Characterization of the L41 gene in Cryptococcus neoformans: its application as a selectable transformation marker for cycloheximide resistance.
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 five 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 microg/ml cycloheximide, at a maximum efficiency of 300 transformants/microg 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.
Varma
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A
Molecular Microbiology Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Kwon-Chung
K J
KJ
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D001483:Base Sequence; D015415:Biomarkers; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003455:Cryptococcus neoformans / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D003513:Cycloheximide / Q000494:pharmacology*; D015342:DNA Probes / Q000737:chemistry; D004352:Drug Resistance, Microbial / Q000235:genetics; D018274:Electroporation; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic
D000935:Antifungal Agents; D015415:Biomarkers; D015342:DNA Probes; D012269:Ribosomal Proteins; D003513:Cycloheximide