J. Mol. Biol. (1991) 218, 293-311 Six Group I Introns and Three Internal Transcribed Spacers in the Chloroplast Large Subunit Ribosomal RNA Gene of the Green Alga Chlamydomonas eugametos Monique Turmel’, Jean Boulangerl, Murray N. Schnare’, Michael W. Gray2 and Claude Lemieuxl 1 Dkpartement de biochimie FacultC des sciences et de ge’nie Universite’ Lava1 Q&bee (Que’bec) GIK 7P4, Canada ‘Department of Biochemistry Dalhousie University Halifax (Nova Scotia) B3H 4H7, Canada (Received 9 July 1990; accepted 6 November 1990) The chloroplast large subunit rRNA gene of Chlamydomonas eugametos and its 5’ flanking region encoding tRNA”” (GAU) and tRNA*‘” (UGC) have been sequenced. The DNA sequence data along with the results of a detailed RNA analysis disclosed two unusual features of this green algal large subunit rRNA gene: (1) the presence of six group I introns (CeLSU .l CeLSU .6) whose insertion positions have not been described previously, and (2) the presence of three short internal transcribed spacers that are post-transcriptionally excised to yield four rRNA species of 280, 52, 810 and 1720 nucleotides, positioned in this order (5’ to 3’) in the primary transcript. Together, these RNA species can assume a secondary structure that is almost identical to that proposed for the 23 S rRNA of Escherichia coli. All three internal transcribed spacers map to variable regions of primary sequence and/or potential secondary structure, whereas all six introns lie within highly conserved regions. The first three introns are inserted within the sequence encoding the 810 nucleotide rRNA species and map within domain II of the large subunit rRNA structure; the remaining introns, found in the sequence encoding the 1720 nucleotide rRNA species, lie within either domain IV or V, as is the case for all other large subunit rDNA introns that have been documented to date. CeLSU .5 and CeLSU .6 each contain a long open reading frame (ORF) of more than 200 codons. While the CeLSU* 6 ORF is not related to any known ORFs, the CeLSU. 5 ORF belongs to a family of ORFs that have been identified in Podospora and Neurospora mitochondrial group I introns. The finding that a polymorphic marker showing unidirectional gene conversion during crosses between C. eugametos and Chlamydomonas moewusii is located within the CeLSU.5 ORF makes it likely that this intron is a mobile element and that its ORF encodes a site-specific endonuclease promoting the transfer of the intron DNA sequence. 1. Introduction RNA intermediate (Michel & Lang, 1985). A Introns in organellar genes can be classified into number of group I introns propagate themselves group I or group II on the basis of several very efficiently by insertion into intron-less alleles conserved sequences that allow the corresponding through site-specific recombination. The first mobile RNA within each group to form a characteristic group I intron that was discovered is the secondary structure required for splicing (Michel & well-characterized rl (or omega) intron in the LSUt Dujon, 1983). Although the origin of these introns remains a debated question, evidence suggests that they evolved from or into mobile elements t Abbreviations used: LSU, large subunit; SSU, small (Lambowitz, 1989). Some group II introns encode subunit; AMV, avian myeloblastosis virus; bp, base- putative reverse transcriptases that are thought to pair(s); cpDKA, chloroplast DNA; ITS, internal transcribed spacer; kb, lo3 base-pairs; nt; nucleotide(s); promote insertion of intron DNA sequences via an ORF, open reading frame. 293 0022-2836/91/060293-19 $03.00/O 0 1991 Academic Press Limited