Isolation of a human genomic fragment, co-amplified with c-Ki-ras, that affects plasmid supercoiling in E. coli.
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.
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002874:Chromosome Mapping; D002881:Chromosomes, Human, Pair 12; D006224:Cricetinae; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D004278:DNA, Superhelical / Q000032:analysis*; D004926:Escherichia coli / Q000378:metabolism*; D005784:Gene Amplification; D006801:Humans; D006822:Hybrid Cells / Q000032:analysis; D008175:Lung Neoplasms / Q000032:analysis / Q000235:genetics; D009710:Nucleotide Mapping; D010957:Plasmids; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras)
D001426:Bacterial Proteins; D004273:DNA, Neoplasm; D004278:DNA, Superhelical; D011518:Proto-Oncogene Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D004264:DNA Topoisomerases, Type I