Fig. 3. Isolation of the CUP gene. (A) Only one of the isolated Antirrhinum NAC-domain genes belonged to the same clade as CUC1, CUC2 and NAM (shaded). Other Arabidopsis NAC-domain proteins are included for comparison. Accession numbers are: NAP, CAA10955; CUC1, BAB20598; CUC2, BAA19529; NAM, CAA63101; CUP, AJ568269; GRAB2, CAA09372; CUC3, AAP82630; AtNAC1, AAF21437; and GRAB1, CAA09371. The tree was constructed by the neighbour-joining method in MacVector, using only the conserved NAC-domains. Bootstrap values are indicated. (B) The CUP gene consists of three exons. Untranslated transcribed regions are shown in grey and the conserved NAC-domain is stippled. The transposon insertion sites are shown for each allele. DNA sequence analysis of PCR products spanning the insertion sites indicated that the transposons were related to Tam-1 (cup-1) (Nacken et al., 1991) and Tam-4 (cup-2) (Luo et al., 1991), respectively. (C) Northern blot hybridised with a truncated CUP probe, lacking the NAC-domain, shows expression in the wild-type control but not in the two cup alleles. Stripping and re-hybridisation of the northern with a constitutive GAPDH probe demonstrates that RNA is present in each lane.