A ROBUST PROTOCOL FOR SITE-DIRECTED MUTAGENESIS OF THE D1 PROTEIN IN CHLAMYDOMONAS-REINHARDTII - A PCR-SPLICED PSBA GENE IN A PLASMID CONFERRING SPECTINOMYCIN RESISTANCE WAS INTRODUCED INTO A PSBA DELETION STRAIN

In this paper, we describe a protocol to obtain a site-directed mutant s in the psbA gene of Chlamydomonas reinhardtii, which overcomes sever al drawbacks of previous protocols, and makes it possible to generate a mutant within a month. Since the large size of the gene, and the pre sence of four large introns has made molecular genetics of the psbA ge ne rather unwieldy, we have spliced all of the exons of the psbA gene by PCR to facilitate genetic manipulation and sequencing of the gene. The resultant construct (plasmid pBA153, with several unique restricti on sites introduced at exon boundaries) carried 1.2 and 1.8 kb intact sequences from the 5'- and 3'-flanking regions, respectively. The plas mid was used to transform a D1-deletion mutant and was found to comple ment the deletion and restore photosynthetic activity. In addition, a bacterial aadA gene conferring spectinomycin resistance (spe(r)) was i nserted downstream of the intron-free psbA gene, to give construct pBA 155. This allowed selection of mutant strains deficient in photosynthe sis by using spectinomycin resistance, and eliminated the possibility of selection for revertant strains which is a consequence of having to use photosynthetic activity as a selection pressure. Finally, pBA155 was used to construct pBA157, in which additional restriction sites we re inserted to facilitate cassette mutagenesis for generation of mutat ions in spans thought to be involved in donor-side interactions. All p sbA deletion strains transformed with intron-free psbA-aadA constructs encoding the wild-type D1 sequence, and screened on spectinomycin pla tes for the spe(r) phenotype, were able to grow photosynthetically, an d all showed identical kinetics for electron transfer from primary (Q( A)) to secondary quinone (Q(B)) in Photosystem II, as assayed by the d ecay of the high fluorescence yield on oxidation of the reduced primar y acceptor (Q(A)(-)).