Complementation of the Chlamydomonas reinhardtii arg7-8 (arg2) Point Mutation by Recombination with a Truncated Nonfunctional ARG7 Gene

Chlamydomonas reinhardtii arg7-8 (arg2) mutant strains carrying a hitherto undescribed mutation in their argininosuccinate lyase gene (ARG7) that leads to arginine auxotrophy have been used together with the corresponding wild-type gene as a very reliable transformation system since 1989. In this study, we finally identify the molecular nature of the arg7-8 mutation as a 6073G to A transition in exon 9 of ARG7 leading to a 288Gly to Ser exchange near the active site of the protein. The same mutation was found in the ARG7 genes of three commonly used C. reinhardtii laboratory strains, namely cw15-302 arg2, CC-48, and CC-1618. We did not observe exact spontaneous reversion of the arg7-8 allele in our study, but did identify two different and rare intragenic suppressor mutations, 27Leu to Phe and 285Tyr to Phe. In our hands, only transformation of the arg7-8 strain with a truncated nonfunctional wild-type ARG7 gene lacking 124 codons at its 5′ end led to exact reversion of the mutant base 6073A to the wild-type 6073G, presumably by recombination. This system offers a positive selection scheme for homologous recombination (HR) and may, therefore, be useful to the methodical improvement of recombination in Chlamydomonas.