Isolation of the URA5 gene from Cryptococcus neoformans var. neoformans and its use as a selective marker for transformation.

Abstract

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.

Comparative gene genealogical analyses of strains of serotype AD identify recombination in populations of serotypes A and D in the human pathogenic yeast Cryptococcus neoformans

“…For strains of serotypes A, B, C and D (and some strains of serotype AD) sequenced previously, direct sequencing using PCR products without cloning revealed no sequence ambiguity, and each strain had only one allele at each of four genes analysed, including URA5 (Xu et al, 2000b; see also Edman & Kwon-Chung, 1990;Casadevall et al, 1992). Since introns in both URA5 and LAC genes from strains of serotypes B and C were difficult to align with those from strains of serotypes A and D (Xu et al, 2000b), strains of serotypes B and C were not included in the analyses here.…”

“…The fragment was amplified using the following oligonucleotide primers (59-39): forward: ACGCCTGCCTGTTACTTAA, reverse: GGACATGATGATTGGAGT. The amplified DNA fragment corresponded to the complete nucleotide sequence (1-779 bp) as reported for a serotype D strain by Edman & Kwon-Chung (1990;GenBank accession number M93026). Because ends of sequences were ambiguous on typical sequencing gels, only the unambiguous nucleotides from position 65 to 706 (a total of 642 nucleotides) were analysed for all strains in this study.…”

“…Because ends of sequences were ambiguous on typical sequencing gels, only the unambiguous nucleotides from position 65 to 706 (a total of 642 nucleotides) were analysed for all strains in this study. The analysed fragment contains two introns, one complete exon, and partial sequences from two other exons (see Edman & Kwon-Chung, 1990).…”

“…In this study, we sequenced a portion (642 nucleotides) of another gene, orotidine monophosphate pyrophosphorylase (URA5) for the same 14 strains and compared genealogies derived from these two genes. URA5 was selected for study because it is present in only one copy in haploid strains of C. neoformans and known to be highly polymorphic (Edman & Kwon-Chung, 1990;Casadevall et al, 1992;Xu et al, 2000b). To strengthen the analyses, we included additional strains of serotypes A and D. The results revealed gene genealogy incongruence, consistent with sexual recombination within populations of both serotypes A and D.…”

The Ess1 prolyl isomerase is dispensable for growth but required for virulence in Cryptococcus neoformans

“…pCn-tel-CnESS1 was constructed by insertion of a 2?1 kb EcoRI-BamHI fragment (consisting of 1?4 kb of upstream untranslated region plus 0?7 kb of the C. neoformans ESS1 gene) into plasmid pCn-tel1. pCn-tel contains a URA5 selection marker and was kindly supplied by Ping Wang and Joseph Heitman (Duke University, Durham, NC, USA; Davidson et al, 2000;Edman & Kwon-Chung, 1990). The 2?1 kb fragment had been obtained by PCR from C. neoformans B-3501 genomic DNA.…”