adenovirus E1A were additive. COS-1 cells, which are derived from CV-1 cells and constitutively express simian virus 40 T antigen, do not support E2 promoter trans activation by T antigen. E1A trans activation of the E2 promoter is efficient in COS-1 cells. These results suggest that although promoter sequence requirements are similar, T antigen and E1A trans activate the E2 promoter by different mechanisms.

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 Stimulation of the adenovirus E2 promoter by simian virus 40 T antigen or E1A occurs by different mechanisms.

1986 ◽  
Vol 6 (6) ◽  
pp. 2020-2026 ◽  
Author(s):  
M R Loeken ◽  
G Khoury ◽  
J Brady
Keyword(s):  
Simian Virus 40 ◽  
Promoter Sequence ◽  
Simian Virus ◽  
Chloramphenicol Acetyltransferase ◽  
T Antigen ◽  
Base Pairs ◽  
Promoter Sequences ◽  
Adenovirus E1a ◽  
E1a Gene ◽  
Stimulation Of

We have examined the ability of simian virus 40 T antigen to stimulate transcription from the adenovirus E2 promoter. T antigen, produced from a cotransfected plasmid, stimulated chloramphenicol acetyltransferase enzyme and mRNA production from an E2 promoter-chloramphenicol acetyltransferase fusion plasmid (pEC113) in monkey kidney CV-1 cells. The level of stimulation of E2 transcription by simian virus 40 T antigen was equal to that observed in cotransfections of pEC113 and the adenovirus E1A gene product. Deletion mutations from the 5' end of the E2 promoter were examined for their ability to express basal, T-antigen, or E1A trans-activated promoter activity. In each case, deletion of upstream promoter sequences to -70 base pairs reduced chloramphenicol acetyltransferase expression to approximately 30% of the level observed with the intact E2 promoter. Deletion to -59 base pairs resulted in chloramphenicol acetyltransferase expression that was 3 to 5% of that observed with the intact E2 promoter. At saturating levels of the stimulatory proteins, the chloramphenicol acetyltransferase levels obtained in response to T antigen and adenovirus E1A were additive. COS-1 cells, which are derived from CV-1 cells and constitutively express simian virus 40 T antigen, do not support E2 promoter trans activation by T antigen. E1A trans activation of the E2 promoter is efficient in COS-1 cells. These results suggest that although promoter sequence requirements are similar, T antigen and E1A trans activate the E2 promoter by different mechanisms.

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