Development of autonomously replicating plasmids for Candida albicans

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Candida albicans is an opportunistic fungal pathogen.
• Efficient genetic manipulation tools are crucial for studying C. albicans.

Purpose of the Study:

• To identify and characterize autonomously replicating sequences (ARS) in Candida albicans DNA.
• To develop a high-efficiency transformation system for C. albicans.

Main Methods:

• Cloning of Candida albicans RsaI fragments into a pBR322 vector containing the ADE2 gene.
• Transformation of a Candida ade2 mutant.
• Selection for adenine prototrophy.
• Plasmid DNA analysis using Southern hybridization and orthogonal field alternation gel electrophoresis.

Main Results:

• A 0.35-kilobase RsaI fragment conferring an ARS phenotype was identified.
• Plasmids containing the ARS demonstrated high-frequency transformation of C. albicans (200-1,000 ADE+ transformants/µg DNA).
• Transforming DNA existed as extrachromosomal oligomers in tandem repeats, with occasional stable integration into chromosomes.

Conclusions:

• The identified 0.35-kb RsaI fragment functions as an ARS in C. albicans.
• This ARS facilitates efficient transformation and stable maintenance of plasmids.
• The ARS offers a valuable tool for genetic studies in Candida albicans.