Insertional mutagenesis in Bacillus subtilis: mechanism and use in gene cloning
| Autor: | Stanislav Dusko Ehrlich, B. Niaudet, A. Goze |
|---|---|
| Rok vydání: | 1982 |
| Předmět: |
Bacillus subtilis
Biology Molecular cloning Insertional mutagenesis chemistry.chemical_compound Plasmid Genetics Escherichia coli Cloning Molecular Base Sequence General Medicine DNA Restriction Enzymes Chromosomes Bacterial biology.organism_classification Molecular biology Restriction enzyme Phenotype chemistry Genes Bacterial Mutation Hybrid plasmid DNA In vitro recombination Plasmids |
| Zdroj: | Gene. 19(3) |
| ISSN: | 0378-1119 |
| Popis: | The plasmid pHV32, which replicates in Escherichia coli but not in Bacillus subtilis , transformed B. subtilis -competent cells efficiently when linked in vitro to Eco RI B. subtilis DNA segments. The transformed clones carried pHV32 inserted in their chromosomes, and often displayed a mutant phenotype. One of the transformed clones carried pHV32 inserted close to the thy B gene. We cleaved the DNA extracted from this clone with Bgl II restriction endonuclease, for which no sites exist on pHV32, ligated the released segments and used them to transform E. coli selecting for pHV32-carried genetic markers. The transformants harbored a hybrid plasmid which carried the B. subtilis thyB gene. Circular molecules composed of pHV32 joined to B. subtilis DNA inserted into the chromosome by a Campbell-like recombination event. Linear molecules, in which pHV32 was flanked by two non-adjacent DNA segments, underwent a double cross-over recombination with the chromosome. In this case the chromosomal sequences between the non-adjacent segments were deleted, and replaced by pHV32 sequences. |
| Databáze: | OpenAIRE |
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