Fig. 5. Molecular details of the Arabidopsis CROOKED gene. (A) The exon-intron structure for At4g01710 (CROOKED) as determined by comparing the genomic DNA with RT-PCR-amplified cDNA fragments and RLM-RACE PCR, respectively. (B) The crooked mutant sequence contains a single point mutation (g>a) at the splice donor site, which leads to premature splicing (arrow) and a 35 bp deletion in exon 2 (nucleotides underlined). (C) RT-PCR using wild-type and crooked cDNA shows that the premature splicing produces a smaller transcript of 360 bp (large arrow) and a weak band (small arrow) corresponding to the 399 bp wild-type transcript. (D) The altered splice site predicts a frame shift resulting in a stop codon 129 bp downstream of the start site and a truncated amino acid sequence of only 44 residues (the last 12 shown). (E) Tissue-specific RT-PCR shows CROOKED to be ubiquitously expressed (R, root; S, inflorescence stem; L, leaves; F, flowers; Si, young siliques). The lower lane shows the elongation factor 1 transcript used as a loading control. (F) The predicted amino acid sequence for CROOKED aligned against ARPC5 subunit sequences from other organisms (Ath, A. thaliana; Dic, D. discoideum (31%); Cel, C. elegans (32%); Dan, D. rerio (29%); Hum, H. sapiens (32%); Spo, S. pombe (25%); Sce, S. cerevisiae (23%). Percentages (%) indicate amino acid identity (black); gray areas show similar amino acids.