Figure 1.

Identification of the CCA1-null line. (A) T DNA insertion site in CCA1. Black boxes, introns; white boxes, coding regions; LB, left border. (B) The cca1-1 line lacks CCA1 RNA. RNA from wild-type (WT) Wassilewskija ecotype and cca1-1 plants was used as a template for RNase protection analysis with probes for CCA1 and a control, UBQ3. (C) CCA1 protein is absent in the cca1-1 plants. Western blots of wild-type (WT) and proteins from cca1-1 plants were assayed with CCA1 antibodies. The upper band (arrowhead) is CCA1; the lower band (asterisk) is a nonspecific band routinely seen in Western blot analyses carried out with CCA1 antibodies. (D–F) Electrophoretic mobility-shift assays showing a major (arrowhead) and a minor (asterisk) CCA1-binding activity; these activities are reduced after preincubation of protein extracts with CCA1 Abs and when a mutated promoter fragment (M1) is used in the binding assay, and the activities are missing completely in protein extracts from cca1-1 plants.