Gene amplification in methotrexate-resistant mouse cells

Publisher Website
Google Scholar
Abstract
Wild-type mouse LtAp20 cells were treated with calcium phosphate-precipitated DNA or chromosomes from two highly Methotrexate (MTX)-resistant mouse lymphoma cell lines — EL4/8 and EL4/11. Transfections with purified MTX-resistant DNA produced colonies of LtAp20 cells resistant to 3×10−8M MTX, at about eight times the frequency with which resistant colonies arose in control transfections. DNA transfectants contained multiple copies of the dihydrofolate reductase (dhfr) gene, but other sequences characteristic of the donor DNA could not be detected. Transfections using isolated chromosomes were twice as efficient as those using purified DNA. Unlike DNA transfectants, over 90% of all chromosome transfectants took up large stretches of donor DNA intact and contained DNA sequences characteristic of donor DNA. Of chromosome transfectants selected for resistance to high levels of MTX (1 mM), 70% amplified a unit of DNA which was indistinguishable from that present in the donor cell. The results showed that large fragments of chromosomes (as opposed to purified DNA) can be taken up by recipient cells without detectable alteration to the fine structure of the DNA they contain. The results also support the notion that all amplified units within a MTX-resistant cell have the same overall complex DNA structure.