Figure 4.

RT-PCR experiments detected RNA molecules containing hairpin ribozymes. RT-PCR experiments were conducted that targeted specific regions of a circular molecule that was predicted based on a metatranscriptome contig (see text). The sequences of these RT-PCR products matched the expected sequence perfectly. Other PCR products presumably reflected sequences of related organisms. (A) Sequences corresponding to the strand containing the hairpin ribozyme. The 612-nucleotide circular molecule is depicted with nucleotide position 1 at the 12 o’clock position. In all cases, PCR primers were at most 25 nucleotides long. One confirmed sequence (P1) started at position 152, ran through position 1 and ended at position 72. The other (P2) went from positions 578 to 484, again via the origin position. These sequences overlap and together span the entire circle. (B) Detection of the strand containing the reverse-complement of the hairpin ribozyme. Some coordinates differ from those in part (A) because of differences in primer locations. Two overlapping RT-PCR products were generated (P3, P4).