Fig. 4. Molecular cloning and expression of VAN3. (A) VAN3 was isolated by positional cloning and mapped to the 89 kb region between the T31B5c and T22N19c markers, which corresponds to two adjoining BAC clones, T31B5 and T22N19. Numbers below the molecular markers indicate the recombination frequency between the marker and the VAN3 locus (recombinant chromosomes/analyzed chromosomes). Black lines and outlines show the VAN3 gene (At5g13300) and putative genes in this region, respectively. (B) Structure of the VAN3 protein and its homologs from the predicted proteins of Arabidopsis and humans. The VAN3 gene encodes an ARF–GTPase-activating protein (ARF–GAP) that includes BAR, PH, ARF–GAP and three-ANK-repeat domains. The mutation in the van3 mutant (TGG [Trp356] to TAG [stop]) is indicated. (C) Comparison of BAR and ARF–GAP domains. Conserved amino acid residues are highlighted by grey boxes. (D) Semi-quantitative RT–PCR analysis of the expression of the VAN3 gene in the wild-type and van3 mutant. RNA was isolated from 7-day-old seedling cotyledons of the wild-type and van3 mutant that had been either mock-treated (0) or treated with 10 µM NAA for 1 hour (10). ACT2 is the control. Numbers of cycles of PCR are indicated on the right.