CLONING AND NUCLEOTIDE-SEQUENCE OF A SPECIFIC DNA FRAGMENT FROM PARACOCCIDIOIDES-BRASILIENSIS

We cloned and sequenced a species-specific 110-bp DNA fragment from Pa racoccidioides brasiliensis. The DNA fragment was generated by PCR wit h primers complementary to the rat beta-actin gene under a low anneali ng temperature. Comparison of the nucleotide sequence, after excluding the primers, with those in the GenBank database identified approximat ely 60% homology with an exon of a major surface glycoprotein gene fro m Pneumocystis carinii and a fragment of unknown function in Saccharom yces cerevisiae chromosome VIII. By Southern hybridization analysis, t he P-32-labelled fragment detected 1.0- and 1.9-kb restriction fragmen ts within whole-cell genomic DNA of P. brasiliensis digested with Hind III and PstI, respectively, but failed to hybridize to genomic DNAs fr om Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans , Aspergillus fumigatus, Saccharomyces cerevisiae, Pneumocystis carini i, rat tissue, or humans under low-stringency hybridization conditions . Additionally, the specific DNA fragment from three different P. bras iliensis isolates (Pb18, RP18, RP17) was amplified by PCR with primers mostly complementary to nonactin sequences of the 110-bp DNA fragment . In contrast, there were no amplified products from other fungus geno mic DNAs previously tested, including Histoplasma capsulatum. To date, this, is the first species-specific DNA fragment cloned from P. brasi liensis which might be useful as a diagnostic marker for the identific ation and classification of different P. brasiliensis isolates.