Abstract
The successful cloning of genes of higher organisms depends on the development of a simple and safe organism for recombination. In the Edinburgh Laboratory of Ken and Noreen Murray the Lambda phage was altered to permit the insertion of eucaryotic DNA. This phage has been modified to prevent lysogeny and recombination with the host E. coli. In addition, we have added three amber mutations to the phage (in genes for lysis, head formation, and capsid protein). This prevents growth of the phage in any wild-type, non-suppressor host. This phage is useful for cloning DNA because of the limited number of endonuclease restriction sites and the presence of a tryptophan gene. It has two targets for the endonuclease Hind III which are on each side of the tryptophan gene. The Hind III nuclease cuts the phage DNA into 3 separate fragments which can be rejoined with DNA ligase. The presence of the trp gene can be detected on a trp deficient E. coli host. The trp gene can also be replaced with a mammalian DNA of a molecular weight up to 15 × 106. In exchanging a new DNA for the trp DNA, the phage phenotype changes to permit easy selection of recombinant phage. We have recombined this phage with Xenopus ribosomal genes. This cloned DNA was labeled and used for mapping the human ribosomal genes and for in situ hybridization with human chromosomes.
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Schmickel, R., Wilson, C. & Hollar, B. THE DEVELOPMENT OF A LAMBDA PHAGE VECTOR FOR RECOMBINATION CLONING OF MAMMALIAN GENES. Pediatr Res 11, 463 (1977). https://doi.org/10.1203/00006450-197704000-00560
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DOI: https://doi.org/10.1203/00006450-197704000-00560
