Figure 6.

GUS Gene Silencing Is Post-Transcriptional.

(A) and (B) Photographs of ethidium bromide–stained agarose gels with the products obtained from semiquantitative RT-PCR analysis of nuclear (A) and total (B) RNA from homozygous active (+A) and silenced (−A) AGCNA-61 plants using primers specific for GUS and actin genes. The lanes labeled M contain the 1-kb DNA ladder (Life Technologies) as a size marker. The lane labeled −RT is the negative control in which reverse transcriptase was omitted from the reaction mixture; the lanes labeled 0.1, 0.25, and 0.5 indicate the different amounts of cDNA (in μL) used to cover the appropriate range for the PCR. The photographs had different exposure times and allow comparison within a panel only.

(C) Phosphorimage of slot-blot hybridization results with nascent RNA synthesized by run-on transcription in nuclei isolated from leaves of active hemizygous (HE) and homozygous (HO) AGCNA-61 plants as well as silenced hemizygous (HE) and homozygous (HO) AGCNA-61(R8) plants. The slot-blot filters contain linearized SK+ plasmids with cloned sequences as indicated: GUS, NPTII, HYG (hygromycin), and CAB (chlorophyl a/b binding protein). The empty plasmid pSK+, indicated by SK+, was added as a negative control as well.