GFP RNA and protein levels from bidirectional promoter constructs.

<p>(A) Schematic presentation of the GFP reporter constructs. The GFP coding region was inserted between lITR and rITR of DIRS–1 and between opposing actin15 promoters (A15P). As a control, GFP with flanking A15P and A8 terminator (A8T) was used. (B) Analysis of GFP mRNA by Northern blots in the indicated strains. Blots were hybridized with probes against GFP sense mRNA (sense gfp, probe P29) and antisense mRNA (antisense gfp, probe P30). Equal loading was verified by rehybridization of the membrane with a ³²P labelled oligonucleotide directed against actin. Samples for A15P GFP A15P were run on a different gel and hybridized separately (see actin loading control). (C) Representative Northern blot of GFP and DIRS–1 siRNAs in the indicated strains. Equal loading was verified by rehybridization of membrane with a ³²P labelled probe directed against snoRNA DdR-6. (D) Representative Northern blot comparing GFP siRNAs generated from opposing ITRs and opposing A15 promoters. DdR-6 was used as a loading control. (E) Representative Western blot of GFP expression under the control of the left and right ITR in the indicated strains. GFP expression by A15P (¼ was loaded to avoid overexposure of the GFP signal) was used as a positive, the untransformed Ax2 as a negative control. CorA was used as a loading control. Quantification of the GFP intensity was performed using ImageJ. GFP expression by A15P was set to 1 (right panel). Ax2 lITR GFP rITR: n = 2. agnA- lITR GFP rITR: n = 4. Error bars: mean with SD. (F) Comparison of GFP silencing by opposing ITRs and A15Ps. GFP expression by A15P (¼ was loaded to avoid overexposure of the GFP signal) was used as a positive control.