A small chloroplast RNA may be required for trans-splicing in Chlamydomonas reinhardtii.

In C. reinhardtii, the mature psaA mRNA is assembled by a process involving trans-splicing of three separate transcripts encoded at three widely scattered loci of the chloroplast genome. At least one additional chloroplast locus (tscA) is required for trans-splicing of exons 1 and 2. We have mapped this gene by transformation of a deletion mutant with a particle gun. The 0.7 kb region of the chloroplast genome that is sufficient to rescue tscA function has been subjected to insertion mutagenesis, showing that it does not contain significant open reading frames. We suggest from these experiments that the product of the tscA gene may be a small chloroplast RNA that acts in trans in the first trans-splicing reaction of psaA. A model for the mode of action of this RNA is presented, in which the characteristic structure of group II introns is assembled from three separate transcripts.

Nuclear and chloroplast mutations affect the synthesis or stability of the chloroplast psbC gene product in Chlamydomonas reinhardtii.

The psbC gene of Chlamydomonas reinhardtii encodes P6, the 43 kd photosystem II core polypeptide. The sequence of P6 is highly homologous to the corresponding protein in higher plants with the exception of the N-terminal region where the first 12 amino acids are missing. Translation of P6 is initiated at GUG in C. reinhardtii. The chloroplast mutant MA16 produces a highly unstable P6 protein. The mutation in this strain maps near the middle of the psbC gene and consists of a 6 bp duplication that creates a Ser-Leu repeat at the end of one transmembrane domain. Two nuclear mutants, F34 and F64, and one chloroplast mutant, FuD34, are unable to synthesize P6. All of these mutants accumulate wild-type levels of psbC mRNA. The FuD34 mutation has been localized near the middle of the 550 bp 5' untranslated region of psbC where the RNA can be folded into a stem-loop structure. A chloroplast suppressor of F34 has been isolated that partially restores synthesis of the 43 kd protein. The mutation of this suppressor is near that of FuD34, in the same stem-loop region. These chloroplast mutations appear to define the target site of a nuclear factor that is involved in P6 translation.

Analysis of the genes of the OEE1 and OEE3 proteins of the photosystem II complex from Chlamydomonas reinhardtii.

The sequences of the nuclear genes of the 33 kDa (OEE1) and the 16 kDa (OEE3) polypeptides of the oxygen evolving complex of Chlamydomonas reinhardtii have been established. Comparison between the OEE1 protein sequences of C. reinhardtii and higher plants and cyanobacteria reveals 67 and 47% homology. In contrast, C. reinhardtii and higher plants have only 28% overall homology for OEE3 which is mostly limited to the central portion of the protein. The transit peptides of the C. reinhardtii proteins consist of 52 (OEE1) and, most likely, 51 (OEE1) amino acids. They have a basic amino terminal region and, at least in the case of OEE1, a hydrophobic segment at their carboxy terminal end typical of thylakoid lumen proteins. Comparison of the genomic and cDNA clones indicates that the OEE1 and OEE3 genes contain five and four introns, respectively, some of which are located within the coding sequences of the transit peptides.

A transposon with an unusual arrangement of long terminal repeats in the green alga Chlamydomonas reinhardtii.

We have isolated a 5.7-kbp dispersed moderately repeated DNA sequence (TOC1) from the mutant OEE1 gene of the Chlamydomonas reinhardtii strain FUD44. The copy number (2 to over 30) and genomic locations of TOC1 elements vary widely in different C. reinhardtii strains. Our standard laboratory photosynthetic strain exhibits a high degree of TOC1 instability during short periods of mitotic growth. TOC1 appears to be a retrotransposon: it contains LTRs and an oligonucleotide stretch that corresponds to a conserved pentapeptide of reverse transcriptase. TOC1 is an unusual retrotransposon: it is not flanked by a target site duplication in the OEE1 gene, the left end of TOC1 only contains a fraction of the LTR the remainder of which is present at its right end and TOC1 does not start with a 5' TG and end with a 3' CA. In most cases, TOC1 excision leaves behind a complete solo LTR sequence (577 bp) and in one case a deleted solo LTR sequence (191 bp). Solo LTR sequences form a separate family of repeated sequences in most of the strains tested.