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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Unstable amplification of an altered dihydrofolate reductase gene associated with double-minute chromosomes.

We have studied a line of 3T6 mouse fibroblasts grown in progressively increasing concentrations of methotrexate. Initially, drug resistance results from amplification of the gene encoding the normal dihydrofolate reductase. Growth of these methotrexate-resistant populations at higher methotrexate concentrations results in the emergence of cells expressing high levels of dihydrofolate reductase with a reduced methotrexate affinity. Using the fluorescence-activated cell sorter, we demonstrate that the variant gene is not present in the population of cells resistant to lower levels of methotrexate, and hence we postulate that the mutational event occurred in cells already containing multiple normal dihydrofolate reductase genes. Growth of the variant cells in the absence of selection is associated with the permanent loss of the altered genes and the disappearance of double-minute chromosomes, on which these genes reside. The pattern of accumulation and loss of double-minute chromosomes is reproduced following transformation of methotrexate-sensitive cells with the altered genes. Our results are consistent with autonomous replication of double-minute chromosomes and a selective advantage of cells with the smallest number of extrachromosomal elements necessary for survival at a given methotrexate concentration.[1]


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