Characterization of the L41 gene in Cryptococcus neoformans: its application as a selectable transformation marker for cycloheximide resistance
✍ Scribed by A. Varma; K. J. Kwon-Chung
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 174 KB
- Volume
- 16
- Category
- Article
- ISSN
- 0749-503X
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✦ Synopsis
A transformation system using resistance to the antibiotic cycloheximide as a dominant selectable marker was developed for the pathogenic yeast Cryptococcus neoformans. A 3.5 kb DNA fragment containing a gene encoding the ribosomal protein L41 was cloned from a wild-type strain of C. neoformans which is sensitive to cycloheximide. The open reading frame of the L41 gene contains ®ve introns and encodes a protein of 107 amino acids, which is similar to those reported for other yeasts. The cycloheximide resistance gene to be used as a marker was constructed by replacing a DNA segment of the wild-type L41 gene, which contained the amino acid proline at its 56th position with a homologous DNA segment from a mutant strain resistant to cycloheximide that contained leucine in that position. Cycloheximide resistant transformants were obtained by electroporation on YEPD plates, supplemented with 10±20 mg/ml cycloheximide, at a maximum ef®ciency of 300 transformants/mg plasmid DNA. While with other genes, most transformants of serotype D in C. neoformans maintain the transforming DNA as episomes, the cycloheximide-resistant transformants were all the result of ectopic genomic integration events.