Fig. 3

Retrozymes in the genome of the physic nut (Jatropha curcas). a Ethidium bromide staining of an RNA extract (~20 μg) from J. curcas leaves and a 100–1000 bp DNA ladder run in a 5 % native polyacrylamide gel (left) and its corresponding Northern blot hybridization (right) using as a probe a digoxigenin-labelled J. curcas retrozyme fragment of negative polarity. Approximate positions of the bands corresponding to the DNA marker are indicated on the Northern blot. b Northern blot analysis of an RNA extract (~30 μg) from J. curcas leaves carried out as in panel a. Doublet bands can be seen for linear as well as circular RNA molecules. 0.1 ng of a (+)RNA transcript of J. curcas retrozyme was included as a marker. Approximate positions of the bands corresponding to the RNA marker are indicated for clarity. c Northern blot analysis of RNA extracts (~30 μg each) from J. curcas leaves, flowers, seedlings and seeds. Samples were run in a 5 % denaturing polyacrylamide gel and detected as in panel a. The positions of the linear and circular RNAs are indicated. Ethidium bromide staining of the 5S rRNA band is shown at the bottom of panels b and c as a loading control. d Minimum free energy secondary structure prediction for a retrozyme RNA from J. curcas leaves (clone Jc_066, 753 nt). Numbering starts at the self-cleavage site of the HHR. The HHR motif is highlighted with purple letters, whereas the conserved 5′ and 3′ ends of the LTRs are shown in blue and red, respectively, and PPT and PBS regions in cyan and orange, respectively. A region of 45 nt usually absent in smaller variants is shown in green letters. Positions showing sequence heterogeneity among cloned variants from different plant tissues are indicated within circles (triangle corresponds to deleted nucleotides, whereas arrows correspond to inserted nucleotides). e Schematic representation of the circular retrozyme RNA and the position of the oligos used for RT and PCR experiments