Stable yeast transformation with chimeric plasmids using a 2 micron-circular DNA-less strain as a recipient

Area of Science:

• Molecular Biology
• Yeast Genetics
• Biotechnology

Background:

• The 2 micron DNA sequence is a key element for stable plasmid maintenance in yeast.
• Previous cloning strategies in yeast faced challenges with plasmid stability and integration.
• The URA3 gene serves as a valuable selection marker in yeast.

Purpose of the Study:

• To develop a yeast strain and chimeric plasmid system for efficient and stable cloning.
• To investigate the maintenance and expression of chimeric plasmids in a 2 micron DNA-deficient yeast strain.
• To assess the stability of the URA3 gene and its encoded enzyme activity in transformed yeast.

Main Methods:

• Transformation of a 2 micron DNA-less yeast strain with chimeric plasmids containing the URA3 gene and bacterial plasmid pCR1.
• Recovery and analysis of plasmids in E. coli to assess autonomous replication and recombination.
• Hybridization experiments to detect chromosomal DNA integration.
• Assay of orotidine 5' monophosphate decarboxylase activity in transformed yeast clones.

Main Results:

• The 2 micron DNA-less yeast strain successfully maintained chimeric plasmids as autonomous replicons with minimal recombination.
• No evidence of URA3 DNA integration into the chromosomal DNA was observed.
• Transformed clones exhibited stable ura+ character and 5-10 fold higher specific activity of orotidine 5' monophosphate decarboxylase compared to wild type.

Conclusions:

• A novel yeast cloning system utilizing chimeric plasmids in a 2 micron DNA-deficient strain provides high plasmid stability.
• The system demonstrates efficient gene expression, indicated by elevated enzyme activity, without chromosomal integration.
• This approach offers promising new avenues for yeast-based cloning and genetic engineering applications.