Multiple origins of replication in the dihydrofolate reductase amplicons of a methotrexate-resistant chinese hamster cell line

1990 ◽  
Vol 10 (4) ◽  
pp. 1338-1346
Author(s):  
C Ma ◽  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Ovary Cell ◽  
Multiple Origins ◽  
Chromosomal Walking ◽  
Chinese Hamster Cell Line ◽  
S Period ◽  
Ovary Cell Line

We recently showed that replication initiates in the early S period at two closely spaced zones in the 240-kilobase (kb) dihydrofolate reductase (DHFR) amplicon of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Both of these initiation loci (ori-beta and ori-gamma) have previously been cloned in a recombinant cosmid. In this study, we identified a third early-firing initiation locus (ori-alpha) in the much larger DHFR amplicon of the independently isolated methotrexate-resistant Chinese hamster cell line DC3F-A3/4K (A3/4K). We describe the molecular cloning of this newly identified locus and demonstrate by chromosomal walking that ori-alpha lies approximately 240 kb upstream from ori-beta. Using overlapping cosmid clones for more than 450 kb of DNA sequence from this region of the DHFR domain, we have monitored the replication pattern of the amplicons in synchronized A3/4K cells. These studies suggest that ori-alpha, ori-beta, and ori-gamma are the only early-firing initiation sites in this 450-kb sequence. In addition, we have been able to roughly localize the termini between ori-alpha and ori-beta and between ori-alpha and the next origin in the 5' direction. Thus, we have now isolated the equivalent of three early-firing replicons (including their origins) from a well-characterized chromosomal domain. With these tools, it should be possible to determine those properties that are shared by the origins and termini of different replicons and which are therefore likely to be functionally significant.

scholarly journals Multiple origins of replication in the dihydrofolate reductase amplicons of a methotrexate-resistant chinese hamster cell line.

1990 ◽  
Vol 10 (4) ◽  
pp. 1338-1346 ◽  
Author(s):  
C Ma ◽  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Ovary Cell ◽  
Multiple Origins ◽  
Chromosomal Walking ◽  
Chinese Hamster Cell Line ◽  
S Period ◽  
Ovary Cell Line

We recently showed that replication initiates in the early S period at two closely spaced zones in the 240-kilobase (kb) dihydrofolate reductase (DHFR) amplicon of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Both of these initiation loci (ori-beta and ori-gamma) have previously been cloned in a recombinant cosmid. In this study, we identified a third early-firing initiation locus (ori-alpha) in the much larger DHFR amplicon of the independently isolated methotrexate-resistant Chinese hamster cell line DC3F-A3/4K (A3/4K). We describe the molecular cloning of this newly identified locus and demonstrate by chromosomal walking that ori-alpha lies approximately 240 kb upstream from ori-beta. Using overlapping cosmid clones for more than 450 kb of DNA sequence from this region of the DHFR domain, we have monitored the replication pattern of the amplicons in synchronized A3/4K cells. These studies suggest that ori-alpha, ori-beta, and ori-gamma are the only early-firing initiation sites in this 450-kb sequence. In addition, we have been able to roughly localize the termini between ori-alpha and ori-beta and between ori-alpha and the next origin in the 5' direction. Thus, we have now isolated the equivalent of three early-firing replicons (including their origins) from a well-characterized chromosomal domain. With these tools, it should be possible to determine those properties that are shared by the origins and termini of different replicons and which are therefore likely to be functionally significant.

Amplification of a cloned Chinese hamster dihydrofolate reductase gene after transfer into a dihydrofolate reductase-deficient cell line

1983 ◽  
Vol 3 (7) ◽  
pp. 1274-1282
Author(s):  
J D Milbrandt ◽  
J C Azizkhan ◽  
J L Hamlin
Keyword(s):  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Genomic Dna ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Ovary Cell ◽  
Dhfr Gene ◽  
Reductase Gene ◽  
Ovary Cell Line

We have transformed a dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary cell line to the DHFR+ phenotype with a recombinant cosmid (cH1) containing a functional Chinese hamster DHFR gene (J.D. Milbrandt et al., Mol. Cell. Biol. 3:1266-1273, 1983). After exposure of cells to successive increases in methotrexate, we have isolated a resistant cell line (JSH-1) that grows in 1 microM methotrexate. We show here that JSH-1 contains 300 to 500 copies of the integrated cosmid and that these copies are located predominantly at one position on a chromosome identified as Z5a. Hybridization analysis of restriction digests of genomic DNA indicates that the cosmid has been integrated intact into the genome and that upon amplification, the original cosmid/genomic junction fragments are also amplified in JSH-1. Furthermore, the pattern of amplified bands observed in ethidium bromide-stained gels indicates that the unit amplified sequence (amplicon) may be as large as 120 to 135 kilobases and therefore includes considerable amounts of flanking DNA in addition to the 45 kilobases of integrated cosmid. We also show that the protein overproduced by the amplified cosmid in JSH-1 comigrates with the 21,000-dalton polypeptide characteristic of the methotrexate-resistant cell line (CHOC 400) from which cH1 was cloned. However, the DHFR mRNA species overproduced in JSH-1 appear to be larger than those detected in CHOC 400, indicating that not all of the normal transcription and processing signals are preserved in the integrated recombinant cosmid.

The dihydrofolate reductase amplicons in different methotrexate-resistant Chinese hamster cell lines share at least a 273-kilobase core sequence, but the amplicons in some cell lines are much larger and are remarkably uniform in structure

1988 ◽  
Vol 8 (12) ◽  
pp. 5268-5279
Author(s):  
J E Looney ◽  
C Ma ◽  
T H Leu ◽  
W F Flintoff ◽  
W B Troutman ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dihydrofolate Reductase ◽  
Dna Sequences ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Resistant Cell ◽  
Pulse Field ◽  
Ovary Cell ◽  
Type I

We have previously cloned and characterized two different dihydrofolate reductase amplicon types from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). The largest of these (the type I amplicon) is 273 kilobases (kb) in length. In the present study, we utilized clones from the type I amplicon as probes to analyze the size and variability of the amplified DNA sequences in five other independently isolated methotrexate-resistant Chinese hamster cell lines. Our data indicated that the predominant amplicon types in all but one of these cell lines are larger than the 273-kb type I sequence. In-gel renaturation experiments as well as hybridization analysis of large SfiI fragments separated by pulse-field gradient gel electrophoresis showed that two highly resistant cell lines (A3 and MK42) have amplified very homogeneous core sequences that are estimated to be at least 583 and 653 kb in length, respectively. Thus, the sizes of the major amplicon types can be different in different drug-resistant Chinese hamster cell lines. However, there appears to be less heterogeneity in size and sequence arrangement within a given methotrexate-resistant Chinese hamster cell line than has been reported for several other examples of DNA sequence amplification in mammalian systems.

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 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.

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