Figure 2

An assay for Zorro3 retrotransposition. (A) The cloned Zorro3 element has a retrotransposition indicator gene (URA3 promoter, URA3 P, and URA3 ORF, disrupted by an antisense intron) inserted into its 3' UTR. The element is preceded by the C. albicans ACT1 promoter and followed by the ACT1 terminator. The transforming vector also contains sequences for selection in bacteria (Ampicillin resistance) and in C. albicans (MPA resistance). The entire construct can be linearised at a unique Pin AI site (not shown) within a copy of the RP10 gene to target integration to the genomic RP10 locus. The first step of retrotranspositon is transcription to give a full-length mRNA (B). The intron in this RNA is then removed by splicing (C). Reverse transcription and integration of the spliced RNA results in a functional and stably integrated URA3 gene and confers a URA3+ phenotype on the host cell (D).