Hygromycin B-resistance phenotype acquired in Paracoccidioides brasiliensis via plasmid DNA integration
| Autor: | Tarcísio A. F. Velho, Maristela O. Azevedo, Lidia Maria Pepe de Moraes, Maria Sueli Soares Felipe, Célia Maria de Almeida Soares, Renata de Bastos Ascenço Soares |
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| Rok vydání: | 2006 |
| Předmět: |
Polymerase Chain Reaction
Paracoccidioides Microbiology chemistry.chemical_compound Plasmid Transformation Genetic Drug Resistance Fungal DNA Fungal Southern blot Paracoccidioides brasiliensis Recombination Genetic biology Electroporation General Medicine biology.organism_classification Molecular biology Yeast Transformation (genetics) Blotting Southern Infectious Diseases chemistry Hygromycin B Plasmids |
| Zdroj: | Medical mycology. 43(8) |
| ISSN: | 1369-3786 |
| Popis: | Yeast cells of the human pathogenic fungus Paracoccidioides brasiliensis strain Pb01 were transformed to hygromycin B resistance using the plasmid pAN7.1. Transformation was achieved by electroporation, with intact or linearized plasmid DNA. The fungus was transformed using 200 mM manitol, 5 or 7 kV/cm field strength, 25 microF capacitance, 400 omega resistance, 5 microg plasmid DNA and 10(7) yeast cells in 400 microl, and selected in BHI medium overlaid with 30 microg/ml hygromycin B (hygB). Mitotic stability was assessed by growing transformants on non-selective BHI medium, followed by plating on hygromycin B (30 microg/ml). Transformants were analyzed by PCR and Southern blotting, confirming the hph gene integration into the transformants genome. A low level of stability of the integrated hph sequence in the transformant genomes was observed, probably because of the multinuclearity of P. brasiliensis yeast cells. |
| Databáze: | OpenAIRE |
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