Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells

F. W. Alt, R. E. Kellems, Joseph Bertino, R. T. Schimke

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Abstract

The rate of dihydrofolate reductase synthesis in the AT-3000 line of methotrexate-resistant murine Sarcoma 180 cells is approximately 200- to 500-fold greater than that of the sensitive, parental line. The authors have purified cDNA sequences complementary to dihydrofolate reductase mRNA and subsequently used this probe to quantitate dihydrofolate reductase mRNA and gene copies in each of these lines. Analysis of the association kinetics of the purified cDNA with DNA from sensitive and resistant cells indicated that the dihydrofolate reductase gene is selectively multiplied approximately 200-fold in the resistant line. A similar analysis of a partially revertant line of resistant cells indicated that the loss of resistance observed when the AT-3000 line is grown in the absence of methotrexate is associated with a corresponding decrease in the dihydrofolate reductase gene copy number. In each of these lines the relative number of dihydrofolate reductase gene copies is proportional to the cellular level of dihydrofolate reductase and dihydrofolate reductase mRNA sequences. The authors have also studied parental and methotrexate-resistant lines of L1210 murine lymphoma cells. Both resistance and an associated 35-fold increase in the level of dihydrofolate reductase appear to be stable properties of the resistant L1210 line since the authors find no decrease in either parameter in over 100 generations of growth in absence of methotrexate. Once again, the authors find that the increased levels of dihydrofolate reductase in the methotrexate-resistant L1210 line are associated with a proportional increase in the number of dihydrofolate reductase gene copies. In this case the dihydrofolate reductase gene copy number appears to be relatively stable in the resistant line. Therefore, the authors conclude that selective multiplication of the dihydrofolate reductase gene can account for the overproduction of dihydrofolate reductase in both stable and unstable lines of methotrexate-resistant cells.

Original languageEnglish (US)
Pages (from-to)1357-1370
Number of pages14
JournalJournal of Biological Chemistry
Volume253
Issue number5
StatePublished - Jan 1 1978
Externally publishedYes

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Tetrahydrofolate Dehydrogenase
Methotrexate
Cultured Cells
Genes
Gene Dosage
Messenger RNA
Complementary DNA
Sarcoma 180

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells",
abstract = "The rate of dihydrofolate reductase synthesis in the AT-3000 line of methotrexate-resistant murine Sarcoma 180 cells is approximately 200- to 500-fold greater than that of the sensitive, parental line. The authors have purified cDNA sequences complementary to dihydrofolate reductase mRNA and subsequently used this probe to quantitate dihydrofolate reductase mRNA and gene copies in each of these lines. Analysis of the association kinetics of the purified cDNA with DNA from sensitive and resistant cells indicated that the dihydrofolate reductase gene is selectively multiplied approximately 200-fold in the resistant line. A similar analysis of a partially revertant line of resistant cells indicated that the loss of resistance observed when the AT-3000 line is grown in the absence of methotrexate is associated with a corresponding decrease in the dihydrofolate reductase gene copy number. In each of these lines the relative number of dihydrofolate reductase gene copies is proportional to the cellular level of dihydrofolate reductase and dihydrofolate reductase mRNA sequences. The authors have also studied parental and methotrexate-resistant lines of L1210 murine lymphoma cells. Both resistance and an associated 35-fold increase in the level of dihydrofolate reductase appear to be stable properties of the resistant L1210 line since the authors find no decrease in either parameter in over 100 generations of growth in absence of methotrexate. Once again, the authors find that the increased levels of dihydrofolate reductase in the methotrexate-resistant L1210 line are associated with a proportional increase in the number of dihydrofolate reductase gene copies. In this case the dihydrofolate reductase gene copy number appears to be relatively stable in the resistant line. Therefore, the authors conclude that selective multiplication of the dihydrofolate reductase gene can account for the overproduction of dihydrofolate reductase in both stable and unstable lines of methotrexate-resistant cells.",
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Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. / Alt, F. W.; Kellems, R. E.; Bertino, Joseph; Schimke, R. T.

In: Journal of Biological Chemistry, Vol. 253, No. 5, 01.01.1978, p. 1357-1370.

Research output: Contribution to journalArticle

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T1 - Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells

AU - Alt, F. W.

AU - Kellems, R. E.

AU - Bertino, Joseph

AU - Schimke, R. T.

PY - 1978/1/1

Y1 - 1978/1/1

N2 - The rate of dihydrofolate reductase synthesis in the AT-3000 line of methotrexate-resistant murine Sarcoma 180 cells is approximately 200- to 500-fold greater than that of the sensitive, parental line. The authors have purified cDNA sequences complementary to dihydrofolate reductase mRNA and subsequently used this probe to quantitate dihydrofolate reductase mRNA and gene copies in each of these lines. Analysis of the association kinetics of the purified cDNA with DNA from sensitive and resistant cells indicated that the dihydrofolate reductase gene is selectively multiplied approximately 200-fold in the resistant line. A similar analysis of a partially revertant line of resistant cells indicated that the loss of resistance observed when the AT-3000 line is grown in the absence of methotrexate is associated with a corresponding decrease in the dihydrofolate reductase gene copy number. In each of these lines the relative number of dihydrofolate reductase gene copies is proportional to the cellular level of dihydrofolate reductase and dihydrofolate reductase mRNA sequences. The authors have also studied parental and methotrexate-resistant lines of L1210 murine lymphoma cells. Both resistance and an associated 35-fold increase in the level of dihydrofolate reductase appear to be stable properties of the resistant L1210 line since the authors find no decrease in either parameter in over 100 generations of growth in absence of methotrexate. Once again, the authors find that the increased levels of dihydrofolate reductase in the methotrexate-resistant L1210 line are associated with a proportional increase in the number of dihydrofolate reductase gene copies. In this case the dihydrofolate reductase gene copy number appears to be relatively stable in the resistant line. Therefore, the authors conclude that selective multiplication of the dihydrofolate reductase gene can account for the overproduction of dihydrofolate reductase in both stable and unstable lines of methotrexate-resistant cells.

AB - The rate of dihydrofolate reductase synthesis in the AT-3000 line of methotrexate-resistant murine Sarcoma 180 cells is approximately 200- to 500-fold greater than that of the sensitive, parental line. The authors have purified cDNA sequences complementary to dihydrofolate reductase mRNA and subsequently used this probe to quantitate dihydrofolate reductase mRNA and gene copies in each of these lines. Analysis of the association kinetics of the purified cDNA with DNA from sensitive and resistant cells indicated that the dihydrofolate reductase gene is selectively multiplied approximately 200-fold in the resistant line. A similar analysis of a partially revertant line of resistant cells indicated that the loss of resistance observed when the AT-3000 line is grown in the absence of methotrexate is associated with a corresponding decrease in the dihydrofolate reductase gene copy number. In each of these lines the relative number of dihydrofolate reductase gene copies is proportional to the cellular level of dihydrofolate reductase and dihydrofolate reductase mRNA sequences. The authors have also studied parental and methotrexate-resistant lines of L1210 murine lymphoma cells. Both resistance and an associated 35-fold increase in the level of dihydrofolate reductase appear to be stable properties of the resistant L1210 line since the authors find no decrease in either parameter in over 100 generations of growth in absence of methotrexate. Once again, the authors find that the increased levels of dihydrofolate reductase in the methotrexate-resistant L1210 line are associated with a proportional increase in the number of dihydrofolate reductase gene copies. In this case the dihydrofolate reductase gene copy number appears to be relatively stable in the resistant line. Therefore, the authors conclude that selective multiplication of the dihydrofolate reductase gene can account for the overproduction of dihydrofolate reductase in both stable and unstable lines of methotrexate-resistant cells.

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