| Summary: | <p>Abstract</p> <p>Background</p> <p><it>Paracoccidioides brasiliensis </it>is a thermo-dimorphic fungus that causes paracoccidiodomycosis (PCM). Glycoprotein gp43 is the fungal main diagnostic antigen, which can also protect against murine PCM and interact with extracellular matrix proteins. It is structurally related to glucanases, however not active, and whose expression varies considerably. We have presently studied polymorphisms in the Pb<it>GP43 </it>flanking regions to help understand such variations.</p> <p>Results</p> <p>we tested the protein-binding capacity of oligonucleotides covering the Pb<it>GP43 </it>proximal 5' flanking region, including overlap and mutated probes. We used electrophoretic mobility shift assays and found DNA binding regions between positions -134 to -103 and -255 to -215. Only mutation at -230, characteristic of <it>P. brasiliensis </it>phylogenetic species PS2, altered binding affinity. Next, we cloned and sequenced the 5' intergenic region up to position -2,047 from <it>P. brasiliensis </it>Pb339 and observed that it is composed of three tandem repetitive regions of about 500 bp preceded upstream by 442 bp. Correspondent PCR fragments of about 2,000 bp were found in eight out of fourteen isolates; in PS2 samples they were 1,500-bp long due to the absence of one repetitive region, as detected in Pb3. We also compared fifty-six Pb<it>GP43 </it>3' UTR sequences from ten isolates and have not observed polymorphisms; however we detected two main poly(A) clusters (1,420 to 1,441 and 1,451 to 1,457) of multiple cleavage sites. In a single isolate we found one to seven sites.</p> <p>Conclusions</p> <p>We observed that the amount of Pb<it>GP43 </it>transcripts accumulated in <it>P. brasiliensis </it>Pb339 grown in defined medium was about 1,000-fold higher than in Pb18 and 120-fold higher than in Pb3. We have described a series of features in the gene flanking regions and differences among isolates, including DNA-binding sequences, which might impact gene regulation. Little is known about regulatory sequences in thermo-dimorphic fungi. The peculiar structure of tandem repetitive fragments in the 5' intergenic region of Pb<it>GP43</it>, their characteristic sequences, besides the presence of multiple poly(A) cleavage sites in the 3' UTR will certainly guide future studies.</p>
|
|---|
