Gene Amplification

Selective amplification of specific regions of the genome, principally in eukaryotic cells, occurs in normal developmental processes and as a consequence of particular metabolic stress situations.

During oogenesis in certain amphibians the genes encoding ribosomal RNAs increase in copy number by some 2000-fold, in preparation for the large amount of protein synthesis that must occur in early development. The amplified DNA is in the form of extrachromosomal circles, each of which contains several copies of the ribosomal DNA repeat and a replication origin. A similar situation has been analyzed in Drosophila, in which genes encoding egg proteins are amplified at a particular developmental stage.

Both types of mechanisms apparently occur during development of certain drug-resistant mammalian cell lines in culture. This process has been studied most widely in cells that become resistant to methotrexate, a dihydrofolate reductase inhibitor. Treatment of leukemia with methotrexate often leads to the emergence of drug-resistant leukemic cell populations, which contain vastly elevated levels of the target enzyme, dihydrofolate reductase (DHFR). Overproduction of DHFR usually results from specific amplification of a large DNA segment that includes the DHFR gene. In one process, tandem duplication of the DNA segment generates a giant chromosome with multiple gene copies, in what is called a homogeneously staining region (HSR), because it lacks the typical chromosome banding pattern.

Alternatively, a DNA segment containing the DhfR gene can be excised, apparently by a recombinational process, to form minichromosomes called double-minute chromosomes. Some resistant cells contain both types of amplified genes. Double-minute chromosomes are maintained within a cell only as long as selective pressure is maintained by growth of the cell in methotrexate. However, the chromosomally amplified phenotype is stable through many generations of cell growth. DHFR sequences were visualized by in situ hybridization with a fluorescent-tagged DNA containing DHFR sequences. This technique is sufficiently sensitive to allow detection of single-copy sequences (white arrows). Note also the giant chromosome containing many gene-equivalents of DhfR gene sequences.

Amplification of genes under selective conditions has been widely observed-for example, in development of pesticide-resistant forms of insects. Such amplified structures could arise either through recombination with unequal sister-chromatid exchange, schematized in Figure 25.41, or by a conservative transposition process. Later, homologous recombination within an amplified region can lead to excision of sequences containing one or more amplified sequences. In order to replicate autonomously, these excised sequences must have a centromere. Such elements probably represent the double-minute chromosomes.

Selective pressure, such as the continuous presence of methotrexate, promotes specifically the survival of cells that can respond to that pressure (i.e., by overproducing DHFR). Once two or more copies of the gene are present on a chromosome, additional copies can be generated by further recombinational events or by abnormalities of replication. Resistance is thus developed in stepwise fashion and occurs over many generations of growth.

See also: Methotrexate