Sodium azide mutagenesis: preferential generation of A.T-->G.C transitions in the barley Ant18 gene.
The molecular basis for the absence of anthocyanins and proanthocyanidins in four independent sodium azide-induced ant18 mutants of barley was examined by sequencing the gene encoding dihydroflavonol 4-reductase in these mutants. Sodium azide generated 21 base substitutions, which corresponds to 0.17% of the 12,704 nucleotides sequenced. Of the substitutions, 86% were nucleotide transitions, and 14% were transversions. A.T-->G.C base pair transitions were about 3 times more frequent than G.C-->A.T transitions. No deletions or mutation hot spots were found. The absence of dihydroflavonol 4-reductase activity in ant18-159, ant18-162, and ant18-164 plants is caused by missense mutations in the respective genes. By using microprojectile bombardment, a plasmid harboring the wild-type Ant18 gene was introduced into ant18-161 mutant cells and resulted in the development of anthocyanin pigmentation, which demonstrates that the mutation is corrected by expression of the introduced gene. On the other hand, a plasmid derivative with the two ant18-161-specific base transitions at the 5' splice site of intron 3 prevented complementation. It is concluded that the absence of detectable mRNA for dihydroflavonol 4-reductase in ant18-161 cells is due to the mutations in the pre-mRNA splice donor site.
Olsen
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O
Department of Physiology, Carlsberg Laboratory, Copenhagen, Denmark.
Wang
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X
von Wettstein
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D
D000429:Alcohol Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D001386:Azides / Q000494:pharmacology*; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D017343:Genes, Plant / Q000187:drug effects*; D001467:Hordeum / Q000166:cytology / Q000187:drug effects / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009153:Mutagens / Q000494:pharmacology*; D010957:Plasmids; D019810:Sodium Azide; D016385:TATA Box
D001386:Azides; D009153:Mutagens; D019810:Sodium Azide; D000429:Alcohol Oxidoreductases; C056570:dihydroflavanol 4-reductase