scholarly journals Rescue of chromosomal T-antigen sequences onto extrachromosomally replicating, defective simian virus 40 DNA by homologous recombination.

1986 ◽  
Vol 6 (4) ◽  
pp. 1320-1325 ◽  
Author(s):  
S Subramani
Keyword(s):  
Homologous Recombination ◽  
Dna Sequences ◽  
Base Pair ◽  
Recombination Event ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Extrachromosomal Dna ◽  
Cos Cells ◽  
Antigen Gene

Recombination between chromosomal and extrachromosomal DNA sequences was analyzed by investigation of the recombinational rescue of a 1,018-base-pair (bp) segment of the T-antigen gene of simian virus 40 from the chromosome of monkey COS cells to two different, extrachromosomally replicating, simian virus 40 DNA molecules lacking this 1,018-bp sequence. The ratio of rescued to unrecombined virus was as high as 10(-3). The rescued molecules, detected optimally 5 to 9 days after transfection of COS cells, had completely recovered the 1,018-bp DNA segment from the chromosome. The recombination event is proposed to occur either by double reciprocal recombination or by gene conversion between the chromosomal T-antigen gene and the extrachromosomal molecules missing the 1,018-bp sequence.

Rescue of chromosomal T-antigen sequences onto extrachromosomally replicating, defective simian virus 40 DNA by homologous recombination

1986 ◽  
Vol 6 (4) ◽  
pp. 1320-1325
Author(s):  
S Subramani
Keyword(s):  
Homologous Recombination ◽  
Dna Sequences ◽  
Base Pair ◽  
Recombination Event ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Extrachromosomal Dna ◽  
Cos Cells ◽  
Antigen Gene

Recombination between chromosomal and extrachromosomal DNA sequences was analyzed by investigation of the recombinational rescue of a 1,018-base-pair (bp) segment of the T-antigen gene of simian virus 40 from the chromosome of monkey COS cells to two different, extrachromosomally replicating, simian virus 40 DNA molecules lacking this 1,018-bp sequence. The ratio of rescued to unrecombined virus was as high as 10(-3). The rescued molecules, detected optimally 5 to 9 days after transfection of COS cells, had completely recovered the 1,018-bp DNA segment from the chromosome. The recombination event is proposed to occur either by double reciprocal recombination or by gene conversion between the chromosomal T-antigen gene and the extrachromosomal molecules missing the 1,018-bp sequence.

Conversion through homologous recombination of the gene encoding Simian virus 40 115,000-molecular-weight super T antigen to a gene encoding a normal-size large T antigen variant

1984 ◽  
Vol 4 (6) ◽  
pp. 1141-1151
Author(s):  
P May ◽  
M Resche-Rigon ◽  
J Borde ◽  
C Breugnot ◽  
E May
Keyword(s):  
Homologous Recombination ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Gene Encoding ◽  
Antigen Gene ◽  
Cell Extracts ◽  
New Variant ◽  
Cloned Gene

We have previously cloned the gene encoding a 115,000-Mr super T antigen (115K super T antigen), an elongated form of the Simian virus 40 large T antigen, originating from the rat cell line V 11 F1 clone 1, subclone 7 (May et al., J. Virol. 45:901-913, 1983). DNA sequence analysis has shown that the 115K super T antigen gene contains notably an in-phase duplication of a sequence located in the region of tsA mutations. We have also shown that the 115K super T antigen gene is able to induce the formation of transformed foci in transfected rat cells. After rat cell cultures were transfected with the cloned gene encoding 115K super T antigen, we obtained a large number of transformants as reported in this paper. In these transformants, we detected a very high frequency of new T antigen variants, as shown by immunoprecipitation of the cell extracts with anti-simian virus 40 tumor serum followed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Based on these results and all of the data presently available, it appears likely that the input plasmid or cosmid DNAs containing the cloned gene were first subjected to recombination events that yield new variant T antigen genes before these recombinant genes become integrated. The new variant T antigens observed in the transformants were predominantly those comigrating with normal-size large T antigen. In fact, these latter variants appeared to be indistinguishable from wild-type large T antigen as judged by restriction mapping by Southern blotting of the total genomic DNA of the transformants. Models of intermolecular or intramolecular homologous recombination occurring between or within the input plasmid or input cosmid DNA molecules are proposed to account for the formation of such revertants.

scholarly journals Conversion through homologous recombination of the gene encoding Simian virus 40 115,000-molecular-weight super T antigen to a gene encoding a normal-size large T antigen variant.

1984 ◽  
Vol 4 (6) ◽  
pp. 1141-1151 ◽  
Author(s):  
P May ◽  
M Resche-Rigon ◽  
J Borde ◽  
C Breugnot ◽  
E May
Keyword(s):  
Homologous Recombination ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Gene Encoding ◽  
Antigen Gene ◽  
Cell Extracts ◽  
New Variant ◽  
Cloned Gene

We have previously cloned the gene encoding a 115,000-Mr super T antigen (115K super T antigen), an elongated form of the Simian virus 40 large T antigen, originating from the rat cell line V 11 F1 clone 1, subclone 7 (May et al., J. Virol. 45:901-913, 1983). DNA sequence analysis has shown that the 115K super T antigen gene contains notably an in-phase duplication of a sequence located in the region of tsA mutations. We have also shown that the 115K super T antigen gene is able to induce the formation of transformed foci in transfected rat cells. After rat cell cultures were transfected with the cloned gene encoding 115K super T antigen, we obtained a large number of transformants as reported in this paper. In these transformants, we detected a very high frequency of new T antigen variants, as shown by immunoprecipitation of the cell extracts with anti-simian virus 40 tumor serum followed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Based on these results and all of the data presently available, it appears likely that the input plasmid or cosmid DNAs containing the cloned gene were first subjected to recombination events that yield new variant T antigen genes before these recombinant genes become integrated. The new variant T antigens observed in the transformants were predominantly those comigrating with normal-size large T antigen. In fact, these latter variants appeared to be indistinguishable from wild-type large T antigen as judged by restriction mapping by Southern blotting of the total genomic DNA of the transformants. Models of intermolecular or intramolecular homologous recombination occurring between or within the input plasmid or input cosmid DNA molecules are proposed to account for the formation of such revertants.

scholarly journals Multiple DNA Damage Signaling and Repair Pathways Deregulated by Simian Virus 40 Large T Antigen

2010 ◽  
Vol 84 (16) ◽  
pp. 8007-8020 ◽  
Author(s):  
Sergei Boichuk ◽  
Liang Hu ◽  
Jennifer Hein ◽  
Ole V. Gjoerup
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Viral Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Productive Infection ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Viral Origin

ABSTRACT We demonstrated previously that expression of simian virus 40 (SV40) large T antigen (LT), without a viral origin, is sufficient to induce the hallmarks of a cellular DNA damage response (DDR), such as focal accumulation of γ-H2AX and 53BP1, via Bub1 binding. Here we expand our characterization of LT effects on the DDR. Using comet assays, we demonstrate that LT induces overt DNA damage. The Fanconi anemia pathway, associated with replication stress, becomes activated, since FancD2 accumulates in foci, and monoubiquitinated FancD2 is detected on chromatin. LT also induces a distinct set of foci of the homologous recombination repair protein Rad51 that are colocalized with Nbs1 and PML. The FancD2 and Rad51 foci require neither Bub1 nor retinoblastoma protein binding. Strikingly, wild-type LT is localized on chromatin at, or near, the Rad51/PML foci, but the LT mutant in Bub1 binding is not localized there. SV40 infection was previously shown to trigger ATM activation, which facilitates viral replication. We demonstrate that productive infection also triggers ATR-dependent Chk1 activation and that Rad51 and FancD2 colocalize with LT in viral replication centers. Using small interfering RNA (siRNA)-mediated knockdown, we demonstrate that Rad51 and, to a lesser extent, FancD2 are required for efficient viral replication in vivo, suggesting that homologous recombination is important for high-level extrachromosomal replication. Taken together, the interplay of LT with the DDR is more complex than anticipated, with individual domains of LT being connected to different subcomponents of the DDR and repair machinery.

Bovine papilloma virus contains an activator of gene expression at the distal end of the early transcription unit

1983 ◽  
Vol 3 (6) ◽  
pp. 1108-1122
Author(s):  
M Lusky ◽  
L Berg ◽  
H Weiher ◽  
M Botchan
Keyword(s):  
Thymidine Kinase ◽  
Dna Sequences ◽  
Base Pair ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Stable Transformation ◽  
High Molecular Weight Dna ◽  
Papilloma Virus ◽  
Recombinant Plasmids ◽  
Bovine Papilloma Virus

Bovine papilloma virus (BPV) contains a cis-acting DNA element which can enhance transcription of distal promoters. Utilizing both direct and indirect transient transfection assays, we showed that a 59-base-pair DNA sequence from the BPV genome could activate the simian virus 40 promoter from distances exceeding 2.5 kilobases and in an orientation-independent manner. In contrast to the promoter 5'-proximal localization of other known viral activators, this element was located immediately 3' to the early polyadenylation signal in the BPV genome. Deletion of these sequences from the BPV genome inactivated the transforming ability of BPV recombinant plasmids. Orientation-independent reinsertion of this 59-base-pair sequence, or alternatively of activator DNA sequences from simian virus 40 or polyoma virus, restored the transforming activity of the BPV recombinant plasmids. Furthermore, the stable transformation frequency of the herpes simplex virus type 1 thymidine kinase gene was enhanced when linked to restriction fragments of BPV DNA which included the defined activator element. This enhancement was orientation independent with respect to the thymidine kinase promoter. The enhancement also appeared to be unrelated to the establishment of the recombinant plasmids as episomes, since in transformed cells these sequences are found linked to high-molecular-weight DNA. We propose that the enhancement of stable transformation frequencies and the activation of transcription units are in this case alternate manifestations of the same biochemical events.

Circular and linear simian virus 40 DNAs differ in recombination

1985 ◽  
Vol 5 (4) ◽  
pp. 869-880
Author(s):  
D Dorsett ◽  
I Deichaite ◽  
E Winocour
Keyword(s):  
Dna Sequences ◽  
Tandem Repeats ◽  
Regulatory Region ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Linear Forms ◽  
Rolling Circle ◽  
Linear Dna ◽  
Sv40 Dna

Linear forms of simian virus 40 (SV40) DNA, when added to transfection mixtures containing circular SV40 and phi X174 RFI DNAs, enhanced the frequency of SV40/phi X174 recombination, as measured by infectious center in situ plaque hybridization in monkey BSC-1 cells. The sequences required for the enhancement of recombination by linear DNA reside within the SV40 replication origin/regulatory region (nucleotides 5,171 to 5,243/0 to 128). Linearization of phi X174 RFI DNA did not increase the recombination frequency. The SV40/phi X174 recombinant structures arising from transfections supplemented with linear forms of origin-containing SV40 DNA contained phi X174 DNA sequences interspersed within tandem head-to-tail repeats derived from the recombination-enhancing linear DNA. Evidence is presented that the tandem repeats are not formed by homologous recombination and that linear forms of SV40 DNA must compete with circular SV40 DNA for the available T antigen to enhance recombination. We propose that the enhancement of recombination by linear SV40 DNA results from the entry of that DNA into a rolling circle type of replication pathway which generates highly recombinogenic intermediates.

Strontium phosphate transfection of human cells in primary culture: stable expression of the simian virus 40 large-T-antigen gene in primary human bronchial epithelial cells

1987 ◽  
Vol 7 (5) ◽  
pp. 2031-2034
Author(s):  
D E Brash ◽  
R R Reddel ◽  
M Quanrud ◽  
K Yang ◽  
M P Farrell ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Primary Culture ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Bronchial Epithelial Cells ◽  
Large T Antigen ◽  
Bronchial Epithelial ◽  
Antigen Gene ◽  
Strontium Phosphate

Strontium ion formed DNA-phosphate precipitates analogous to those formed by calcium but lacking the lethal and differentiation-inducing effects of calcium on many epithelial cell types in primary culture. Human primary bronchial epithelial cells were transiently and stably transfected by using strontium phosphate; the frequency of stable transformation with a plasmid carrying the simian virus 40 large-T-antigen gene was greater than 10(-4).

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.

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