ABSTRACT
The self-splicing group II intron (rl1) from Scenedesmus obliquus mitochondria together with its 6 bp intron binding site (IBS1) were inserted in the correct and inverse orientation into the chloroplast tscA gene from C.reinhardtii. Precursor RNA derived from the chimeric tscA-rl1 gene can be used to demonstrate in vitro self-splicing of the rl1 intron RNA. Using the particle bombardment technique, the tscA-rl1 construct was transferred into the chloroplast of the unicellular alga Chlamydomonas reinhardtii. We recovered transformants which contain the chimeric tscA-rl1 gene as shown by Southern analysis. Hybridization and PCR analysis of transcripts confirmed that the heterologous intron is correctly spliced in vivo. From sequencing of cDNA clones we conclude that the IBS1 sequence is sufficient for correct splicing of the mitochondrial intron in C. reinhardtii chloroplasts. Using specific probes, we demonstrate by Northern hybridization that the mature RNA, as well as an intron-3' exon intermediate, accumulate in transformants containing the rl1 intron, correctly inserted into the tscA gene. As expected, no RNA splicing at all was observed when the intron had an inverted orientation within the tscA gene. In addition, a mutated intron RNA with an altered 3' terminal nucleotide was tested in vivo. In contrast to similar mutants examined in vitro, this mutated RNA shows accumulated intron and intron-3' exon intermediates, but no ligated exons at all. Our approach should prove useful for elucidating nucleotide residues involved in splicing of organelle introns in vivo.
Subject(s)
Chlamydomonas reinhardtii/genetics , Chlorophyta/genetics , Chloroplasts/chemistry , Introns , RNA Splicing , RNA/genetics , Animals , Base Sequence , Blotting, Southern , Gene Transfer Techniques , Molecular Sequence Data , Mutation , Nucleic Acid Hybridization , Plasmids , Polymerase Chain Reaction , RNA Precursors/genetics , RNA, Mitochondrial , Restriction MappingABSTRACT
We have determined the nucleotide sequence of a 6.9 kbp BamHI-XbaI fragment of broad bean chloroplasts. Part of this fragment (subfragment BglII-ClaI) is known to contain three tRNA genes (trnL-CAA, trnL-UAA and trnF). We have now further identified a gene coding for the third tRNA(Leu) isoacceptor (trnL-UAG) which is located close to trnF. The BamHI-XbaI fragment also contains the gene for subunit 5 of NADH dehydrogenase (ndhF) and two unidentified open reading frames (ORFx and ORF48). ORFx shares a high sequence homology with the long reading frames of tobacco (ORF1708), spinach (ORF2131), and liverwort (ORF2136), while ORF48 shares sequence homology with ORF69 of liverwort and ORF55 of tobacco.