Electrophoretic karyotypes of clinical isolates of Coccidioides immitis

Abstract

Chromosomes of the fungal respiratory pathogen, Coccidioides immitis, were separated by contour-clamped homogeneous electric field gel electrophoresis. Twelve isolates were examined, the majority of which showed four chromosomes with a range of molecular size from 11.5 to 3.2 Mb. Three isolates (C634, C735, and L) revealed three chromosomal bands under the conditions employed for electrophoretic separation. However, in two of these isolates (C634 and C735), four chromosomes were visible on membrane transfers of pulsed-field gels after Southern hybridization between the chromosomal DNA and selected DNA probes. The probes included a conserved ribosomal gene and three previously described cDNAs isolated from C. immitis expression libraries. The L isolate was determined to have the same genome size as a typical four-chromosome isolate on the basis of microspectrophotometric comparison of fluorescence intensity of the ethidium bromide-stained nuclear DNA. The genome size of C. immitis determined by microspectrophotometry was approximately 28.2 ± 2.6 Mb. The calculated genome size based on addition of the average molecular weights of chromosomal bands separated by contour-clamped homogeneous electric field gel electrophoresis was approximately equal to the estimate derived from the spectrophotometric analyses. This is the first report of the electrophoretic 4872 on July 16, 2020 by guest http://iai.asm.org/ Downloaded from on July 16, 2020 by guest http://iai.asm.org/ Downloaded from on July 16, 2020 by guest http://iai.asm.org/ Downloaded from on July 16, 2020 by guest

Electrophoretic Karyotypes and Genome Sizing of the Pathogenic Fungus Paracoccidioides brasiliensis

“…The electrophoretic conditions established in this study permitted the clear separation of four chromosomal bands in the range of 2.0 to 10.0 Mbp, and the karyotypes obtained under these conditions were very reproducible. Our results suggest that the chromosomes from P. brasiliensis are comparable in size and number to Coccidioides immitis, Neurospora crassa, and Dictyostelium discoideum chromosomes (10,22,23). We did not observe chromosomes smaller than 2.0 Mbp under a variety of electrophoretic conditions.…”

“…To determine the DNA content and to examine the ploidy of other fungi, optical and photometric methods (flow microfluorometry, fluorescence microscopy, photometry, and cytofluorimetry) have been employed (21,23,28,34,35). Our results of microfluorometry with P. brasiliensis DAPI-stained nuclei by confocal fluorescence microscopy indicated a DNA content in the range of 45.7 to 60.9 Mbp.…”

“…Erlenmeyer flasks containing 50 ml of YPD broth medium were inoculated with the entire growth of two culture slants, placed in a reciprocating shaker at 120 rpm, and grown for 5 days at 35°C. Approximately 10 8 yeast cells of P. brasiliensis B-339 and 113 were immobilized in 2% (wt/vol) low-melting-point agarose blocks, and spheroplasts were obtained at 30°C by lysing the cell wall in PBS (pH 7.5) containing 10 U of chitinase (Sigma) ml Ϫ1 and 30 U of lyticase (Sigma) ml Ϫ1 for 1 to 2 h (22,23,30). The blocks were dialyzed three times against 250 mM EDTA, pH 8.0, at 37°C to inactivate the enzymes.…”

“…In order to obtain intact chromosome-sized DNA molecules for PFGE studies, yeast cells were embedded in low-meltingpoint agarose blocks and spheroplasts were obtained at 30°C by lysing the cell wall in PBS (pH 7.5) containing chitinase and lyticase for 1 to 2 h (22,23,30). Chromosomal DNA prepared by this method could be stored in EDTA at 4°C for several months without apparent degradation, as assessed by conventional electrophoresis and PFGE.…”

Developments in Fungal Taxonomy

“…Techniques such as electron microscopy, pulsed-field gel electrophoresis (354,413,493), and its recent modification, contour-clamped homogeneous electric field electrophoresis (535) can contribute to the precise determination of karyotypes. The high intraspecific variability of the electrophoretic karyotypes of fungi makes this technique especially applicable for studies of populations in pathogenic yeasts (133,279,318,421,427) and filamentous fungi (68,535).…”