Fig. 4.

Detection of polyribosome-associated RNA. Cytoplasmatic extract from hairy root line PO10d was loaded onto a sucrose cushion gradient and RNA was extracted from the resulting polysomal pellet and corresponding supernatant. The RNA panel shows that large and small ribosomal RNAs (28S and 18S) were effectively concentrated in the pellet, indicating the presence of polyribosomes. RNA was used for the reverse transcription reaction and the subsequent PCR was performed with undiluted (+) or 10× diluted [(0.1x)+] cDNA with primers GUS-F and GUS-R (unspliced and spliced transcripts are indicated as U and S, respectively) or act-F and act-R designed for amplification of part of the actin transcript (used as a control). Eventual false-positive results caused by contamination with genomic DNA were excluded in the RT− reaction (–). The result of RT-PCR with total RNA is also presented (tot). The marker is lambda DNA digested with PstI