Polymorphism in the flanking regions of the PbGP43 gene from the human pathogen Paracoccidioides brasiliensis: search for protein binding sequences and poly(A) cleavage sites.
BACKGROUND
Paracoccidioides brasiliensis 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 PbGP43 flanking regions to help understand such variations.


RESULTS
we tested the protein-binding capacity of oligonucleotides covering the PbGP43 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 P. brasiliensis phylogenetic species PS2, altered binding affinity. Next, we cloned and sequenced the 5' intergenic region up to position -2,047 from P. brasiliensis 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 PbGP43 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.


CONCLUSIONS
We observed that the amount of PbGP43 transcripts accumulated in P. brasiliensis 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 PbGP43, their characteristic sequences, besides the presence of multiple poly(A) cleavage sites in the 3' UTR will certainly guide future studies.
Rocha
Antonio A
AA
Department of Microbiology, Federal University of São Paulo, São Paulo, Brazil. rocha14@unifesp.br
Morais
Flávia V
FV
Puccia
Rosana
R
D020413:3' Untranslated Regions; D000946:Antigens, Fungal / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D006023:Glycoproteins / Q000235:genetics*; D010228:Paracoccidioides / Q000235:genetics*; D011061:Poly A / Q000235:genetics; D011110:Polymorphism, Genetic; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D020080:Tandem Repeat Sequences
D020413:3' Untranslated Regions; C103180:43 kDa protein, Paracoccidioides; D000946:Antigens, Fungal; D004271:DNA, Fungal; D005656:Fungal Proteins; D006023:Glycoproteins; D011061:Poly A