Amplification of the dihydrofolate reductase gene in methotrexate-resistant Chinese hamster cells

1992 ◽  
Vol 276 (3) ◽  
pp. 179-187 ◽  
Author(s):  
Joyce L. Hamlin
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster ◽  
Chinese Hamster Cells ◽  
Reductase Gene

Similar 150-kilobase DNA sequences are amplified in independently derived methotrexate-resistant Chinese hamster cells

1985 ◽  
Vol 5 (4) ◽  
pp. 619-627
Author(s):  
M Montoya-Zavala ◽  
J L Hamlin
Keyword(s):  
Dna Sequence ◽  
Cell Lines ◽  
Dihydrofolate Reductase ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Chinese Hamster Cells ◽  
Reductase Gene ◽  
Reductase Domain

We have isolated overlapping recombinant cosmids that represent 150 kilobases of contiguous DNA sequence from the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). This sequence includes the 25-kilobase dihydrofolate reductase gene and an origin of DNA synthesis. Eight cosmids that span this domain have been utilized as radioactive hybridization probes to analyze the similarities among the dihydrofolate reductase amplicons in four independently derived methotrexate-resistant Chinese hamster cell lines. We have observed no significant differences among the four cell lines within the 150-kilobase DNA sequence that we have examined, except for polymorphisms that result from the amplification of one or the other of two possible alleles of the dihydrofolate reductase domain. We also show that the restriction patterns of the amplicons in these four resistant cell lines are virtually identical to that of the corresponding, unamplified sequence in drug-susceptible parental cells. Furthermore, measurements of the relative copy numbers of fragments from widely separated regions of the amplicon suggest that all fragments in this 150-kilobase region may be amplified in unison. Our data show that in methotrexate-resistant Chinese hamster cells, the amplified unit is large relative to the dihydrofolate reductase gene itself. Furthermore, within the 150-kilobase amplified consensus sequence that we have examined, significant rearrangements do not seem to occur during the amplification process.

scholarly journals Similar 150-kilobase DNA sequences are amplified in independently derived methotrexate-resistant Chinese hamster cells.

1985 ◽  
Vol 5 (4) ◽  
pp. 619-627 ◽  
Author(s):  
M Montoya-Zavala ◽  
J L Hamlin
Keyword(s):  
Dna Sequence ◽  
Cell Lines ◽  
Dihydrofolate Reductase ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Chinese Hamster Cells ◽  
Reductase Gene ◽  
Reductase Domain

We have isolated overlapping recombinant cosmids that represent 150 kilobases of contiguous DNA sequence from the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). This sequence includes the 25-kilobase dihydrofolate reductase gene and an origin of DNA synthesis. Eight cosmids that span this domain have been utilized as radioactive hybridization probes to analyze the similarities among the dihydrofolate reductase amplicons in four independently derived methotrexate-resistant Chinese hamster cell lines. We have observed no significant differences among the four cell lines within the 150-kilobase DNA sequence that we have examined, except for polymorphisms that result from the amplification of one or the other of two possible alleles of the dihydrofolate reductase domain. We also show that the restriction patterns of the amplicons in these four resistant cell lines are virtually identical to that of the corresponding, unamplified sequence in drug-susceptible parental cells. Furthermore, measurements of the relative copy numbers of fragments from widely separated regions of the amplicon suggest that all fragments in this 150-kilobase region may be amplified in unison. Our data show that in methotrexate-resistant Chinese hamster cells, the amplified unit is large relative to the dihydrofolate reductase gene itself. Furthermore, within the 150-kilobase amplified consensus sequence that we have examined, significant rearrangements do not seem to occur during the amplification process.

scholarly journals Assignment of the human dihydrofolate reductase gene to the q11----q22 region of chromosome 5.

1984 ◽  
Vol 4 (10) ◽  
pp. 2010-2016 ◽  
Author(s):  
V L Funanage ◽  
T T Myoda ◽  
P A Moses ◽  
H R Cowell
Keyword(s):  
Dihydrofolate Reductase ◽  
Hybrid Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Chromosome 5 ◽  
Dhfr Gene ◽  
Reductase Gene ◽  
Ovary Cell Line ◽  
Biochemical Analyses ◽  
Human Dihydrofolate Reductase

Cells from a dihydrofolate reductase-deficient Chinese hamster ovary cell line were hybridized to human fetal skin fibroblast cells. Nineteen dihydrofolate reductase-positive hybrid clones were isolated and characterized. Cytogenetic and biochemical analyses of these clones have shown that the human dihydrofolate reductase (DHFR) gene is located on chromosome 5. Three of these hybrid cell lines contained different terminal deletions of chromosome 5. An analysis of the breakpoints of these deletions has demonstrated that the DHFR gene resides in the q11----q22 region.

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.

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