Abstract
Targeting mutations to specific genomic loci is invaluable for assessing in vivo the effect of these changes on the biological role of the gene in study. Here, we attempted to introduce a mutation that was previously implicated in an increased heat stability of the mesophilic cyanobacterium Synechocystis sp. PCC6803 via homologous recombination to the psbA gene of Chlamydomonas reinhardtii. For that, we established a strategy for targeted mutagenesis that was derived from the efficient genome-wide homologous-recombination-based methodology that was used to target individual genes of Saccharomyces cerevisiae. While the isolated mutants did not show any benefit under elevated temperature conditions, the new strategy proved to be efficient for C. reinhardtii even in the absence of direct positive selection.
Highlights
The unicellular green alga Chlamydomonas reinhardtii has served as a primary platform for studies of photosynthesis in eukaryotes[1,2,3], making it a favorable target for studying the phenotypic consequences of the mutations in the D1 protein
We wished to determine whether the replacement of serine at positions 209 and 212 of C. reinhardtii’s D1 protein with alanine and cysteine, respectively, would promote thermophilic behavior of cell cultures
As the two serine residues are located in exon 4 of the psbA gene of C. reinhardtii (Fig. 1a), we decided on an experimental approach that will introduce the two mutations simultaneously by targeted replacement of exon 4 of the psbA gene of C. reinhardtii 2137a plastome (Fig. 1b)
Summary
The unicellular green alga Chlamydomonas reinhardtii has served as a primary platform for studies of photosynthesis in eukaryotes[1,2,3], making it a favorable target for studying the phenotypic consequences of the mutations in the D1 protein. Modified or heterologous gene sequences that are flanked by plastome homologous DNA sequences are introduced into the plastome by the particle bombardment methodology This approach was used to restore photosynthetic competence of the non-photosynthetic mutant of C. reinhardtii[6], or to confer herbicide resistance to the D1 protein[9]. PCR-amplified DNA fragments with limited flanking homologous sequences proved sufficient for complementing a psbA-deletion mutant of C. reinhardtii and restoring autotrophic growth[15]. This raised the possibility of adapting to C. reinhardtii the highly efficient gene targeting protocol, in which PCR-amplified linear DNA fragments were used to tag each known open reading frame in S. cerevisiae with high-affinity epitope[16]. Co-transformation with amplified fragments of psbA gene allowed fast and effective selection of mutants in the absence of positive selection
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