Consequences of T‐DNA insertion on SWP expression in swp mutant.

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Consequences of T‐DNA insertion on SWP expression in swp mutant. Consequences of T‐DNA insertion on SWP expression in swp mutant. (A) Northern blot analysis of SWP expression in the swp mutant showing overproduction of aberrant transcripts in swp. A northern blot with ∼2 μg of poly(A)+ RNA from swp homozygote plants (swp) or wild‐type plants (ws) was hybridized with a SWP RNA‐specific probe corresponding to the 3′ end of the cDNA (represented in C). RNA size was estimated using a RNA ladder run on the same gel. (B) RT–PCR experiment showing fusion of SWP mRNA with T‐DNA left border region in swp mutant. Total RNA from swp plants (swp) or wild‐type plants (ws) were reverse transcribed and amplified using primers designed to the 5′ end of the SWP cDNA (swpUp) and to the T‐DNA left border (LB1) (represented in C). A 0.7 kb fragment could be amplified from swp RNA (lane 4) and not from WS RNA (lane 3). Amplification using primers from the SWP cDNA only (swpUp/swpLow; see C) provides the loading control (lanes 1 and 2). SWP1 and SWP2 primers are located downstream and upstream of the first intron of the SWP gene, respectively (see C), excluding genomic contamination of the reverse transcribed RNA samples, which would have led to an 1 kb amplification product as shown in lane 6. (C) Schematic representation of the T‐DNA insertion site in the swp mutant line. The T‐DNA caused a 6 bp deletion at the predicted transcription start site of the SWP gene (arrow, WT transcription start). In swp, initiation of transcription may occur at upstream functional sites existing within the T‐DNA and could be driven by the cauliflower mosaic virus promoter (35S) as suggested by the high expression level detected in swp. The sequence of the region of fusion between the SWP mRNA and T‐DNA left border (LB) is given. In the T‐DNA LB sequence (underlined), several putative start codons (bold letters) are detected that could compete with the wild‐type SWP start codon, potentially leading to transcriptional attenuation in swp mutant, as suggested by the recessive nature of the swp mutation. bar, basta (ammonium glufosinate) herbicide resistance gene; tnos, nos gene terminator. Daphné Autran et al. EMBO J. 2002;21:6036-6049 © as stated in the article, figure or figure legend