A novel repeat sequence (CKRS-1) containing a tandemly repeated sub-element (kre) accounts for differences between Candida krusei strains fingerprinted with the probe CkF1,2

Abstract

CkF1,2 has been reported as an effective DNA fingerprinting probe of Candida krusei. It is composed of two genomic EcoRI-restriction fragments, F1 and F2, which are approximately 5.4 and 5.2 kb, respectively. Sequence analysis of F1 reveals that it is 5261 bp-long, has a GC content of 42.2 mol%, and originates from the intergenic region of the ribosomal RNA cistrons (IGR). F1 comprises 488 bp of the 3′ end of a 25s rRNA gene, a non-transcribed spacer region 1 (NTS1), a 5s gene (121 bp), and a major portion of the non-transcribed spacer region 2 (NTS2). A 1256 bp-long repeated sequence, CKRS-1, with a GC content of 35 mol%, has been identified in NTS2. CKRS-1 contains eight tandemly repeated sub-elements, kre-0 to kre-7. The first two, kre-0 and kre-1, are 164 bp-long, the next five sub-elements, kre-2 to kre-6, are 165 bp-long, and the last element, kre-7, is 103 bp-long. The eight sub-elements share nucleotide-sequence homologies between 66 to 100%, with kre-2, kre-3 and kre-4 identical, and kre-0 the most divergent. Shorter repeated sequences were also identified in three regions of F1, which were named domains ``a'', ``b'' and ``c''. Restriction mapping, cross hybridization, and direct comparison of sequences show that F1 and F2 are polymophic forms of the IGR and their size difference is due both to the number of kre sub-elements in CKRS-1 and to a 24-bp deletion in domain ``b''. While F1 contains eight kre sub-elements, F2 contains seven. In C. krusei strain K31, four polymorphic forms of CKRS-1 have been identified containing five, six, seven and eight kre sub-elements. CKRS-1 is dispersed on three of the chromosomes of highest molecular weights separated by transverse alternating-field electrophoresis. CKRS-1 does not hybridize significantly to any transcription product. Polymorphisms in single DNA fingerprints and differences between the DNA fingerprints of strains of C. krusei based upon CkF1,2 hybridization patterns therefore appear to be based, at least in part, on the variable number of tandemly repeated kre sub-elements in CKRS-1.