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Nucleic Acids Research, 1981, Vol. 9, No. 18 4459-4474
© 1981

MOLECULAR BIOLOGY

Identification of gene products programmed by restriction endonuclease DNA fragments using an E. coli in vitro system

Julie M. Pratt, Graham J. Boulnois, Valerie Darby+, Elisha Orr, Elmar Wahle* and I.Barry Holland

Department of Genetics, University of Leicester LEI 7RH, UK +Department of Genetics, University of Madison WI 53706, USA *Institute of Zoology, University of Munster D-4400, GFR

Received July 8, 1981. DNA restriction enzyme fragments have been used to programme the synthesis of polypeptides in an in vitro system without apparent loss in fidelity compared with supercoiled templates. The system is extremely sensitive, less than 1 µg of DNA can be used to direct the synthesis of 35S-labelled polypeptides of sufficiently high specific activity such that products can be identified by SDS-PAGE after a few hours autoradiography. The ability to analyse fragments can be used to readily assign specific proteins to small regions of the coding template, to identify cloned gene products distinct from those of the vector, and to identify cloned genes expressed from their own promoters. The in vitro system can be used successfully with bacterial DNA from other species and efficient extracts can be prepared from any E. coli K-12 strain, which should greatly facilitate the purification of factors controlling the expression of specific genes by complementation assay.


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