Figure 3

Diagram of the pΔγ-1 vector, the genomic insertion site, the size-selected genomic DNA clone with flanking sequences, and demonstration that uni3–1 is a deletion allele. (A) The vector pΔγ-1 carries the ARG7 gene (Debuchy et al., 1989), which is diagrammed as the striped box, and pBR329 sequences, which are diagrammed as black lines. At the ends, when the plasmid is linearized with EcoRI, are 1049-bp and 750-bp segments of the Chlamydomonas γ-tubulin gene, which are diagrammed as open boxes. (B) The orientation of the plasmid at the insertion site in uni3–1 cells. Most of the pBR329 and all of the γ-tubulin sequences were deleted from the right side as diagrammed. The 1,049 bp of γ-tubulin sequences from the left side of the pΔγ-1 plasmid remained in the transformant. (C) The cloned genomic DNA from the size-selected library made from AvaI–BamHI digested DNA from uni3–1 cells. Probe b was used to screen the phage libraries and contains single-copy DNA from the site of the insertion. (D) Restriction map of uni3–1 and wild-type DNA with the enzyme NheI. The location of probes a–f on the wild-type map are shown. Probe b in parts C and D are the same probe. (E) Southern blots of genomic DNA from uni3–1 and wild-type cells hybridized with probes a–f. The left lane in each panel contains wild-type DNA (+) and the right lane contains uni3–1 DNA (−).