Fig. 4.

Splicing defects in wtf1 mutants. Assays used seedling leaf RNA from plants of the indicated genotypes. Introns are designated as subgroup IIA or IIB, according to ref. 35. (A) Poisoned-primer extension assays. Oligonucleotides complementary to exon sequences near the 3′-splice junction of the indicated introns were used to prime reverse transcription in the presence of a dideoxynucleotide that terminates cDNA synthesis after different distances on spliced and unspliced templates. The asterisk marks a product terminating at the branchpoint adenosine formed during the first splicing step. (B) RNA gel blots probed with exon sequences from the tRNA gene indicated at bottom. Asterisks identify unspliced precursors. (C) Ribonuclease-protection assay. The probe was body-labeled and spanned the splice junction diagrammed below. The lengths in nucleotides of probe segments corresponding to intron and exon are indicated in the diagram. S, spliced; U, unspliced; pg, pale green seedlings; iv, ivory seedlings.