scholarly journals Hairpin structures are the primary amplification products: a novel mechanism for generation of inverted repeats during gene amplification.

1994 ◽  
Vol 14 (12) ◽  
pp. 7782-7791 ◽  
Author(s):  
S Cohen ◽  
D Hassin ◽  
S Karby ◽  
S Lavi
Keyword(s):  
Gene Amplification ◽  
De Novo ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Dna Amplification ◽  
Simian Virus ◽  
Inverted Repeats ◽  
Template Switching ◽  
Two Dimensional Gel Electrophoresis

Early events of DNA amplification which occur during perturbed replication were studied by using simian virus 40 (SV40)-transformed Chinese hamster cells (CO60) as a model system. The amplification is observed shortly after carcinogen treatment, and the amplified sequences contain molecules organized as inverted repeats (IRs). SV40 amplification in vitro was studied by using extracts from carcinogen-treated CO60 cells. In the amplified DNA the SV40 origin region was rereplicated, while more distal sequences were not replicated even once. Using several experimental procedures such as sucrose gradients, "snap-back" assay, and two-dimensional gel electrophoresis, we show that the overreplicated DNA contains IRs which are synthesized de novo as hairpins or stem-loop structures which were detached from the template molecules. The fully replicated SV40 molecules synthesized by the HeLa extracts do not contain such IRs. We propose "U-turn replication" as a novel mechanism for gene amplification, accounting for the generation of extrachromosomal inverted duplications as a result of perturbed replication and template switching of the DNA polymerases.

Hairpin structures are the primary amplification products: a novel mechanism for generation of inverted repeats during gene amplification

1994 ◽  
Vol 14 (12) ◽  
pp. 7782-7791
Author(s):  
S Cohen ◽  
D Hassin ◽  
S Karby ◽  
S Lavi
Keyword(s):  
Gene Amplification ◽  
De Novo ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Dna Amplification ◽  
Simian Virus ◽  
Inverted Repeats ◽  
Template Switching ◽  
Two Dimensional Gel Electrophoresis

Early events of DNA amplification which occur during perturbed replication were studied by using simian virus 40 (SV40)-transformed Chinese hamster cells (CO60) as a model system. The amplification is observed shortly after carcinogen treatment, and the amplified sequences contain molecules organized as inverted repeats (IRs). SV40 amplification in vitro was studied by using extracts from carcinogen-treated CO60 cells. In the amplified DNA the SV40 origin region was rereplicated, while more distal sequences were not replicated even once. Using several experimental procedures such as sucrose gradients, "snap-back" assay, and two-dimensional gel electrophoresis, we show that the overreplicated DNA contains IRs which are synthesized de novo as hairpins or stem-loop structures which were detached from the template molecules. The fully replicated SV40 molecules synthesized by the HeLa extracts do not contain such IRs. We propose "U-turn replication" as a novel mechanism for gene amplification, accounting for the generation of extrachromosomal inverted duplications as a result of perturbed replication and template switching of the DNA polymerases.

Carcinogen-induced DNA amplification in vitro: overreplication of the simian virus 40 origin region in extracts from carcinogen-treated CO60 cells

1990 ◽  
Vol 10 (1) ◽  
pp. 75-83
Author(s):  
Y Berko-Flint ◽  
S Karby ◽  
D Hassin ◽  
S Lavi
Keyword(s):  
De Novo ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Dna Molecules ◽  
Viral Origin ◽  
Cell Extracts ◽  
Origin Region

An in vitro system to study carcinogen-induced amplification in simian virus 40 (SV40)-transformed Chinese hamster (CO60) cells is described. SV40 amplification in this system resembled in many aspects the viral overreplication observed in drug-treated CO60 cells. Cytosolic extracts from N-methyl-N'-nitro-N-nitrosoguanidine-treated cells supported de novo DNA synthesis in the presence of excess exogenous T antigen and the SV40-containing plasmid pSVK1. The pattern of viral replication in these extracts was unique, since only the 2.4-kilobase-pair region spanning the origin was overreplicated, whereas distal sequences were not replicated significantly. Extracts from control cells supported only marginal levels of replication. In HeLa extracts, complete SV40 DNA molecules were replicated efficiently. The overreplication of the origin region in CO60 cell extracts was bidirectional and symmetrical. A fraction of the newly synthesized DNA molecules underwent a second round of replication, yielding MboI-sensitive fragments representing the 2.4-kilobase-pair region around the origin. The mechanisms controlling the amplification of the viral origin region, the nature of the cellular factors induced in the carcinogen-treated cells, and their putative association with general drug-induced SOS-like responses are discussed.

Carcinogen-mediated methotrexate resistance and dihydrofolate reductase amplification in Chinese hamster cells

1986 ◽  
Vol 6 (6) ◽  
pp. 1958-1964
Author(s):  
T Kleinberger ◽  
S Etkin ◽  
S Lavi
Keyword(s):  
Gene Amplification ◽  
Dihydrofolate Reductase ◽  
Alkylating Agents ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Dna Amplification ◽  
Simian Virus ◽  
Dhfr Gene ◽  
Cellular Mechanisms ◽  
Methotrexate Resistance

We have investigated different parameters characterizing carcinogen-mediated enhancement of methotrexate resistance in Chinese hamster ovary (CHO) cells and in simian virus 40-transformed Chinese hamster embryo (C060) cells. We show that this enhancement reflects dihydrofolate reductase (dhfr) gene amplification. The carcinogens used in this work are alkylating agents and UV irradiation. Both types of carcinogens induce a transient enhancement of methotrexate resistance which increases gradually from the time of treatment to 72 to 96 h later and decreases thereafter. Increasing doses of carcinogens decrease cell survival and increase the enhancement of methotrexate resistance. Enhancement was observed when cells were treated at different stages in the cell cycle, and it was maximal when cells were treated during the early S phase. These studies of carcinogen-mediated dhfr gene amplification coupled with our earlier studies on viral DNA amplification in simian virus 40-transformed cells demonstrate that the same parameters characterize the amplification of both genes. Possible cellular mechanisms responsible for the carcinogen-mediated gene amplification phenomenon are discussed.

scholarly journals Carcinogen-induced DNA amplification in vitro: overreplication of the simian virus 40 origin region in extracts from carcinogen-treated CO60 cells.

1990 ◽  
Vol 10 (1) ◽  
pp. 75-83 ◽  
Author(s):  
Y Berko-Flint ◽  
S Karby ◽  
D Hassin ◽  
S Lavi
Keyword(s):  
De Novo ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Dna Molecules ◽  
Viral Origin ◽  
Cell Extracts ◽  
Origin Region

An in vitro system to study carcinogen-induced amplification in simian virus 40 (SV40)-transformed Chinese hamster (CO60) cells is described. SV40 amplification in this system resembled in many aspects the viral overreplication observed in drug-treated CO60 cells. Cytosolic extracts from N-methyl-N'-nitro-N-nitrosoguanidine-treated cells supported de novo DNA synthesis in the presence of excess exogenous T antigen and the SV40-containing plasmid pSVK1. The pattern of viral replication in these extracts was unique, since only the 2.4-kilobase-pair region spanning the origin was overreplicated, whereas distal sequences were not replicated significantly. Extracts from control cells supported only marginal levels of replication. In HeLa extracts, complete SV40 DNA molecules were replicated efficiently. The overreplication of the origin region in CO60 cell extracts was bidirectional and symmetrical. A fraction of the newly synthesized DNA molecules underwent a second round of replication, yielding MboI-sensitive fragments representing the 2.4-kilobase-pair region around the origin. The mechanisms controlling the amplification of the viral origin region, the nature of the cellular factors induced in the carcinogen-treated cells, and their putative association with general drug-induced SOS-like responses are discussed.

scholarly journals Carcinogen-mediated methotrexate resistance and dihydrofolate reductase amplification in Chinese hamster cells.

1986 ◽  
Vol 6 (6) ◽  
pp. 1958-1964 ◽  
Author(s):  
T Kleinberger ◽  
S Etkin ◽  
S Lavi
Keyword(s):  
Gene Amplification ◽  
Dihydrofolate Reductase ◽  
Alkylating Agents ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Dna Amplification ◽  
Simian Virus ◽  
Dhfr Gene ◽  
Cellular Mechanisms ◽  
Methotrexate Resistance

We have investigated different parameters characterizing carcinogen-mediated enhancement of methotrexate resistance in Chinese hamster ovary (CHO) cells and in simian virus 40-transformed Chinese hamster embryo (C060) cells. We show that this enhancement reflects dihydrofolate reductase (dhfr) gene amplification. The carcinogens used in this work are alkylating agents and UV irradiation. Both types of carcinogens induce a transient enhancement of methotrexate resistance which increases gradually from the time of treatment to 72 to 96 h later and decreases thereafter. Increasing doses of carcinogens decrease cell survival and increase the enhancement of methotrexate resistance. Enhancement was observed when cells were treated at different stages in the cell cycle, and it was maximal when cells were treated during the early S phase. These studies of carcinogen-mediated dhfr gene amplification coupled with our earlier studies on viral DNA amplification in simian virus 40-transformed cells demonstrate that the same parameters characterize the amplification of both genes. Possible cellular mechanisms responsible for the carcinogen-mediated gene amplification phenomenon are discussed.

Transfer of nucleosomes from parental to replicated chromatin

1991 ◽  
Vol 11 (12) ◽  
pp. 6257-6267
Author(s):  
T Krude ◽  
R Knippers
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Replication Process ◽  
Two Dimensional Gel Electrophoresis ◽  
Sv40 Origin ◽  
Dna Strands ◽  
Topological State ◽  
High Concentrations ◽  
Sv40 Dna

Simian virus 40 (SV40) minichromosomes were used as the substrate for in vitro replication. Protein-free SV40 DNA or plasmids, carrying the SV40 origin of replication, served as controls. Replicated minichromosomal DNA possessed constrained negative superhelicity indicative of the presence of nucleosomes. The topological state of replicated minichromosomal DNA was precisely determined by two-dimensional gel electrophoresis. We show that most or all nucleosomes, present on the replicated minichromosomal DNA, were derived from the parental minichromosome substrate. The mode and the rate of nucleosome transfer from parental to minichromosomal daughter DNA were not influenced by high concentrations of competing replicating and nonreplicating protein-free DNA, indicating that nucleosomes remain associated with DNA during the replication process. The data also show that parental nucleosomes were segregated to the replicated daughter DNA strands in a dispersive manner.

scholarly journals Transfer of nucleosomes from parental to replicated chromatin.

1991 ◽  
Vol 11 (12) ◽  
pp. 6257-6267 ◽  
Author(s):  
T Krude ◽  
R Knippers
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Replication Process ◽  
Two Dimensional Gel Electrophoresis ◽  
Sv40 Origin ◽  
Dna Strands ◽  
Topological State ◽  
High Concentrations ◽  
Sv40 Dna

Simian virus 40 (SV40) minichromosomes were used as the substrate for in vitro replication. Protein-free SV40 DNA or plasmids, carrying the SV40 origin of replication, served as controls. Replicated minichromosomal DNA possessed constrained negative superhelicity indicative of the presence of nucleosomes. The topological state of replicated minichromosomal DNA was precisely determined by two-dimensional gel electrophoresis. We show that most or all nucleosomes, present on the replicated minichromosomal DNA, were derived from the parental minichromosome substrate. The mode and the rate of nucleosome transfer from parental to minichromosomal daughter DNA were not influenced by high concentrations of competing replicating and nonreplicating protein-free DNA, indicating that nucleosomes remain associated with DNA during the replication process. The data also show that parental nucleosomes were segregated to the replicated daughter DNA strands in a dispersive manner.

scholarly journals Sequence-specific interaction of histones with the simian virus 40 enhancer region in vitro.

1985 ◽  
Vol 260 (23) ◽  
pp. 12394-12397
Author(s):  
M F Clarke ◽  
P C FitzGerald ◽  
J M Brubaker ◽  
R T Simpson
Keyword(s):  
Specific Interaction ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Enhancer Region
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