Novel submicroscopic extrachromosomal elements containing amplified genes in human cells

Area of Science:

• Genetics
• Molecular Biology
• Cell Biology

Background:

• Methotrexate (MTX)-resistant cell lines often exhibit dihydrofolate reductase (DHFR) gene amplification.
• Previous MTX-resistant HeLa BU25 variants showed high DHFR gene amplification without typical homogeneously staining regions (HSRs) or numerous minute chromosomes.

Purpose of the Study:

• To characterize the physical nature of amplified DHFR genes in MTX-resistant HeLa BU25-10B3 cells.
• To investigate the potential for novel karyotypic abnormalities associated with gene amplification.

Main Methods:

• Isolation of DNA structures containing amplified DHFR genes using pulsed-field gradient or field-inversion gel electrophoresis.
• Characterization of these structures as homogeneous DNA molecules of approximately 650 kb.
• Electron microscopy of metaphase spreads to visualize related chromatin structures.

Main Results:

• Successfully isolated ~650 kb homogeneous DNA molecules containing amplified DHFR genes from HeLa BU25-10B3 cells.
• Observed chromatin fibers in electron microscopy consistent with these extrachromosomal DNA structures.
• Demonstrated a novel type of extrachromosomal structure facilitating gene amplification.

Conclusions:

• The amplified DHFR genes in HeLa BU25-10B3 cells exist on novel, extrachromosomal DNA molecules.
• These findings expand the understanding of mechanisms underlying gene amplification in mammalian cells.
• Suggests a new class of extrachromosomal elements involved in drug resistance.