Stable transformation and regulated expression of an inducible reporter construct in Candida albicans using restriction enzyme-mediated integration.
To allow the regulated expression of cloned genes in Candida albicans, a plasmid was constructed using the inducible promoter of the C. Albicans MAL2 gene. To demonstrate that the MAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of the C. albicans URA3 gene. This plasmid was introduced into a Ura- strain of C. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of the BamHI-linearized plasmid in the presence of BamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomal BamHI sites. All transformants examined were inducible for URA3 expression, which was determined by growth analysis and by measuring the level of URA3 gene product activity. The URA+ phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes in C. albicans.
Brown
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DH
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111, USA.
Slobodkin
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IV
Kumamoto
C A
CA
D001483:Base Sequence; D002176:Candida albicans / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D017931:DNA Primers; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D010641:Phenotype; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D014170:Transformation, Genetic
D017931:DNA Primers; D005656:Fungal Proteins; D011994:Recombinant Proteins