Fungal molecular biology

Abstract

Introduction and Objectives: Paracoccidioides brasiliensis is a thermodimorphic fungus and is the ethiologic agent of Paracoccidioidomicose. This dimorphic fungus undergoes a complex differentiation in vivo and is responsible for one of the most prevalent mycosis in Latin America. During in vitro growth, phase transitions are triggered when the incubation temperature is shifted from 26°C to 37°C or in the reverse direction. When exposed to environmental stress such as temperature elevation, all living organisms respond by rapidly producing increasing amounts of heat shock proteins (Hsps), which presumably protect cells against the effects of the stress agent. Although a number of heat shock proteins have been identified, Hsp70 is one of the most abundant. Because our group is interested in studying genes involved in the transition from mycelium to yeast, we decided to analyze sequences of the hsp70 gene in different isolates of P. brasiliensis. Methods and Results: We amplified and sequenced the hsp70 genomic sequences of 21 isolates. We also aligned the hsp70 sequences of the 21 isolates, using the program Clustal W, to verify differences or similarities between them. We found the following additional sequences within the hsp70 gene of isolate Pb01 in comparison to the homologs in other isolates: three regions with 16, 23 and 22 base pairs located in the first intron of this gene. We also confirmed that the third region (22 bp) correspond to a microsatellite (CT) region. To verify the uniqueness of these sequences in the isolate Pb01, we designed a reverse primer based in the specific region (23 bp) located in the first intron and a forward primer based in the 5' UTR region common to all isolates of P. brasiliensis used in this analysis. A fragment of 400 bp was amplified by PCR only in the Pb01 isolate. Conclusion: We found intrinsic differences in the hsp70 gene of P.