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Free keywords:
expression/immunoenzymatic; assay/lac; operon/molecular cloning
Abstract:
We describe a method which permits the detection of exon fragments. Such DNA was cloned and expressed in the promoter proximal part of the lac Z gene of Escherichia coli. The resulting antigen-beta-galactosidase chimeras are bound to their respective antibodies fixed to polyvinyl sheets. The beta-galactosidase part of the chimera permits detection of such clones by histochemical staining. As model DNA, we used the lac I gene cleaved with HaeIII, HhaI, or HpaII. Fragments were tailed with poly(dC) and inserted into the poly(dG)-tailed promoter proximal part of the lac Z gene. Recombinant clones, isolated on lactose-agar plates, were replica-plated and lysed with chloroform. Polyvinyl sheets coated with antibody against lac repressor were placed onto the top of the lysed colonies for immunoadsorption. The immune complexes were made visible after washing by incubation with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside in buffered agar. The beta-galactosidase activity of the chimera cleaves the colourless histochemical compound to a blue dye at those positions where clones produce the antigen. In the case of the lac I gene two types of clones were isolated, carrying the NH2-terminal part of the lac repressor up to codons 27 and 75.
