Chlamydomonas Reinhardii, A Potential Model System for Chloroplast Gene Manipulation
Studies on the structure, function and regulation of genes coding for chloroplast proteins are important for understanding the biosynthesis of the photosynthetic apparatus and the integration of chloroplasts within plant cells. Chlamydomonas reinhardii is particularly well suited for solving these problems because this green unicellular alga can be manipulated with ease both at the biochemical and genetic level. Several genes have been identified on the physical map of the chloroplast genome. They include genes coding for ribosomal RNA, tRNA and several proteins including the large subunit of ribulose 1,5 bisphosphate carboxylase (RubisCo) and several thylakoid polypeptides. The nuclear gene for the small subunit of RubisCo has also been cloned. Because chloroplast DNA recombination occurs in C. reinhardii, a rare property among plants, chloroplast genes can be analyzed by genetic means. Numerous chloroplast photosynthetic mutations have been isolated and several of them have been shown to be part of a single linkage group (Gillham, 1978). We have reached the stage where the genetic and biochemical approaches can be coupled efficiently in C. reinhardii; in particular, it has been possible to correlate the physical and genetic chloroplast DNA maps at a few sites.
A nuclear transformation system has been developed for C. reinhardii by using a cell wall deficient arginine auxotroph which can be complemented with a plasmid containing the yeast ARG4 locus. Transformation vectors have been constructed by inserting random nuclear and chloroplast DNA fragments into a plasmid containing the yeast ARG4 locus and by testing the recombinant plasmids for their ability to promote autonomous replication in yeast (ARS sites) and C. reinhardii (ARC sites). Several plasmids have been recovered that act as shuttle vectors between E. coli, C. reinhardii and yeast. Four ARS sites and four ARC sites have been mapped on the chloroplast genome of C. reinhardii. One plasmid replicates both in C. reinhardii and yeast. Because C. reinhardii cells contain a single large chloroplast they offer interesting possibilities for attempts of chloroplast transformation by microinjection. Since appropriate selective markers and transformation vectors are available, this approach can now be explored.
KeywordsRecombination Polypeptide Arginine Ampicillin Tetracycline
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