Construction of stable episomes in Cryptococcus neoformans.
We report the generation of stable plasmids constructed by inserting specific DNA sequences into previously known unstable vectors. These sequences were obtained from a DNA library recovered from a previously reported stable minichromosome created by electroporative transformation in Cryptococcus neoformans (Varma and Kwon-Chung 1994). A 6-kb insert from this minichromosome significantly enhanced both the frequencies at which URA5 transformants were obtained as well as the stability of their uracil prototrophy on non-selective media. A 1.5-kb sequence of this insert contained telomeric sequence repeats which when introduced into plasmids resulted in significant increases in transformation frequency. A 1081-bp sequence (STAB), present in the remainder of the insert, had an ARS-like function enhancing the episomal maintenance of plasmids in the transformants regardless of the gene (ADE2/URA5) used as a selection marker.
Molecular Microbiology Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, Building 10, Room 11C304, National Institutes of Health, Bethesda, MD 20892, USA.
A
A
Varma
K J
KJ
Kwon-Chung
D015139:Blotting, Southern; D015825:Chromosomes, Fungal; D003455:Cryptococcus neoformans / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D014170:Transformation, Genetic
D004271:DNA, Fungal; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases