Novel use of synthetic oligonucleotide insertion mutants for the study of homologous recombination in mammalian cells.

G. Shapira, J. L. Stachelek, A. Letsou, L. K. Soodak, R. M. Liskay

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Abstract

Thymidine kinase-deficient mouse L cells have been transformed with plasmid DNAs carrying 8-base-pair Xho I linker insertion mutations in the coding region of the herpes simplex virus type 1 thymidine kinase gene. When the mutant plasmids are introduced individually into LTK- cells, transformation efficiencies are greatly reduced relative to the wild type. However, when two mutant plasmids are cotransferred into the same LTK- recipients, significantly higher frequencies of transformation are observed (30-300 times). Here we demonstrate the usefulness of linker insertions for the study of homologous recombination in detecting the existence of normal thymidine kinase gene sequences (i.e., sequences lacking the insertions after recombination are substantiated by DNA . DNA hybridization). In addition, the frequencies of recombination in the various "crosses" are consistent with the known positions of the mutations.

Original languageEnglish (US)
Pages (from-to)4827-4831
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume80
Issue number15
DOIs
StatePublished - Aug 1983

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