ABSTRACT
Figure 8 (A) Strategy for targeting and replacing the WI-I locus in B. dermatitidis. Anticipated homologous crossover leading to a gene replacement event at the WI-I locus. Linearized pQWhph can direct the hph gene to the WI-l locus through homologous crossover in two ways. X's depict crossing over at 1 kB of WI-I 5' flanking sequence (all WI-I coding, designated in black) and at 2.5 kB of WI-l 3' flanking sequence (881 bp of WI-I coding in black and 1395 bp of noncoding in horizontal lines). Alternatively, the broken line depicts crossing over at a short region of the 375 bp WI-I minipromoter in front of the hph gene. Either crossover event would replace most of the WI-I coding region with the hph cassette. (B) PeR analysis for targeted gene replacement of WI-I. Candidates for a WI-l null phenotype were analyzed for targeted recombination by PeR amplification of a novel junction fragment spanning the WI-I 5' region (not present on the targeting vector) and the hph gene. The forward primer is present only in the endogenous locus, whereas the reverse primer is present in the hph gene on transforming DNA. The resulting 675-bp fragment is amplified if WI-l sequences have been interrupted by hph in the manner depicted by a dashed line in (A), yielding an interrupted locus shown at the bottom of (A) and in (B). The product's authenticity is confirmed by the presence of EcoRV and Aat II restriction sites in the WI-I upstream segment of the product. as shown in an agarose gel, and in accord with published WI-I genomic sequence [51]. (From Ref. 60.)
phages poorly (Fig. 9A). The number of knockout yeasts bound to or inside of macrophages in vitro was far lower than that for wild-type yeasts at all time points between 15 min and 6 hr of incubation. At 6 hr, the association index for the knockout yeast was one-sixth the value for the wild-type yeast, and the ingestion index was one-eighth as high. The defect in binding and entering macrophages was restored fully in the knockout when 10% serum was added as a source of complement (Fig. 9B) or after WI-I was reexpressed in strain 4/55 (Fig. 9C). The findings imply that WI-I mediates binding and entry of yeast into macrophages in the opsoninpoor environment of the lung early in the course of infection, before tissues are inflamed and serum exudes into the alveolus.
