Fig. 7. DIP13 antisense RNA experiments. (A) pVW1 antisense construct. The AR-promoter (AR-P), DIP13 inverse reading frame (DIP13-ORF) and the RBCS2 gene 3' sequences (RBCS2-3') are indicated. Single restriction sites XbaI, BamHI, and KpnI are given for orientation. Primers (P1, P2) for amplification of a specific 560-bp fragment (open box) are indicated at their respective positions. (B) Result of PCR analysis with primers P1 and P2 and genomic DNA of three putative transformants (#33, 45 and 67) and control genomic DNA from the untransformed parent strain (wt). Controls were done without added template (– control) or 1 ng pVW1-DNA (+ control). St, DNA size standard. For orientation, some fragment sizes (in bp) are indicated to the right. (C) Phenotypic comparison of antisense transformants (C1-3) and untransformed cells (C4). Bar, 10 µm. (D) Analysis of DIP13 protein reduction in antisense strains #33, 45 and 67. Left panel: Coomassie-stained gel as loading control. Lane A: extract from the untransformed strain. Middle panel: immunoblot with the same amounts of protein per lane of the same four strains probed successively with anti-DIP13 antibody (DIP13), anti--tubulin antibody (-Tub) and anti-L23 antibody (L23). Right panel: result of densitometric analysis of DIP13 protein levels derived from the immunoblots shown. For standardization of loading, the signals obtained with anti L23 antibody were used. (E) Indirect immunofluorescence with anti-DIP13 antibody (E1) and DAPI staining (E3) of a transformed cell of strain #45 showing typical labeling (compare Fig. 4) at the two basal body spots (E1, white arrows) and two nuclei (E3, white arrows) at opposite poles. E2, corresponding phase image. Bar in C4 (10 µm) applies to all microscopic images.