A novel type of unstable homogeneously staining region with a head-to-tail arrangement: spontaneous decay and reintegration of DNA elements into a plethora of new chromosomal sites.
After transfection of amplification-promoting DNA elements into mammalian cells, homogeneously staining regions (HSRs) are formed by high copy numbers of transfected DNA arranged in head-to-tail polymers. Here, we wanted to evaluate the stability of this type of HSR during prolonged cultivation of transfected cells in selective medium. Thymidine kinase-deficient mouse L cells were transfected with pAPR4tk DNA harboring the amplification-promoting element 4 (APR4) linked to the gene for thymidine kinase (TK) or, alternatively, transfected with a DNA construct (pARP4t-PA) carrying, in addition, the expression cassette for human tissue-type plasminogen activator (t-PA). After transfection, one or two HSRs per cell were formed that disintegrated spontaneously after 25-40 wk of continuous cultivation in the presence of selective HAT (hypoxanthine-aminopterin-thymidine) medium. Unexpectedly, plasmid DNA reinserted into a plethora of new chromosomal sites, as revealed by in situ hybridization and Southern blot analysis. Coincidently, secretion of t-PA decreased to 10-20% of its original level. After transfection of pAPR4tk DNA lacking the t-PA expression cassette, HSR decay and reintegration of plasmid constructs into multiple chromosomal sites were also observed, whereas the ptk vector without an amplification-promoting DNA element did not form an HSR after transfection. We conclude that, in contrast to the pattern of known structures with head-to-tail arrangements, the HSR formed by amplification-promoting DNA elements represents a novel type of HSR that disintegrates by transposition into a plethora of new chromosomal integration sites. This process is mediated by the amplification-promoting DNA element itself and can be observed even when selective pressure is maintained.
Institut für Anthropologie und Humangenetik, Universität München, Germany.
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U
Koehler
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Abken
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Grummt
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Wienberg
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UH
Weidle
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002874:Chromosome Mapping; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D005784:Gene Amplification; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D004247:DNA; D013937:Thymidine Kinase; D010959:Tissue Plasminogen Activator