Figure 5

RNA expression at the ONSEN insertion and DNA methylation of the 5′ and 3′ flanking regions of the ONSEN insertion.

(a) RT-PCR analyses of transcripts for ABI5. A PCR product of the ONSEN insertion site was not amplified in the 13–7 ibm2 and 13–7 parent line because of transcription elongation defects. In contrast, the first intron was spliced out in 13–7 and 13–7 nrpd1 of F3, similarly to the wild type. ABI5 1^st intron; the first intron that ONSEN was inserted in 13–7. 18S; 18S rRNA gene. Genomic DNA (gDNA) was used as a template. The red arrowhead indicates an ONSEN insertion. (b) A 3′ Rapid Amplification of cDNA Ends (RACE) of ABI5 in the F3 progeny. The red arrowhead indicates an ONSEN insertion and black arrows indicate primers used in 3′ RACE. (c) Bisulfite sequence analysis of 5′ and 3′ flanking regions of the ONSEN insertion in F3 progenies (derived from the progeny of a cross between 13–7 and an ibm2). 13–7: F3 progeny that contains an ONSEN insertion in ABI5. 13–7 ibm2: F3 progeny that contains an ONSEN insertion in ABI5 and a mutation in IBM2. 13–7 nrpd1: F3 progeny that contains an ONSEN insertion in ABI5 and a mutation in NRPD1.