A REORGANIZED CANDIDA-ALBICANS DNA-SEQUENCE PROMOTING HOMOLOGOUS NONINTEGRATIVE GENETIC-TRANSFORMATION

In order to develop plasmids adequate for non-integrative genetic transformation of Candida albicans, a DNA fragment of 15.3 kb was cloned from this organism on the basis of its capacity to convert the integrative Saccharomyces cerevisiae vector YIp5 into a non-integrative one. Southern hybridization analysis, carried out with a labelled DNA probe of 3.6 kb derived from the cloned fragment, showed that it consisted of C. albicans DNA, the hybridization pattern indicating that the corresponding sequences were homologous to several chromosomal regions. The size of the C. albicans DNA promoting autonomous replication in S. cerevisiae was substantially reduced by subcloning. A 5.1 kb subfragment, defined by BamHI and SalI restriction sites, retained autonomous replication sequences (ARS) functional in the heterologous S. cerevisiae system and in C. albicans, when inserted in plasmid constructions that carried a S. cerevisiae trichodermin-resistance gene (tcm1) as selection marker. C. albicans transformants were both of the integrative and the non-integrative type and the plasmids recovered from the latter very often carried a reorganized ARS, indicating that recombination of the inserted ARS DNA had occurred in the homologous host. Successive reorganizations of the ARS insert in C. albicans eventually led to a more stable and much smaller fragment of 687 bp that was subsequently recovered unchanged from transformants. Sequence analysis of the 687 bp fragment revealed four 11-base blocks, rich in A+T, that carried the essential consensus sequence considered relevant for yeast ARS elements in addition to other features also described as characteristic of yeast replication origins.