24 Results
for author "Bruce L. Miller"
Figure 8Schematic depiction of matA flanking sequence duplication and restoration of wild-type matA gene structure at the endogenous locus. Genetic organization of the matA locus after 3′ homologous integration of pWP3 carrying the matA transgene (solid box). 5′ and 3′ flanking regions are shown with the silencer element (shaded box). The DNA fragment containing pyrG and the matA transgene allows alignment of duplicated 5′ flanking regions and rearrangement during mitotic recombination. The matA(0) allele and pWP3 carrying pyrG are excised and wild-type matA allele is restored at chromosome III. Recombinant events containing wild-type matA and the pyrG89 mutation (chr I) were selected after plating on media supplemented with 5′-FOA.
Figure 9Developmental expression of matA at the resident locus. Expression of matA was analyzed in undifferentiated hyphae, H, and in reproductive tissue 6 days postinduction of sexual development, D. Expression of the matA transgene at the matA(0) locus is suppressed in the reproductive tissue (before 5′-FOA). Wild-type expression of matA is restored at the resident locus (after 5′-FOA) upon removal of matA(0) and plasmid sequences and the recovery of wild-type genomic structure.
Figure 10Duplication of 830 bp of matA 5′ flanking sequences at the resident matA locus interferes with the expression and molecular function of the matA transgene. Structure of the complemented endogenous matA(0) locus and associated phenotype are shown. Bars represent matA with flanking regions and pyrG/pyrG89 gene. Solid line represents vector sequence of pWP3; dashed line represents deleted 5′ flanking sequences from −1001 to −171. Shaded box represents silencer element. (A) Integration of the pWP3 plasmid via 3′ homology introduces a duplication of 830 bp of 5′ flanking sequences at the resident matA locus and interferes with fertility. (B) Integration of the pWP3 (∆830 bp), via 3′ homology at the resident matA locus results in wild-type fertility.