Open AccessIdentification ofin vivoprocessing intermediates and of splice junctions of tRNAs from maize chloroplasts by amplification with the polymerase chain reaction
Author(s) -
Gabriele Delp,
Gabor L. Igloi,
Hans Kössel
Publication year - 1991
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/19.4.713
Subject(s) - biology , transfer rna , intron , rna splicing , splice , rnase p , gene , cleavage (geology) , microbiology and biotechnology , chloroplast , genetics , exon , polymerase chain reaction , ribosomal rna , primer (cosmetics) , rna , biochemistry , chemistry , fracture (geology) , paleontology , organic chemistry
Total RNA from chloroplasts of maize seedlings was used for polymerase chain reaction (PCR) mediated amplification of tRNA precursors and of mature tRNAs encoded by the two split tRNA genes of the ribosomal spacer (tRNA(lle)GAU and tRNA(Ala)UGC) and the single intron-containing tRNA(Gly)UCC 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 amplification 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 endonucleolytic cleavage by RNase P occurs as one of the final steps in the tRNA processing pathway of which the endonucleolytic cleavage at the 3' side probably occurs prior to the splicing of the intron sequences.