scholarly journals Evolutionarily selected replication origins: functional aspects and structural organization.

1986 ◽  
Vol 6 (9) ◽  
pp. 3077-3085 ◽  
Author(s):  
G J Lee-Chen ◽  
M Woodworth-Gutai
Keyword(s):  
Inverted Repeat ◽  
Replication Origin ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Inverted Repeats ◽  
Regulatory Sequences ◽  
Functional Aspects ◽  
Cellular Sequence ◽  
Transfection Mixture

A selective replicative pressure occurs during the evolution of simian virus 40 variants. When the replication origin is duplicated as an inverted repeat, there is a dramatic enhancement of replication. Having regulatory sequences located between the inverted repeat of ori magnifies their enhancing effect on replication. A passage 20 variant and a passage 45 variant containing three pairs of an inverted repeat of ori replicated more efficiently than a passage 13 variant containing nine copies of ori arranged in tandem. A 69-base-pair cellular sequence inserted between inverted repeats of ori of both passage 40 and 45 variants enhanced simian virus 40 DNA replication. Differences in replication efficiencies became greater as the total number of replicating species was increased in the transfection mixture, under conditions where T antigen is limiting. In a competitive environment, sequences flanking the replication origin may be inhibitory to replication.

Evolutionarily selected replication origins: functional aspects and structural organization

1986 ◽  
Vol 6 (9) ◽  
pp. 3077-3085
Author(s):  
G J Lee-Chen ◽  
M Woodworth-Gutai
Keyword(s):  
Inverted Repeat ◽  
Replication Origin ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Inverted Repeats ◽  
Regulatory Sequences ◽  
Functional Aspects ◽  
Cellular Sequence ◽  
Transfection Mixture

A selective replicative pressure occurs during the evolution of simian virus 40 variants. When the replication origin is duplicated as an inverted repeat, there is a dramatic enhancement of replication. Having regulatory sequences located between the inverted repeat of ori magnifies their enhancing effect on replication. A passage 20 variant and a passage 45 variant containing three pairs of an inverted repeat of ori replicated more efficiently than a passage 13 variant containing nine copies of ori arranged in tandem. A 69-base-pair cellular sequence inserted between inverted repeats of ori of both passage 40 and 45 variants enhanced simian virus 40 DNA replication. Differences in replication efficiencies became greater as the total number of replicating species was increased in the transfection mixture, under conditions where T antigen is limiting. In a competitive environment, sequences flanking the replication origin may be inhibitory to replication.

scholarly journals DNA-binding properties of simian virus 40 T-antigen mutants defective in viral DNA replication.

1983 ◽  
Vol 3 (11) ◽  
pp. 1958-1966 ◽  
Author(s):  
C Prives ◽  
L Covey ◽  
A Scheller ◽  
Y Gluzman
Keyword(s):  
Amino Acid ◽  
Dna Sequences ◽  
Simian Virus 40 ◽  
Amino Acid Position ◽  
Simian Virus ◽  
T Antigen ◽  
Regulatory Sequences ◽  
Viral Dna ◽  
Viral Origin ◽  
Specific Affinity

Three simian virus 40 (SV40)-transformed monkey cell lines, C2, C6, and C11, producing T-antigen variants that are unable to initiate viral DNA replication, were analyzed with respect to their affinity for regulatory sequences at the viral origin of replication. C2 and C11 T antigens both bound specifically to sequences at sites 1 and 2 at the viral origin region, whereas C6 T antigen showed no specific affinity for any viral DNA sequences under all conditions tested. Viral DNA sequences encoding the C6 T antigen have recently been cloned out of C6 cells and used to transform an established rat cell line. T antigen from several cloned C6-SV40-transformed rat lines failed to bind specifically to the origin. C6 DNA contains three mutations: two located close to the amino terminus of T antigen at amino acid positions 30 and 51 and a third located internally at amino acid position 153. Two recombinant SV40 DNA mutants were prepared containing either the amino-terminal mutations at positions 30 and 51 (C6-1) or the internally located mutation at position 153 (C6-2) and used to transform Rat 2 cells. Whereas T antigen from C6-2-transformed cells lacked any specific affinity for these sequences. Therefore, the single mutation at amino acid position 153 (Asn leads to Thr) is sufficient to abolish the origin-binding property of T antigen. A T antigen-specific monoclonal antibody, PAb 100, which had been previously shown to immunoprecipitate an immunologically distinct origin-binding subclass of T antigen, recognized wild-type or C6-1 antigens, but failed to react with C6 or C6-2 T antigens. These results indicate that viral replication function comprises properties of T antigen that exist in addition to its ability to bind specifically to the SV40 regulatory sequences. Furthermore, it is concluded from these data that specific viral origin binding is not a necessary feature of the transforming function of T antigen.

scholarly journals The Gonadotropin-Releasing Hormone Receptor Gene Promoter Directs Pituitary-Specific Oncogene Expression in Transgenic Mice*

Endocrinology ◽  
1999 ◽  
Vol 140 (5) ◽  
pp. 2415-2421 ◽  
Author(s):  
Constance T. Albarracin ◽  
Matthew P. Frosch ◽  
William W. Chin
Keyword(s):  
Transgenic Mice ◽  
Pituitary Gland ◽  
Gene Promoter ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Regulatory Sequences ◽  
Pituitary Cells ◽  
R Gene ◽  
Flanking Region

Abstract Our previous work has shown that 1.2 kb of the 5′ flanking region of the mouse GnRH receptor (mGnRH-R) gene is sufficient to direct tissue-specific expression in vitro. In this study, we have used the cell-specific regulatory sequences of the mGnRH-R gene promoter to target the expression of the simian virus 40 virus T antigen (TAg) to the pituitary gland of transgenic mice. A hybrid transgene, GnRH-R/TAg, was prepared using the −1164/+52 region of the mGnRH-R gene and +2533/+5234 sequences encoding the large T antigen of the simian virus 40. Two founders developed tumors of apparent pituitary origin at 44 (M28, female) and 50 (M25, male) days of age. M28 and M25 mice were about 50% underweight, and their gonads were grossly underdeveloped compared with wild-type litter mates. A third male founder, M29, developed a tumor at a later time (109 days). M29 was able to breed successfully and stably transmit the GnRH-R/TAg transgene. Mice of the M29 transgene line developed tumors at 4–5 months of age. Gross examination showed that the tumors extend from the sella and infiltrate into the inferior surface of the brain. In small tumors collected from young transgenic animals, normal pituitary cells as well as transition areas of increasing cellular atypia are evident. Frankly malignant cells are seen in all tumors. The pituitary tumors express the α-, FSHβ-, and LHβ-subunits and the GnRH-R messenger RNA, all markers of a gonadotrope but not of other anterior pituitary cell lineages. In summary, our studies indicate that 1.2 kb of the 5′-flanking region of the mGnRH-R gene can be used to target expression specifically to the gonadotropes of the pituitary gland in transgenic mice. The GnRH-R gene promoter-directed expression appears to be cell-specific and results in the formation of tumors that are primarily of gonadotropic origin.

scholarly journals Relationship among Location of T-Antigen-Induced DNA Distortion, Auxiliary Sequences, and DNA Replication Efficiency

2003 ◽  
Vol 77 (19) ◽  
pp. 10651-10657 ◽  
Author(s):  
Susan Okuley ◽  
Mindy Call ◽  
Tara Mitchell ◽  
Bugen Hu ◽  
Mary E. Woodworth
Keyword(s):  
Dna Replication ◽  
Regulatory Region ◽  
Simian Virus 40 ◽  
Single Copy ◽  
Simian Virus ◽  
T Antigen ◽  
Replication Efficiency ◽  
Cellular Sequence ◽  
In The Wild ◽  
Sv40 Dna

ABSTRACT T-antigen-induced DNA distortion was studied in a series of simian virus 40 (SV40) plasmid constructs whose relative replication efficiency ranges from 0.2 to 36. Bending was detected in the wild-type SV40 regulatory region consisting of three copies of the GC-rich 21-bp repeat but not in constructs with only one or two copies of the 21-bp repeat. In a construct with enhanced replication efficiency, bending occurred in a 69-bp cellular sequence located upstream of a single copy of the 21-bp repeat. Bending occurred both upstream of ori and in the three 21-bp repeats located downstream of ori in a construct with reduced replication efficiency. In a construct with no 21-bp repeats, DNA distortion occurred downstream of ori. The results indicate that SV40 DNA replication is enhanced when the structure of the regulatory region allows the DNA to form a bent structure upstream of the initial movement of the replication fork.

DNA-binding properties of simian virus 40 T-antigen mutants defective in viral DNA replication

1983 ◽  
Vol 3 (11) ◽  
pp. 1958-1966
Author(s):  
C Prives ◽  
L Covey ◽  
A Scheller ◽  
Y Gluzman
Keyword(s):  
Amino Acid ◽  
Dna Sequences ◽  
Simian Virus 40 ◽  
Amino Acid Position ◽  
Simian Virus ◽  
T Antigen ◽  
Regulatory Sequences ◽  
Viral Dna ◽  
Viral Origin ◽  
Specific Affinity

Three simian virus 40 (SV40)-transformed monkey cell lines, C2, C6, and C11, producing T-antigen variants that are unable to initiate viral DNA replication, were analyzed with respect to their affinity for regulatory sequences at the viral origin of replication. C2 and C11 T antigens both bound specifically to sequences at sites 1 and 2 at the viral origin region, whereas C6 T antigen showed no specific affinity for any viral DNA sequences under all conditions tested. Viral DNA sequences encoding the C6 T antigen have recently been cloned out of C6 cells and used to transform an established rat cell line. T antigen from several cloned C6-SV40-transformed rat lines failed to bind specifically to the origin. C6 DNA contains three mutations: two located close to the amino terminus of T antigen at amino acid positions 30 and 51 and a third located internally at amino acid position 153. Two recombinant SV40 DNA mutants were prepared containing either the amino-terminal mutations at positions 30 and 51 (C6-1) or the internally located mutation at position 153 (C6-2) and used to transform Rat 2 cells. Whereas T antigen from C6-2-transformed cells lacked any specific affinity for these sequences. Therefore, the single mutation at amino acid position 153 (Asn leads to Thr) is sufficient to abolish the origin-binding property of T antigen. A T antigen-specific monoclonal antibody, PAb 100, which had been previously shown to immunoprecipitate an immunologically distinct origin-binding subclass of T antigen, recognized wild-type or C6-1 antigens, but failed to react with C6 or C6-2 T antigens. These results indicate that viral replication function comprises properties of T antigen that exist in addition to its ability to bind specifically to the SV40 regulatory sequences. Furthermore, it is concluded from these data that specific viral origin binding is not a necessary feature of the transforming function of T antigen.

Functional organization of the simian virus 40 origin of DNA replication

1986 ◽  
Vol 6 (4) ◽  
pp. 1117-1128 ◽  
Author(s):  
J J Li ◽  
K W Peden ◽  
R A Dixon ◽  
T Kelly
Keyword(s):  
Dna Replication ◽  
Inverted Repeat ◽  
Functional Organization ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Origin Of Replication ◽  
Antigen Binding Site

To define the sequence elements involved in initiation of DNA synthesis at the simian virus 40 origin of replication, we determined the relative replication efficiencies in vitro and in vivo of templates containing a variety of mutations within the origin region. Replication of the mutants in vitro was assayed by the cell-free DNA replication system that we recently described (J.J. Li and T.J. Kelly, Proc. Natl. Acad. Sci. USA 81:6973-6977, 1984; J.J. Li and T.J. Kelly, Mol. Cell. Biol. 5:1238-1246, 1985), and replication in vivo was assayed after transfection of the mutant templates into COS-1 cells. The minimal origin of replication defined by both assays included a 15-base-pair (bp) imperfect inverted repeat, a 27-bp perfect inverted repeat, and a 17-bp A/T-rich region. T-antigen binding site I was not required for DNA replication, but its presence increased replication efficiency severalfold both in vitro and in vivo. Although SP1 binding sites and enhancers had little or no effect on replication in vitro, the presence of either element markedly increased replication in vivo. Thus, the biological role of these elements is not restricted to stimulating transcription but may be more general.

scholarly journals Cooperative assembly of simian virus 40 T-antigen hexamers on functional halves of the replication origin.

1991 ◽  
Vol 65 (6) ◽  
pp. 2798-2806 ◽  
Author(s):  
R E Parsons ◽  
J E Stenger ◽  
S Ray ◽  
R Welker ◽  
M E Anderson ◽  
...  
Keyword(s):  
Replication Origin ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Cooperative Assembly

scholarly journals Functional organization of the simian virus 40 origin of DNA replication.

1986 ◽  
Vol 6 (4) ◽  
pp. 1117-1128 ◽  
Author(s):  
J J Li ◽  
K W Peden ◽  
R A Dixon ◽  
T Kelly
Keyword(s):  
Dna Replication ◽  
Inverted Repeat ◽  
Functional Organization ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Origin Of Replication ◽  
Antigen Binding Site

To define the sequence elements involved in initiation of DNA synthesis at the simian virus 40 origin of replication, we determined the relative replication efficiencies in vitro and in vivo of templates containing a variety of mutations within the origin region. Replication of the mutants in vitro was assayed by the cell-free DNA replication system that we recently described (J.J. Li and T.J. Kelly, Proc. Natl. Acad. Sci. USA 81:6973-6977, 1984; J.J. Li and T.J. Kelly, Mol. Cell. Biol. 5:1238-1246, 1985), and replication in vivo was assayed after transfection of the mutant templates into COS-1 cells. The minimal origin of replication defined by both assays included a 15-base-pair (bp) imperfect inverted repeat, a 27-bp perfect inverted repeat, and a 17-bp A/T-rich region. T-antigen binding site I was not required for DNA replication, but its presence increased replication efficiency severalfold both in vitro and in vivo. Although SP1 binding sites and enhancers had little or no effect on replication in vitro, the presence of either element markedly increased replication in vivo. Thus, the biological role of these elements is not restricted to stimulating transcription but may be more general.

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