Electrophoretic Karyotypes and Genome Sizing of the Pathogenic Fungus Paracoccidioides brasiliensis

Abstract

Here we present the karyotype analysis and genome sizing ofParacoccidioides brasiliensis, a pathogen refractory to conventional genetic analysis. We have established pulsed-field gel electrophoresis (PFGE) conditions to resolve the high-molecular-weight chromosomal bands of two clinical isolates ofP. brasiliensis. Both isolates showed four megabase-sized bands, ranging from 2.0 to 10.0 Mbp. Significant differences in chromosome sizes and in the chromosomal location of genes for the gp43 antigen and chitin synthase were found. Different technical approaches were employed to estimate the DNA content and to define the ploidy ofP. brasiliensis. An estimated genome size in the range of 45.7 to 60.9 Mbp was provided by the analysis of data generated by measuring the amplitude of fluorescence intensity of DAPI (4′,6-diamidino-2-phenylindole)-stained nuclei (by confocal microscopy). The nuclear genome size estimated by confocal microscopy is twice that estimated by the average sum of the molecular weight of chromosome-sized DNA molecules by PFGE, suggesting that each separatedP. brasiliensis chromosomal band is diploid.

Electrophoretic karyotype of environmental isolates of Paracoccidioides brasiliensis

“…In a previous report, Nogueira et al . [14] presented differences in the electrophoretic mobilities of bands in their isolates of P. brasiliensis , as compared with our previous publication [13], although this variation can also be explained by the difference in the electrophoresis techniques used, PFGE vs. CHEF, both reports indicate that chromosome polymorphism does exist in P .…”

“…Chromosomal aberrations and rearrangements may play an important role in the genetic variability and evolution of the different isolates. Similar structural aberrations may have caused the small difference observed in the patterns of the clinical and non‐clinical isolates of P. brasiliensis [13,14].…”

“…The above studies indicate that there are variations in P. brasiliensis electrophoretic patterns [13,14]. Comparisons among such patterns, could furnish important information concerning genetic relatedness and epidemiological trends of the fungus.…”

“…Recently two clinical isolates of P. brasiliensis , B‐339 and 113, were analysed by PFGE. The electrophoretic conditions employed in this study allowed separation of four chromosomal bands in the range of 2 to 10 Mb and the haploid genome size was estimated to be in the range of 23–27·6 Mb [14].…”

Updates in Paracoccidioides Biology and Genetic Advances in Fungus Manipulation

“…The first study to evaluate genetic aspects of the genus Paracoccidioides demonstrated that the conidia are uninucleate, becoming multinucleated as soon as differentiation for yeasts is initiated [197]. Another approach using pulsed-field gel electrophoresis (PFGE) to separate chromosomes and labelling with 4 ,6 -diamino-2-phenyl-indole (DAPI) to quantify nucleotide fluorescence suggested that the genome of Paracoccidioides (isolates B-339 and 113) would have four chromosomes and would probably be diploid [198]. A subsequent karyotype study, using similar methods, evaluated 12 isolates of Paracoccidioides and suggested that there should be four or five chromosomes and a general diploid status.…”