Yeast Yeast 2005; 22: 739–743. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1248 Yeast Sequencing Report Cloning and functional analysis of the orotidine-5 ′ - phosphate decarboxylase gene (PbrURA3) of the pathogenic fungus Paracoccidioides brasiliensis Cristina Reinoso 1 , Fran¸ coise Sorais 2 , Gustavo A. Ni˜ no-Vega 2 *, Encarnaci ´ on Fermi˜ n´ an 1 , Gioconda San-Blas 2 and Angel Dom´ ınguez 1 1 Departamento de Microbiolog´ ıa y Gen´ etica, Universidad de Salamanca, Salamanca, Spain 2 Instituto Venezolano de Investigaciones Cient´ ıficas, Centro de Microbiolog´ ıa y Biolog´ ıa Celular, Apartado 21827, Caracas 1020A, Venezuela *Correspondence to: Gustavo A. Ni˜ no-Vega, Instituto Venezolano de Investigaciones Cient´ ıficas, Centro de Microbiolog´ ıa y Biolog´ ıa Celular, Apartado 21827, Caracas 1020A, Venezuela. E-mail: gnino@ivic.ve Received: 11 January 2005 Accepted: 7 May 2005 Abstract A genomic clone encoding the Paracoccidioides brasiliensis orotidine monophosphate decarboxylase gene (PbrURA3 ) was isolated by screening a subgenomic plasmid DNA library of this fungus, using a PCR amplification product of the gene as a probe. Sequence analysis revealed that the gene contains an open reading frame of 855 bp with a single intron (162 bp), and encodes a putative 285 amino acids polypeptide of estimated molecular weight 31.1 kDa and isoelectric point 6.5. The deduced amino acid sequence predicted a 73.4% identity with orotidine monophosphate decarboxylase of Aspergillus nidulans. Functionality of the gene was demonstrated by transformation into a Saccharomyces cerevisiae ura3 null mutant. The PbrURA3 gene sequence has been submitted to GenBank database under Accession No. AJ133782. Copyright  2005 John Wiley & Sons, Ltd. Keywords: Paracoccidioides brasiliensis; URA3; complementation Introduction Orotidine-5 ′ -monophosphate decarboxylase is an enzyme responsible for the conversion of orotidine- 5 ′ -phosphate (OMP) to uridine-5 ′ -phosphate (UMP), the final step in the pyrimidine biosynthetic pathway. It is encoded by URA3, a gene that was initially used to derive a construct (the ura-blaster cassette; Alani et al., 1987) for the disruption of Saccharomyces cerevisiae genes. In order to study the molecular mechanisms underlying pathogenicity and other fungal biolog- ical processes, the disruption technique became particularly attractive in the study of Candida albi- cans (Fonzi and Irwin, 1993; Goshorn et al., 1992; Lay et al., 1998; Bain et al., 2001; Sundstrom et al., 2002). Also, in non-C. albicans species the disruption of alleles in diploid strains and mul- tiple gene disruption has been reported (Sakai et al., 1991; Zhou et al., 1994; Rodr´ ıguez et al., 1998). The pathogenic dimorphic fungus Para- coccidioides brasiliensis is the causative agent of paracoccidioidomycosis, one of the most frequent systemic mycoses in the rural population of Cen- tral and South America. This fungus can grow as hyphae at 20–23 ◦ C and change to yeast if a thermal stimulus at 37 ◦ C occurs (San-Blas and Ni˜ no-Vega, 2001, 2004; San-Blas et al., 2002). Genomic studies (Reinoso et al., in press) and par- tial transcriptome analysis of P. brasiliensis (Felipe et al., 2003; Goldman et al., 2003) identified genes involved in cellular metabolism, information stor- age and cellular processes. Some of them were differentially expressed during the dimorphic tran- sition, others were potentially involved in drug resistance and virulence. Additionally, P. brasilien- sis genes related to dimorphism have been studied (for review, see San-Blas and Ni˜ no-Vega, 2004). Functional studies on these genes by single and multiple gene disruptions could help to estab- lish their real involvement in dimorphism and pathogenicity, since the development of efficient transformation systems is essential to characterize Copyright  2005 John Wiley & Sons, Ltd.