genetic strategy to identify mutations in CCH and RAN1 (Kry- san et al. 1999). Mutant alleles of CCH (cch-1) and RAN1 (ran1-4) were identified from a population of 72,960 inde- pendent T-DNA insertion lines.
In cch-1, the T-DNA is inserted six bases upstream of the CCH start codon (Fig. 3 a). CCH mRNA is not detectable in cch-1 by RNA blot analysis (Fig. 3 b) and no CCH protein is detected in extracts of cch-1 analyzed by immunoblotting (Lola Peñarrubia, personal communication). cch-1 is indistin- guishable from wildtype under all growth conditions tested (data not shown). Nutrient analysis was performed on the dis- tal 15 mm of leaves 5 and 6 from cch-1 as described