Figure 9.

Disruption of the racF1 gene. (A) Diagram of the Dictyostelium racF1 gene and its disruption by the blasticidin resistance cassette (bsr) in the racF1⁻ mutant. A construct was made in which the blasticidin resistance cassette was inserted into a unique BglII site of a 4.4-kb genomic clone. H, HindIII; K, KpnI restriction sites. Arrows indicate oligonucleotides used for PCR to confirm the insertion of the resistance cassette. (B) Southern blot analysis demonstrated that a gene replacement event had occurred. Genomic DNA was digested with HindIII, and the blot was probed with the ³²P-labeled probe 2. Insertion of the bsr cassette causes a shift of a 3.3-kb band to a 4.7-kb band in the mutant. No signal was observed after probing with pUC19 vector. (C) Replacement of the racF1 gene results in an altered mRNA. Northern blot containing 30 μg RNA per lane was hybridized with probe 1. RacF1⁻ mutant expresses a slightly larger mRNA that corresponds to a truncated racF1 plus sequences of the actin-8 terminator provided by the bsr cassette.