Use of lambda phasmids for deletion mapping of non-selectable markers cloned in plasmids |
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Authors: | D W Grogan J E Cronan |
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Institution: | Department of Microbiology, University of Illinois, 131 Burrill Hall, 407 S. Goodwin St., Urbana, 1L 61801 U.S.A., Tel. (217) 333-0425 |
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Abstract: | A nonselectable gene carried on a poorly selectable recombinant plasmid has been physically mapped by deletion analysis. Our method involved cloning the plasmid into a coliphage lambda vector and treating the recombinant phage with a chelator. Virtually all particles surviving this treatment carried large deletions within the plasmid insert. Further deletion analysis was done by inserting a selectable lambda sequence into one such deletion derivative and repeating the chelator selection. Chelator selection was also used to isolate deletions constructed in vitro. The deleted phage are readily characterized by restriction mapping, and the gene in question scored after infection of a mutant host strain. These techniques have enabled us to physically assign the cyclopropane fatty acid synthase gene of Escherichia coli to 0.8 kb of a 16-kb segment after characterizing only a small number of isolates. This approach should be generally useful in the mapping of plasmids for which no convenient method exists for selecting or scoring the gene in question. |
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Keywords: | Recombinant DNA phage X vectors chelator selection restriction mapping cyclopropane fatty acid synthase Abbreviations: CFA cyclopropane fatty acid 4 deletion kb kilobase pairs PFU plaque-forming units R broth see MAT & METH section (a) SDS sodium dodecyl sulfate ( ) ndicates prophage genome [ ] indicates plasmid-carrier state |
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