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Hygromycin B-resistance phenotype acquired in Paracoccidioides brasiliensis via plasmid DNA integration

Renata De B A Soares1, Tarcísio A F Velho, Lidia M P De Moraes

  • 1Departamento de Biologia Celular IB, Campus Darcy Ribeiro, Universidade de Brasília, Brasília, DF, Brasil.

Medical Mycology
|January 21, 2006
PubMed

Insights

This study successfully transformed Paracoccidioides brasiliensis yeast cells using electroporation, integrating the hygromycin B resistance gene (hph). However, the transformed cells showed low mitotic stability, likely due to the fungus

Area of Science:

  • Medical Mycology
  • Molecular Biology
  • Fungal Genetics

Background:

  • Paracoccidioides brasiliensis is a human pathogenic fungus.
  • Genetic manipulation of P. brasiliensis is crucial for understanding its biology and developing antifungal strategies.
  • Hygromycin B resistance is a common selectable marker in fungal transformation.

Purpose of the Study:

  • To establish a reliable method for transforming P. brasiliensis yeast cells.
  • To confirm the integration of the hygromycin B resistance gene (hph) into the fungal genome.
  • To assess the mitotic stability of the transformants.

Main Methods:

  • Transformation of P. brasiliensis strain Pb01 using electroporation with plasmid pAN7.1.
  • Selection of transformants on BHI medium supplemented with hygromycin B (hygB).
  • Analysis of transformants using Polymerase Chain Reaction (PCR) and Southern blotting.
  • Assessment of mitotic stability by serial subculturing on selective and non-selective media.

Main Results:

  • Successful transformation of P. brasiliensis yeast cells was achieved via electroporation.
  • Integration of the hph gene into the P. brasiliensis genome was confirmed by PCR and Southern blotting.
  • A low level of mitotic stability was observed in the transformants.

Conclusions:

  • Electroporation is an effective method for transforming P. brasiliensis.
  • The observed low mitotic stability is likely attributed to the multinuclear nature of P. brasiliensis yeast cells.
  • Further studies are needed to improve the genetic stability of P. brasiliensis transformants.

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