Ty3 targeting of novel loci requires Pol III promoter element. (A) Box A–box B pairs at iSCS3 and iYIL100W target loci. (Top row) Approximate 20-nt spacing between integration sites (i) and box A and the consensus box A and box B sequence of fungal tDNAs, with an average spacing of 29 bp (Marck et al. 2006). Lowercase indicates divergence from consensus. (Right column, box B mut.) Box B mutations described in B and C. (B) Target activity of novel Ty3 target iYIL100W. YMA1322 transformed with pKN3050 and plasmids containing 500-bp iYIL100W-flanking sequence and a box B mutant derivative was induced to express Ty3 for 36 h. Integration was monitored by PCR (+/−). (Triangles) Insertion sites; (double line) chromosomal DNA; (TATA) upstream TATA-box consensus; (m) mutated box B (mutation sequence listed in A). (C) Target activity of iSCS3. As described in B, 500-bp iSCS3-flanking sequence and variants were tested for targeting. (Filled triangles) Insertion sites determined from sequence analysis; (empty triangles) those recovered from target plasmids; (lm and rm) mutated box B in loci shown; (lh and rh) half truncation as shown.
