Nucleic Acids Research, 1991, Vol. 19, No. 4 713-716
© 1991
MOLECULAR BIOLOGY |
Identification of in vivo processing intermediates and of splice junctions of tRNAs from maize chloroplasts by amplification with the polymerase chain reaction
Institut für Biologie III, Universität Freiburg Schänzle-Straße 1, D-7800 Freiburg, FRG
* To whom correspondence should be addressed
Received December 18, 1990. Accepted January 21, 1991.
Total RNA from chloroplasts of maize seedlings was used for polymerase chain reaction (PCR) mediated amplification of tRNA precursors and of mature tRNAs encaded by the two split tRNA genes of the ribosomal spacer (tRNAlleGAU and tRNAAlaUGC) and the single intron-containing tRNAGlyUCC gene. Sequence analysis of DNAs amplified from the mature tRNAs by combinations of exon specific primers allows unambiguous identification of the respective splice junctions. Primer combinations In which 5'- or 3'-flanking precursor tRNA sequences are included, leads to the ampitfication of processing intermediates in which 5'-terminal extensions are still present, whereas no PCR products corresponding to 3'-terminal extensions could be detected. From this it is concluded that in chloroplasts the 5'-terminal endonucieoiytic cleavage by RNase P occurs as one of the final steps in the tRNA processing pathway of which the endonucleoiytic cleavage at the 3' side probably occurs prior to the splicing of the intron sequences.