Organization and genesis of dihydrofolate reductase amplicons in the genome of a methotrexate-resistant Chinese hamster ovary cell line.
We have recently isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase (dhfr) amplicon types from the methotrexate-resistant Chinese hamster ovary (CHO) cell line CHOC 400. In the work described in this report, we used pulse-field gradient gel electrophoresis to analyze large SfiI restriction fragments arising from the amplified dhfr domains. The junction between the 260-kilobase type I amplicons (which are arranged in head-to-tail configurations in the genome) has been localized, allowing the construction of a linear map of the parental dhfr locus. We also show that the 220-kilobase type II amplicons are arranged as inverted repeat structures in the CHOC 400 genome and arose from the type I sequence relatively early in the amplification process. Our data indicate that there are a number of minor amplicon types in the CHOC 400 cell line that were not detected in previous studies; however, the type II amplicons represent ca. 75% of all the amplicons in the CHOC 400 genome. Both the type I and type II amplicons are shown to be composed entirely of sequences that were present in the parental dhfr locus. Studies of less resistant cell lines show that initial amplicons can be larger than those observed in CHOC 400. Once established, a given amplicon type appears to be relatively stable throughout subsequent amplification steps. We also present a modification of an in-gel renaturation method that gives a relatively complete picture of the size and variability of amplicons in the genome.
Ma
C
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Department of Biochemistry and Cell, University of Virginia School of Medicine, Charlottesville 22908.
Looney
J E
JE
Leu
T H
TH
Hamlin
J L
JL
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D003360:Cosmids; D006224:Cricetinae; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004274:DNA, Recombinant / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D005784:Gene Amplification; D008727:Methotrexate / Q000494:pharmacology; D009693:Nucleic Acid Hybridization; D010053:Ovary / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
D004274:DNA, Recombinant; D013762:Tetrahydrofolate Dehydrogenase; D004262:DNA Restriction Enzymes; D008727:Methotrexate