Isolation of the URA5 gene from Cryptococcus neoformans var. neoformans and its use as a selective marker for transformation.
A cDNA encoding Cryptococcus neoformans orotidine monophosphate pyrophosphorylase (OMPPase) has been isolated by complementation of the cognate Escherichia coli pyrE mutant. The cDNA was used as a probe to isolate a genomic DNA fragment encoding the OMPPase gene (URA5). By using electroporation for the introduction of plasmid DNA containing the URA5 gene, C. neoformans ura5 mutants could be transformed at low efficiency. Ura+ transformants obtained with supercoiled plasmids containing the URA5 gene showed marked mitotic instability and contained extrachromosomal URA5 sequences, suggesting limited ability to replicate within C. neoformans. Transformants obtained with linear DNA were of two classes: stable transformants with integrated URA5 sequences, and unstable transformants with extrachromosomal URA5 sequences.
Hormone Research Institute, University of California, San Francisco 94143-0534.
J C
JC
Edman
K J
KJ
Kwon-Chung
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003454:Cryptococcus / Q000235:genetics*; D003455:Cryptococcus neoformans / Q000201:enzymology / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics; D015183:Restriction Mapping; D014170:Transformation, Genetic
D004271:DNA, Fungal; D009962:Orotate Phosphoribosyltransferase