In organelles, the posttranscriptional steps of gene expression are tightly controlled by nucleus-encoded factors, most often acting in a gene-specific manner. Despite the molecular identification of a growing number of factors, their mode of action remains largely unknown. In the green alga Chlamydomonas reinhardtii, expression of the chloroplast petA gene, which codes for cytochrome f, depends on two specific nucleus-encoded factors. MCA1 controls the accumulation of the transcript, while TCA1 is required for its translation. We report here the cloning of MCA1, the first pentatricopeptide repeat protein functionally identified in this organism. By chloroplast transformation with modified petA genes, we investigated the function of MCA1 in vivo. We demonstrate that MCA1 acts on the very first 21 nucleotides of the petA 5 untranslated region to protect the whole transcript from 533 degradation but does not process the 5 end of the petA mRNA. MCA1 and TCA1 recognize adjacent targets and probably interact together for efficient expression of petA mRNA. MCA1, although not strictly required for translation, shows features of a translational enhancer, presumably by assisting the binding of TCA1 to its own target. Conversely, TCA1 participates to the full stabilization of the transcript through its interaction with MCA1.Organelle genomes have retained a limited set of genes from their prokaryotic ancestor, the expression of which is tightly controlled by the nucleus. Within organelles, mRNAs may undergo cis or trans splicing, editing, endo-and exonucleolytic cleavage, and 5Ј-and 3Ј-end processing. Their stabilization, translation, and degradation are highly regulated (reviewed in references 3, 5, 23, 26, and 63). Each of these posttranscriptional steps depends on nucleus-encoded factors. Strikingly, most are gene specific, one factor being required for the expression of one or a few organelle mRNAs. Altogether, several hundreds of nucleus-encoded factors may be required for the proper expression of the organelle genome (3, 65).The green alga Chlamydomonas reinhardtii and the yeast Saccharomyces cerevisiae have been instrumental in the identification of these factors. Genetic analyses of nuclear mutants, defective for the expression of single organelle genes, have emphasized the existence of two major classes of trans-acting factors. Some are required for the proper maturation and stabilization of specific organellar transcripts (reviewed in references 3, 44, and 48), while others are required for their translation (reviewed in references 3, 23, and 78). Among the numerous nucleus-encoded factors involved in the stabilization of specific chloroplast transcripts identified in Chlamydomonas (14,16,28,33,35,36,39,45,66), only three have been cloned up to now-NAC2, MCD1, and MBB1-that, respectively, control psbD, petD, and psbB transcript stability (6,46,71). They act on the 5Ј untranslated regions (5ЈUTRs) of their target transcripts and protect them from 5Ј33Ј ribonucleolytic degradation (14,50,71), but the molecular bas...
The introduction of exogenous DNA into the nuclear genome of Chlamydomonas reinhardtii occurs predominantly via non-homologous (illegitimate) recombination and results in integration at apparently-random loci. Using truncated and modified versions of the C. reinhardtii ARG7 gene in a series of transformation experiments, we demonstrate that homologous recombination between introduced DNA molecules occurs readily in C. reinhardtii, requires a region of homology of no more than 230 bp, and gives rise to intact copies of ARG7 in the nuclear genome. Evidence is presented for homologous recombination between introduced ARG7 DNA and the resident copy of the gene, and for the de-novo synthesis of the ARG7 sequence during transformation.
The random integration of transforming DNA into the nuclear genome of Chlamydomonas has been employed as an insertional mutagen to generate a collection of photosynthetic mutants that display abnormal steady-state fluorescence levels and an acetate-requiring phenotype. Electron paramagnetic resonance spectroscopy was then used to identify those mutants that specifically lack a functional cytochrome b6f complex. Our analysis of RNA and protein synthesis in five of these mutants reveals four separate phenotypes. One mutant fails to accumulate transcript for cytochrome f, whilst a second displays a severely reduced accumulation of the cytochrome b6 transcript. Two other mutants appear to be affected in the insertion of the haem co-factor into cytochrome b6. The fifth mutant displays no detectable defect in the synthesis of any of the known subunits of the complex. Genetic analysis of the mutants demonstrates that in three cases, the mutant phenotype co-segregates with the introduced DNA. For the mutant affected in the accumulation of the cytochrome f transcript, we have used the introduced DNA as a tag to isolate the wild-type version of the affected gene.
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