A novel episomal shuttle vector for transformation of Cryptococcus neoformans with the ccdB gene as a positive selection marker in bacteria

Authors

  • Philippe Mondon,

    1. Molecular Microbiology Section, LCI, National Institute of Allergy and Infectious Diseases, N.I.H., Bldg. 10, 11C304, Bethesda, MD 20892, USA
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  • Yun C Chang,

    1. Molecular Microbiology Section, LCI, National Institute of Allergy and Infectious Diseases, N.I.H., Bldg. 10, 11C304, Bethesda, MD 20892, USA
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  • Ashok Varma,

    1. Molecular Microbiology Section, LCI, National Institute of Allergy and Infectious Diseases, N.I.H., Bldg. 10, 11C304, Bethesda, MD 20892, USA
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  • Kyung J Kwon-Chung

    Corresponding author
    1. Molecular Microbiology Section, LCI, National Institute of Allergy and Infectious Diseases, N.I.H., Bldg. 10, 11C304, Bethesda, MD 20892, USA
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*Corresponding author. Tel.: +1 (301) 496-1602; Fax: +1 (301) 402-1003, E-mail address: june_kwon-chung@nih.gov

Abstract

We report the engineering of a new shuttle vector featuring its episomal maintenance in Cryptococcus neoformans and the lethal Escherichia coli ccdB gene for positive selection in bacteria. Telomere-like sequences from C. neoformans and the STAB fragment confer episomal maintenance to the vector (pPM8) upon transformation in C. neoformans. The vector generated high transformation frequencies and each transformant was estimated to harbor thirty copies of the plasmid. The plasmids recovered in E. coli from the C. neoformans transformants showed no evidence of rearrangement. This construct will be very useful for cloning and studying the regulation of genes in C. neoformans.

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