Figure 4.

Positional Cloning of the FRA8 Gene.

(A) The fra8 locus was mapped to a small region in the BAC clone F24D13 on chromosome 2. The FRA8 gene is composed of four exons and three introns. The fra8 mutation results in a C-to-T transition in the fourth exon. Black boxes in the FRA8 gene diagram indicate exons, and lines between exons indicate introns. Chr, chromsome; cM, centimorgan.

(B) Nucleotide and amino acid sequences around the fra8 mutation site. The fra8 mutation changes a wild-type codon encoding Pro into a codon encoding Leu.

(C) Elimination of a BanII site in the fra8 mutant gene. The single nucleotide mutation in fra8 occurs at a BanII restriction endonuclease cleavage site. This is revealed by digestion of the PCR-amplified DNA fragments with BanII, which shows that a BanII site is missing in the fra8 mutant DNA compared with the wild type.

(D) and (E) Complementation of the fra8 mutant by the wild-type FRA8 gene. The thin fiber wall and collapsed vessel phenotypes exhibited by the fra8 mutant (D) were rescued by introduction of the wild-type FRA8 gene into the fra8 mutant (E). co, cortex; if, interfascicular fiber; ve, vessel. Bars = 116 μm.