Multiple Modification of Chromosomal Loci Using URA5.3 Selection Marker in the Unicellular Red Alga Cyanidioschyzon merolae.

The unicellular red alga Cyanidioschyzon merolae has been used as a eukaryotic photosynthetic model for various basic and applied studies. Although the nuclear genome of C. merolae can be modified by homologous recombination with exogenously introduced DNA, it has been difficult to modify multiple chromosome loci within the same strain because of the limited number of available positive selection markers. Recently, we reported a modified URA5.3 gene cassette (URA5.3T), which can be used repeatedly for nuclear genome transformation using the pMKT plasmid vectors for epitope tagging (3x FLAG- or 3x Myc-) of nuclear-encoded proteins. In addition, these plasmid vectors can also be used to knock out multiple genes one by one. This report describes the construction of DNA fragments for transformation and the detailed transformation procedure.