Structure and organization of a stable extrachromosomal element in human cells

We have determined the structure and organization of a 630-kb extrachromosomal element (amplisome) containing the dihydrofolate reductase-encoding gene (DHFR) in a methotrexate (MTX)-resistant human cell line, HeLa-Bu25-10B3. The size and copy number of amplisomes have previously been found to remain remarkably stable with or without selection. Both linear and open circular 630-kb amplisomes are present in these cells. We have been able to isolate the linear amplisomes after pulsed-field gel electrophoresis (PFGE), and transfect the amplisomes into MTX-sensitive recipient cells by electroporation, thus demonstrating that DNA as large as 630 kb can be transfected into mammalian cells. The NotI restriction site immediately upstream from DHFR on the circular amplisome is devoid of methylation, suggesting that it is transcriptionally active. Restriction mapping by PFGE reveals that there is only one copy of DHFR per amplisome and no repetitive structure is observed. The small size of the amplisomes, their stability and our ability to transfect large DNA molecules provide the necessary ingredients for the development of mammalian cloning vectors for large DNA fragments.