Sterol mutants of Chlamydomonas reinhardtii: Characterisation of three strains deficient in C24(28) reductase

Three mutants of Chlamydomonas reinhardtii (strain arg7cw15) were obtained using the strategy of insertional mutagenesis by random plasmid integration with subsequent selection for resistance against the polyene antibiotic ny statin. Sterols were isolated by precipitation with digitonin, fractionated by both normal and argentation TLC, and then analysed by GLC and GC-MS. Al l the mutants accumulated ergosta-5,7,22,24(28)-tetraenol, ergosta-5,7,24(2 8)-trienol, ergosta-7,24(28)-dienol, stigmasta-5,7,22,24(28)-tetraenol, sti gmasta-5,7,24(28)-trienol, stigmasta-8,24(28)-dienol and stigmasta-7,24(28) -dienol, while ergosterol and 7-dehydroporiferasterol which are the only ma jor sterol components of the original strain were absent in the mutants. It is concluded that all these mutants are impaired in this C24(28) reductase which catalyses the reduction of the C24(28) tetraenol to the correspondin g 24-alkyl sterol. There is strong evidence that the same enzyme acts on bo th the C-28 and C-29 sterol series. This view is also supported by Southern blot hybridisation analysis revealing that in all three mutants, plasmid i nsertion occurred at the same site indicating the disruption of the same ge ne. Due to the insertional nature of the mutations, the strains can be used for cloning the corresponding gene. (C) Elsevier, Paris.