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.
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.
Looney
J E
JE
Department of Biochemistry, University of Virginia School of Medicine, Charlottesville 22908.
Ma
C
C
Leu
T H
TH
Flintoff
W F
WF
Troutman
W B
WB
Hamlin
J L
JL
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004351:Drug Resistance / Q000235:genetics; D005784:Gene Amplification; D005796:Genes; D008727:Methotrexate / Q000494:pharmacology*; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D009695:Nucleic Acid Renaturation; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate