Fig. 8. CRS2 co-sediments with group II introns during sucrose gradient centrifugation of chloroplast stroma. (A) Immunoblot showing the distribution of CRS2 in sucrose gradient fractions. Stromal extract (2 mg protein) was sedimented through a sucrose gradient. Protein from each gradient fraction was analyzed on immunoblots by probing with the CRS2 antibody. Twenty micrograms of stromal protein (str) were included for reference. (B) RNA gel blots showing distribution of intron RNA in sucrose gradient fractions. RNA gel blots of RNA purified from the same gradient fractions analyzed in (A) were hybridized with radiolabeled probes specific for ndhB, petB or atpF intron. The remaining fractions (1–7 and 18–30) did not contain significant amounts of CRS2 or intron RNA (not shown). The intron RNAs detected migrated with RNA markers of 700–800 nt, the size of the excised introns. Although unspliced pre-mRNAs accumulate to high levels in chloroplasts (Jenkins et al., 1997), no other transcripts were detected with these probes in any gradient fraction (data not shown), presumably due to partial degradation of unspliced mRNA precursors during this experimental protocol.