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. 1999 May;10(5):1353–1366. doi: 10.1091/mbc.10.5.1353

Figure 5.

Figure 5

Preparation and characterization of the GSA7 knockout. The S. cerevisiae ARG4 gene was inserted into HindIII and BglII sites within the ORF of GSA7. The ARG4 insert included the ORF of ARG4 (arrow) and 1300 bp of 5′ and 500 bp of 3′ noncoding regions. The 5′ noncoding region includes the ARG4 promoter, whereas the 3′ noncoding region presumably lacks a promoter sequence, because the next gene downstream of ARG4 is 2200 bp away. In addition, the 3′ noncoding region likely contains a transcription terminator. The knockout fragment (5.1 kb) was cut out of the shuttle vector by ApaI and ScaI digestion and used to transform PPF1 (his4, arg4). Stable Arg+ transformants were isolated, gsa mutants were identified by direct colony assay, and the recombination of this construct with the genomic DNA was confirmed by PCR. Genomic DNA was isolated from GS115 and gsa7Δ, and the site of insertion of the ARG4 gene was verified by PCR analysis using the primers inside (b and d) and outside (a and c) the insert. The expected fragments of 5.0 kb (a → b), 6.1 kb (a → c), and 3.3 kb (a → d) were consistent with the ARG4 gene being inserted into the GSA7 locus of gsa7Δ cells.