A novel method for the cloning of chromosomal mutations in a single step: Isolation of two mutant alleles of envZ, an osmoregulatory gene from Escherichia coli |
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Authors: | Dorothy E. Comeau and Masayori Inouye |
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Affiliation: | (1) Department of Biochemistry, State University of New York at Stony Brook, 11794-5215, NY, USA;(2) Department of Biochemistry, Robert Wood Johnson School of Medicine and Dentistry at Rutgers University, 08854 Piscataway, NJ, USA;(3) Present address: College of Physicians and Surgeons of Columbia University, 630 West 168th Street, Box 361, 10032 New York, NY, USA |
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Abstract: | Summary We have developed a simple, rapid and powerful method for the cloning of chromosomal mutations from total cellular DNA in a single step using a plasmid carrying the clined wild-type locus of interest and a convenient selectable marker such as antibiotic resistance. This method relies upon the ability of the cloned wild-type gene to form a heteroduplex with the mutant chromosomal locus. The plasmid from primary transformants can be screened rapidly by size; more than 50% of plasmids of the correct size contained the mutant locus. When this method was used to clone two chromosomal mutations in the envZ gene of Escherichia coli, a locus which encodes a membrane-bound sensory protein involved in the osmoregulation of outer membrane porin biosynthesis, more than 50% of the retransformants from the plasmids selected by size were found to exhibit the mutant phenotype. Preliminary characterization of these mutant alleles is discussed. This novel and powerful method should be generally applicable in any system where the cloned locus is available.This work was presented at the 86th Annual Meeting of the American Society for Microbiology, March 1986, Washingnton, D.C. |
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Keywords: | Cloning method envZ gene ompB locus Osmoregulation Gene expression |
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