Atypical URA5 gene restriction fragment length polymorphism banding profile in Cryptococcus neoformans strains

Abstract

URA5-RFLP is one of the most widely used genotyping methods relating to Cryptococcus neoformans and C. gattii consensus genotype nomenclature. In order to identify a molecular type, this method uses a visual comparison of digested PCR products of tested and reference strains, therefore any anomaly in RFLP patterns of studied isolates makes recognition difficult or impossible. This report describes a strain of VNIV type showing an atypical URA5-RFLP pattern as well as a group of AD hybrids displaying the same anomaly. The atypical RFLP pattern is the result of a point mutation and emergence of a new restriction site. Emergence of the allele presenting a new banding pattern may lead to misidentification using the URA5-RFLP technique; the results of this study as well as the literature data may suggest the spread of the allele in the environment.

Fig. 1

URA5-RLFP profiles of reference and atypical strains. Lanes: 1 and 6—molecular marker (Gene Ruler 100 bp Plus DNA Ladder, Thermo Scientific Waltham, MA, USA); 2–5 reference strain profiles (2—VNI, 3—VNII, 4—VNIII, 5—VNIV); 7–11 atypical strains (7—atypical VNIV, 8–11—atypical VNIII)

Fig. 2

Restriction map of K1b strain URA5 gene prepared using BioEdit v7.2.0 (top); comparison of CBS 10079 and K1b strains’ sequence fragments. In K1b sequence point mutation (bold; position 374) as well as recognition sequences in the new restriction site are indicated (bottom)