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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 nystatin. Sterols were isolated by precipitation with digitonin, fractionated by both normal and argentation TLC, and then analysed by GLC and GC-MS. All the mutants accumulated ergosta-5,7,22,24(28)-tetraenol, ergosta-5,7,24(28)-trienol, ergosta-7,24(28)-dienol, stigmasta-5,7,22,24(28)-tetraenol, stigmasta-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 major 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 corresponding 24-alkyl sterol. There is strong evidence that the same enzyme acts on both the C28 and C29 sterol series. This view is also supported by Southern blot hybridisation analysis revealing that in all three mutants, plasmid insertion occurred at the same site indicating the disruption of the same gene. Due to the insertional nature of the mutations, the strains can be used for cloning the corresponding gene. |
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