Hygromycin B-resistance phenotype acquired in Paracoccidioides brasiliensis via plasmid DNA integration.
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.
Soares
Renata De B A
Rde B
Departamento de Biologia Celular IB, Campus Darcy Ribeiro, Universidade de Brasília, Brasília, DF, Brasil.
Velho
Tarcísio A F
TA
De Moraes
Lidia M P
LM
Azevedo
Maristela O
MO
Soares
Célia M De A
CM
Felipe
Maria Sueli S
MS
D015139:Blotting, Southern; D004271:DNA, Fungal / Q000032:analysis; D025141:Drug Resistance, Fungal / Q000235:genetics*; D018274:Electroporation; D006921:Hygromycin B / Q000494:pharmacology*; D010228:Paracoccidioides / Q000187:drug effects* / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D014170:Transformation, Genetic
D004271:DNA, Fungal; D006921:Hygromycin B