Difference between revisions of "PMID:2851704"

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'''Comeau, DE  and Inouye, M '''  (1988) 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. ''Mol. Gen. Genet.'' '''213''':166-9
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!align=left  |Abstract
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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 cloned 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.
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[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=2851704 PubMed]
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!align=left  |Keywords
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Alleles; Chromosomes, Bacterial/physiology; Cloning, Molecular; DNA Restriction Enzymes; Escherichia coli/genetics; Genes, Bacterial; Mutation; Plasmids; Water-Electrolyte Balance
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==Main Points of the Paper ==
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{{LitSignificance}}
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== Materials and Methods Used ==
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{{LitMaterials}}
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==Phenotype Annotations==
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==Notes==
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==References==
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[[Category:Publication]]

Latest revision as of 18:48, 16 September 2012

Citation

Comeau, DE and Inouye, M (1988) 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. Mol. Gen. Genet. 213:166-9

Abstract

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 cloned 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.

Links

PubMed

Keywords

Alleles; Chromosomes, Bacterial/physiology; Cloning, Molecular; DNA Restriction Enzymes; Escherichia coli/genetics; Genes, Bacterial; Mutation; Plasmids; Water-Electrolyte Balance

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Main Points of the Paper

Please summarize the main points of the paper.

Materials and Methods Used

Please list the materials and methods used in this paper (strains, plasmids, antibodies, etc).

Phenotype Annotations

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Phenotype of Taxon Information Genotype Information (if known) Condition Information OMP ID OMP Term Name ECO ID ECO Term Name Notes Status
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Notes

References

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