A novel pathway for transversion mutation induced by dCTP misincorporation in a mutator strain of CHO cells

1989 ◽  
Vol 9 (4) ◽  
pp. 1810-1812
Author(s):  
G Phear ◽  
M Meuth
Keyword(s):  
Dna Replication ◽  
Cho Cells ◽  
Mutator Strain ◽  
Transversion Mutation

Intracellular imbalances of dCTP produce both T----C transitions and an unusual class of transversions (A----C) at the aprt locus of CHO cells. Our data suggest that this transversion pathway is the consequence of dCTP:T mispairs which are not efficiently proofread during DNA replication.

scholarly journals A novel pathway for transversion mutation induced by dCTP misincorporation in a mutator strain of CHO cells.

1989 ◽  
Vol 9 (4) ◽  
pp. 1810-1812 ◽  
Author(s):  
G Phear ◽  
M Meuth
Keyword(s):  
Dna Replication ◽  
Cho Cells ◽  
Mutator Strain ◽  
Transversion Mutation

Intracellular imbalances of dCTP produce both T----C transitions and an unusual class of transversions (A----C) at the aprt locus of CHO cells. Our data suggest that this transversion pathway is the consequence of dCTP:T mispairs which are not efficiently proofread during DNA replication.

Replication in the amplified dihydrofolate reductase domain in CHO cells may initiate at two distinct sites, one of which is a repetitive sequence element

1989 ◽  
Vol 9 (2) ◽  
pp. 532-540
Author(s):  
B Anachkova ◽  
J L Hamlin
Keyword(s):  
Dna Replication ◽  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Replication Initiation ◽  
Ovary Cell ◽  
Sequence Element ◽  
Hybridization Probes ◽  
Ovary Cell Line

To study initiation of DNA replication in mammalian chromosomes, we have established a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) that contains approximately 1,000 copies of the early replicating dihydrofolate reductase (DHFR) domain. We have previously shown that DNA replication in the prevalent 243-kilobase (kb) amplicon type in this cell line initiates somewhere within a 28-kb region located downstream from the DHFR gene. In an attempt to localize the origin of replication with more precision, we blocked the progress of replication forks emanating from origins at the beginning of the S phase by the introduction of trioxsalen cross-links at 1- to 5-kb intervals in the parental double-stranded DNA. The small DNA fragments synthesized under these conditions (which should be centered around replication origins) were then used as hybridization probes on digests of cosmids and plasmids from the DHFR domain. These studies suggested that in cells synchronized by this regimen, DNA replication initiates at two separate sites within the previously defined 28-kb replication initiation locus, in general agreement with results described in the accompanying paper (T.-H. Leu and J. L. Hamlin, Mol. Cell. Biol. 9:523-531, 1989). One of these sites contains a repeated DNA sequence element that is found at or near many other initiation sites in the genome, since it was also highly enriched in the early replicating DNA isolated from cross-linked CHO cells that contain only two copies of the DHFR domain.

scholarly journals Chromosome architecture can dictate site-specific initiation of DNA replication in Xenopus egg extracts.

1996 ◽  
Vol 135 (5) ◽  
pp. 1207-1218 ◽  
Author(s):  
S J Lawlis ◽  
S M Keezer ◽  
J R Wu ◽  
D M Gilbert
Keyword(s):  
Dna Replication ◽  
Nuclear Envelope ◽  
Cho Cells ◽  
Chromosome Condensation ◽  
G1 Phase ◽  
Unusual Site ◽  
Chromosome Architecture ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Xenopus egg extracts initiate DNA replication specifically at the dihydrofolate reductase (DHFR) origin locus with intact nuclei from late G1-phase CHO cells as a substrate, but at nonspecific sites when purified DNA is assembled by the extract into an embryonic nuclear structure. Here we show that late G1-phase CHO nuclei can be cycled through an in vitro Xenopus egg mitosis, resulting in the assembly of an embryonic nuclear envelope around G1-phase chromatin. Surprisingly, replication within these chimeric nuclei initiated at a novel specific site in the 5' region of the DHFR structural gene that does not function as an origin in cultured CHO cells. Preferential initiation at this unusual site required topoisomerase II-mediated chromosome condensation during mitosis. Nuclear envelope breakdown and reassembly in the absence of chromosome condensation resulted in nonspecific initiation. Introduction of condensed chromosomes from metaphase-arrested CHO cells directly into Xenopus egg extracts was sufficient to elicit assembly of chimeric nuclei and preferential initiation at this same site. These results demonstrate clearly that chromosome architecture can determine the sites of initiation of replication in Xenopus egg extracts, supporting the hypothesis that patterns of initiation in vertebrate cells are established by higher order features of chromosome structure.

scholarly journals Contribution of DNA polymerase δ to DNA replication in permeable CHO cells synchronized in S phase

1989 ◽  
Vol 17 (12) ◽  
pp. 4757-4767 ◽  
Author(s):  
Alexei Basnakian ◽  
Gaspar Banfalvi ◽  
Nilima Sarkar
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Cho Cells ◽  
S Phase ◽  
Dna Polymerase Δ

scholarly journals Replication in the amplified dihydrofolate reductase domain in CHO cells may initiate at two distinct sites, one of which is a repetitive sequence element.

1989 ◽  
Vol 9 (2) ◽  
pp. 532-540 ◽  
Author(s):  
B Anachkova ◽  
J L Hamlin
Keyword(s):  
Dna Replication ◽  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Replication Initiation ◽  
Ovary Cell ◽  
Sequence Element ◽  
Hybridization Probes ◽  
Ovary Cell Line

To study initiation of DNA replication in mammalian chromosomes, we have established a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) that contains approximately 1,000 copies of the early replicating dihydrofolate reductase (DHFR) domain. We have previously shown that DNA replication in the prevalent 243-kilobase (kb) amplicon type in this cell line initiates somewhere within a 28-kb region located downstream from the DHFR gene. In an attempt to localize the origin of replication with more precision, we blocked the progress of replication forks emanating from origins at the beginning of the S phase by the introduction of trioxsalen cross-links at 1- to 5-kb intervals in the parental double-stranded DNA. The small DNA fragments synthesized under these conditions (which should be centered around replication origins) were then used as hybridization probes on digests of cosmids and plasmids from the DHFR domain. These studies suggested that in cells synchronized by this regimen, DNA replication initiates at two separate sites within the previously defined 28-kb replication initiation locus, in general agreement with results described in the accompanying paper (T.-H. Leu and J. L. Hamlin, Mol. Cell. Biol. 9:523-531, 1989). One of these sites contains a repeated DNA sequence element that is found at or near many other initiation sites in the genome, since it was also highly enriched in the early replicating DNA isolated from cross-linked CHO cells that contain only two copies of the DHFR domain.

scholarly journals DNA replication in Chinese hamster ovary cells made permeable to nucleotides by tween-80 treatment.

1976 ◽  
Vol 69 (3) ◽  
pp. 732-736 ◽  
Author(s):  
D Billen ◽  
A C Olson
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Tween 80 ◽  
S Phase ◽  
Ovary Cells ◽  
Permeabilized Cells

We have developed a method for permeabilizing CHO cells to nucleotides under conditions which allow most cells to remain viable. Permeabilized cells can carry out ATP-dependent, semiconservative synthesis of DNA. The data are consistent with the continuation of DNA synthesis in those cells in S phase at the time of treatment, possibly limited to completion of replicon synthesis without new initiations.

scholarly journals Spatial distribution and specification of mammalian replication origins during G1 phase

2003 ◽  
Vol 161 (2) ◽  
pp. 257-266 ◽  
Author(s):  
Feng Li ◽  
Jianhua Chen ◽  
Eduardo Solessio ◽  
David M. Gilbert
Keyword(s):  
Spatial Distribution ◽  
Dna Replication ◽  
Cho Cells ◽  
G1 Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Temporal Program

We have examined the distribution of early replicating origins on stretched DNA fibers when nuclei from CHO cells synchronized at different times during G1 phase initiate DNA replication in Xenopus egg extracts. Origins were differentially labeled in vivo versus in vitro to allow a comparison of their relative positions and spacing. With nuclei isolated in the first hour of G1 phase, in vitro origins were distributed throughout a larger number of DNA fibers and did not coincide with in vivo origins. With nuclei isolated 1 h later, a similar total number of in vitro origins were clustered within a smaller number of DNA fibers but still did not coincide with in vivo origins. However, with nuclei isolated later in G1 phase, the positions of many in vitro origins coincided with in vivo origin sites without further change in origin number or density. These results highlight two distinct G1 steps that establish a spatial and temporal program for replication.

Replication of Bacteriophage 186 DNA

1972 ◽  
Vol 30 ◽  
pp. 194-195
Author(s):  
Dhruba K. Chattoraj ◽  
Ross B. Inman
Keyword(s):  
Electron Microscopy ◽  
Dna Replication ◽  
Frame Of Reference ◽  
Dna Molecules ◽  
E Coli ◽  
Phage Dna ◽  
Partial Denaturation

Electron microscopy of replicating intermediates has been quite useful in understanding the mechanism of DNA replication in DNA molecules of bacteriophage, mitochondria and plasmids. The use of partial denaturation mapping has made the tool more powerful by providing a frame of reference by which the position of the replicating forks in bacteriophage DNA can be determined on the circular replicating molecules. This provided an easy means to find the origin and direction of replication in λ and P2 phage DNA molecules. DNA of temperate E. coli phage 186 was found to have an unique denaturation map and encouraged us to look into its mode of replication.

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