Construction and characterization of autonomously replicating plasmids in the green unicellular alga Chlamydomonas reinhardii

Abstract

Plasmids that replicate autonomously in Chlamydomonas reinhardii were constructed by inserting random DNA fragments from this alga into a plasmid containing the yeast ARG4 locus. Arginine prototrophy was used as a selective marker. The presence of free plasmids in the DNA of the transformants was demonstrated by hybridization with a specific plasmid probe and by recovering these plasmids in E. coli after transformation. Four of them were characterized. Their inserts of 415, 257, 153, and 102 bp all hybridize to chloroplast DNA and were localized on the physical map of the chloroplast genome. One of these plasmids also promotes autonomous replication in yeast. Sequence analysis of the inserts of the plasmids reveals several short direct and inverted repeats and two semiconserved AT-rich elements of 19 and 12 bp that may play a role in promoting autonomous replication in C. reinhardii.