A Positive Selection Vector for the Analysis of Structural Plasmid Instability inBacillus subtilis
| Autor: | Gerard Venema, Sierd Bron, Rob Meima |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Exodeoxyribonuclease V
Sequence analysis Genetic Vectors Molecular Sequence Data Mutant Chloramphenicol Resistance Bacillus subtilis Gene product Plasmid Genes Reporter Selection Genetic Promoter Regions Genetic Molecular Biology Gene Sequence Deletion Terminator Regions Genetic Genetics Base Sequence biology Tetracycline Resistance Promoter biology.organism_classification Molecular biology Transformation (genetics) Exodeoxyribonucleases Transformation Bacterial |
| Zdroj: | Plasmid. 35:14-30 |
| ISSN: | 0147-619X |
| DOI: | 10.1006/plas.1996.0002 |
| Popis: | A system for the positive selection of structural plasmid rearrangements in Bacillus subtilis was developed. Random deletions removing a transcription terminator structure in the assay plasmid, designated pGP100, resulted in expression of the cat-86 gene, under control of a constitutive bacteriophage promoter. The resulting chlorampenicol-resistant colonies were analyzed for plasmid contents and were shown, by restriction analysis, to contain initially both the intact parental plasmid and a deletion variant. Sequence analysis of deletion derivatives revealed a consensus target site (5'-A-T-T-A-A/T-3') at or near deletion termini, which resembles topoisomerase I target sites. Endpoints on one side of the deletions were found to be clustered in the promoter region of the tetracycline resistance gene present on pGP100, the gene product of which is an integral membrane protein. Furthermore, deletion of the genes encoding the ATP-dependent exonuclease, AddAB, severely reduced the structural stability of pGP100. The data indicate that similar mechanisms underlie deletion formation in pGP100, and a different plasmid-based system, pGP1, which we have analyzed previously. |
| Databáze: | OpenAIRE |
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