Figure 2.

Molecular isolation and characterization of Asc-1. (A) Isolation of Asc-1. Genetic distance of chromosome markers are in centimorgans (cM); overlapping YACs are light gray boxes, in the detail of YAC442A (dark gray) are the EcoRI fragments positioned at zero recombinants. The corresponding chromosome segment in the asc, asc isoline (asc-1) contains deletions (blank gaps). Tomato DNA fragments used in the A. rhizogenes-mediated transformation are black lines. Only DNA constructs which give functional complementation in the hairy root bioassay (+) are depicted. ORF1 is Asc-1 (2.5 kb), which contains six exons (black boxes). The second exon of the asc-1 gene contains a 2-bp deletion (ΔAG). (B) Hairy root bioassay with two representative transformants of pGreen029 (empty binary vector) and pBAsc404 (containing Asc-1) on SAMs. (C) Genomic DNA blot of the Asc isolines and YAC442A hybridized with the coding region of the Asc-1 gene (dashed line in A). The deletions in the asc-1 promoter region are detected by a shift of a 4.2- (Asc) to a 3.9-kb (asc) HindIII fragment. (D) Competitive RT-PCR for visualizing relative mRNA levels of Asc-1 and asc-1 in leaves of the Asc isolines. Experiments were repeated three times from individual plants with similar results. The upper fragment represents the 457-bp fragment amplified from added competitor genomic Asc-1 DNA; the lower band represents the Asc-1-specific 289-bp cDNA fragment.