CLONING OF A GENE FOR CHLOROPLAST OMEGA-6 DESATURASE OF A GREEN-ALGA,CHLAMYDOMONAS-REINHARDTII

A gene for chloroplast omega 6 desaturase, which catalyzes the desatur ation of monoenoic to dienoic acids in chloroplasts, was isolated from Chlamydomonas reinhardtii. We first performed reverse transcriptase-p olymerase chain reaction with oligonucleotide primers corresponding to regions conserved among plastid omega 6 desaturases of higher plants and Delta 12 desaturases of cyanobacteria, using C. reinhardtii poly(A )(+) RNA, An amplified DNA fragment of 0.5 kb, containing a frame for a protein homologous to these desaturases, was used as a probe for scr eening cDNA and genomic DNA libraries of C. reinhardtii, The cDNA clon e of 2.2 kb obtained contained an open reading frame encoding a protei n of 424 amino acids with a putative molecular mass of 48.4 kDa, the a mino acid sequence of which showed 46-51% homology to those of higher plant plastid omega 6 and cyanobacterial Delta 12 desaturases, Introdu ction of the cloned genomic counterpart of this cDNA, designated as de s6, into a Chlamydomonas mutant with defects in chloroplast omega 6 de saturation and in the activities of photosystems I and II (PSI and PSI I) complemented the desaturation mutation, indicating that the des6 ge ne codes for chloroplast omega 6 desaturase, The complemented strains did not recover from the photosynthetic lesions, but showed lower PSII activity at 45 degrees C than the desaturation mutant, proving that t he photosynthetic lesions in hf-9 are not caused by the desaturation m utation, and that the lowered unsaturation level of chloroplast lipids in the mutant is responsible for the expression at this high temperat ure of PSII activity, one of the thylakoid membrane functions.