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.

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We have recently isolated and characterized several flower-specific clones from a sunflower cDNA library [ 1]. Two of these clones, SF2 and SF18, were shown to hybridize exclusively to RNA from anthers in the late developmental stages (none of the two RNA species is detected in anthers from a closed sunflower inflorescence). The two mRNAs, which are abundant RNAs in anthers from male-fertile plants, are barely detectable in anthers from male-sterile plants. In situ hybridization studies using the labeled SF2 cDNA clone as probe have shown that the SF2 mRNA is present only in the epidermal anther cells (Evrard et al., submitted). The lack of crosshybridization between the two cDNAs suggested that the SF2 and SF18 mRNAs code for different proteins.We describe here the nucleotide sequence of the two cDNAs SF2 and SF18. The sequence analysis revealed a size of 653 bp for SF2 cDNA and 656 bp for SF18 cDNA (Fig. 1); this is a little smaller than that of the corresponding mRNAs estimated in northern experiments (800 nucleotides for SF2 and 750 nucleotides for SF18) and indicates that the two cDNAs are incomplete. Translation of the cDNA sequence of SF2 revealed an open reading frame starting with a methionine and extending over 121 amino acids. That translation must start at this specific methionine residue in vivo has recently been confirmed by the study of the corresponding gene (C. Domon, unpublished). SF18 cDNA carries a 161 amino acid-long reading frame, which is incomplete at the amino terminus. The first 8 amino acids of this incomplete SF18 reading frame are also found in the amino terminal region of SF2 (only one valine substituting for a leucine). In SF2, this region carries a typical signal peptide sequence (21 amino acids, 16 of which are hydrophobic).Not only are the SF2 and SF18 mRNAs similar in size, they also share short segments of significant sequence homology: one of these is found in the region coding for the signal peptide (where 25 out of 28 nucleotides are identical); another one is found in the untranslated 3' region which contains the polyadenylation site (AATAAA) and a short poly(A) stretch (21 A residues in SF2 and 18 A residues in SF18). This region is considered to be involved in mRNA stability [2].Although no additional sequence similarity can be detected in the SF2 and SF18 polypeptides (except for the signal peptide sequence), they share some features suggesting that they are functionally related. One of these features is a pattern of 7 cysteine residues with a nearly identical spacing in the amino moiety of the two mature polypeptides. This 'cysteine domain' (Fig. 2A), is alsoxich in charged amino acids (35 ~o in SF2 andThe nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X53374 (SF2) and X53375 (SF18).

No abstract

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.