Identification of differentially expressed transcripts in the human pathogenic fungus Paracoccidioides brasiliensis by differential display.
Paracoccidioides brasiliensis is a dimorphic human pathogenic fungus that is the causal agent of paracoccidioidomycosis, a systemic disease that predominantly affects rural communities in South and Central America. Dimorphism is a common characteristic of systemic human pathogenic fungi. Here we describe the use of differential display (DD) to isolate and identify differentially expressed genes of P. brasiliensis, in the two cell types, yeast (Y) and mycelium (M), as well as at different time intervals during temperature-induced M to Y transition. Using two oligo-deoxythymidine-anchored primers combined with 10 arbitrary ones, we were able to detect the presence of at least 20 differentially transcribed cDNA fragments. Some of these fragments were further analysed by reverse-northern blot and northern blot in order to confirm their differential expression. The M32, M51 and M73 cDNA fragments were specific for the mycelial form of P. brasiliensis. Furthermore, we found two cDNA fragments (M-Y1 and M-Y2) that were upregulated during M-Y transition. This method was efficient and useful in the detection of differentially expressed genes in P. brasiliensis.
Venancio
E J
EJ
Universidade de Brasilia, Instituto de Biologia, Departamento de Biologia Celular, DF, Brasil.
Kyaw
C M
CM
Mello
C V
CV
Silva
S P
SP
Soares
C M A
CM
Felipe
M S S
MS
Silva-Pereira
I
I
D018076:DNA, Complementary / Q000032:analysis* / Q000302:isolation & purification; D004271:DNA, Fungal / Q000032:analysis*; D020869:Gene Expression Profiling; D010228:Paracoccidioides / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D004271:DNA, Fungal