Nucleic Acids Research, 1987, Vol. 15, No. 8 3411-3420
© 1987
Articles |
Isolation of a human genomic fragment, co-amplified with c-Ki-ras, that affects plasmid supercoiling in E. coli
1Department of Cell Biology, The Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute Wilmslow Road, Withington, Manchester M20 9BX, UK 2Department of Cell Biology and Genetics, Erasmus University Rotterdam, The Netherlands
*To Whom correspondence should be addressed
Received March 2, 1987. Accepted March 20, 1987.
Amplification of cellular proto-oncogenes has been implicated In the development of human malignancies. A library was constructed from genomic DNA extracted from a lung tumour, previously shown to carry an amplified c-Ki-ras 2 gene. Using a v-Ki-ras probe, a fragment with ras homology was isolated and shown to be amplified in the original tumour DNA to the same level as c-Ki-ras. Studies with human hamster hybrids demonstrated that it is normally located on human chromosome 12 (as is c-Ki-ras). The restriction map of the fragment is different from that of the known Ha, Ki or N-ras genes and its sequence shows evolutionary conservation, as demonstrated by hybridisation to the genomic DNA of several mammalian species. A pUC19 subclone (pK42), carrying a 1.3kb insert, shows supercoil heterogeneity in plasmid preparations, as does a second compatible plasmid introduced into the same bacterial host with pK42. It appears therefore that the subclone is encoding a product that affects DNA topoisomerase activity in E.coli.