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Biomedical subjects

J Wienberg

Publications and source records attributed to J Wienberg.

76 records · Page 5Linked to original sources
PubMed1989

Distinct mouse DNA sequences enable establishment and persistence of plasmid DNA polymers in mouse cells.

Distinct elements isolated from mouse genomic DNA confer on plasmid DNA the ability to persist at high copy numbers in mouse L fibroblasts (1). Field inversion gel electrophoresis demonstrated that - in contrast to our previous assumption - the persisting plasmid DNA does not exist extrachromosomally but as clusters of tandem repeats integrated into genomic DNA. Digestion with restriction endonucleases that do not cut within the plasmid DNA results in fragments of 50-300 kb in length indicating reiteration of 10-50 plasmid DNA molecules. Restriction with several enzymes that cut once or twice within the plasmid sequences lead to fragment(s) indicative for head-to-tail tandem repeats. In situ hybridization revealed signals for a long homogeneously staining region (HSR) in one or two chromosomes per cell nucleus. Possibilities how these elements could act in the establishment and/or maintenance of the head-to-tail polymers of plasmid DNA in mouse cells are discussed.

Animals↗
PubMed1988

Amplified expression constructs for human tissue-type plasminogen activator in Chinese hamster ovary cells: instability in the absence of selective pressure.

By linking an expression cassette for human tissue-type plasminogen activator (t-PA) to an amplifiable marker gene, its introduction into Chinese hamster ovary dhfr- cells and subsequent amplification with methotrexate, we have generated cell lines that overproduce the heterologous protein and contain 300-1100 copies of the expression constructs integrated into the hamster genome. We present a detailed investigation of the fate of amplified sequences in the presence and absence of selective pressure by parallel examination of three producer cell lines with respect to relevant parameters. These include the determination of t-PA production upon continuous propagation in culture, the genomic organization of the integrated expression constructs by Southern blotting, and the localization of homogeneously staining regions by in-situ hybridization with biotinylated probes and visualization by interference reflection microscopy. We conclude that in the three cell lines examined, the decrease in production of t-PA in the absence of methotrexate selection is accompanied by decreases in the number of integrated expression constructs and the size of the amplified regions, whereas all these parameters are stable when selective pressure is maintained. The instability is probably due to the head-to-tail mode of integration of the expression constructs in the hamster genome, which increases the frequency of homologous recombination between the integrated plasmids in recombination-proficient cells in the absence of selective pressure.

Animals↗