Extrachromosomal maintenance and amplification of yeast artificial chromosome DNA in mouse cells.
Linear and circular forms of a 660-kb yeast artificial chromosome (YAC) containing the human hypoxanthine phosphoribosyltransferase gene were introduced into mouse L A-9 cells by fusion with yeast spheroplasts, and in situ hybridization was used to determine the location and fate of the yeast- and YAC-derived DNA in 25 fusion cell lines. Human and yeast DNAs were observed as extrachromosomal DNA molecules, present at least 27 cell divisions after fusion, in half the cell lines. The extrachromosomal molecules replicate extrachromosomally but segregate poorly like plasmids that contain an autonomously replicating sequence element in yeast. This system may allow analysis of DNA requirements for replication and segregation in mammalian cells. The extrachromosomal elements resemble double minutes (DMs), and in five cell lines human and yeast DNAs were present as very large integrated blocks of DNA resembling homogeneously staining regions (HSRs). Thus, the fusion cell lines contain the two characteristic forms of amplified DNA observed in cancer cell lines, DMs and HSRs, indicating that these can be formed from fragments of DNA introduced by cell fusion.
Featherstone
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Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110.
Huxley
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C
D000818:Animals; D002455:Cell Division; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000032:analysis / Q000096:biosynthesis; D004270:DNA, Circular / Q000378:metabolism; D004271:DNA, Fungal / Q000032:analysis / Q000096:biosynthesis; D003850:Deoxyribonuclease I; D005784:Gene Amplification; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000096:biosynthesis / Q000235:genetics*; D017403:In Situ Hybridization; D007621:Karyotyping; D007739:L Cells; D008677:Metaphase; D051379:Mice; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014162:Transfection / Q000379:methods
D004270:DNA, Circular; D004271:DNA, Fungal; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D003850:Deoxyribonuclease I