Figure 2.

Determination of the initial cleavage sites in the degradation of psbA mRNA. (A) Characterization of psbA degradation intermediates by high-resolution RNA blot analysis. Lysed chloroplasts from spinach leaves were incubated at 25°C for 180 min, and the RNA was recovered and separated in denaturing polyacrylamide gels, which were electroblotted to nylon membranes and hybridized with a ³²P-radiolabeled oligonucleotide complementary to the 5′ or 3′ end of psbA mRNA. (B) Determination of the initial psbA decay intermediate cleavage sites by primer extension analysis. The 5′ ends of the degradation intermediates were determined by primer extension analysis. Oligonucleotide primers complementary to positions 801–823 (Upper) or complementary to positions 1084–1105 (Lower) of the coding region of psbA mRNA were used. Lanes G, A, T, and C show the sequencing reactions of the corresponding cloned fragments. Nucleotide numbers of reverse transcriptase stops in the psbA gene are indicated. Positions of the three corresponding poly(A)-rich addition sites are given in parentheses.