Sterol mutants of Chlamydomonas reinhardtii: Characterisation of three strains deficient in C24(28) reductase |
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| Institution: | 1. Department of Genetics, State University of St. Petersburg, Universitetskaya nab. 7/9, St. Petersburg 199034, Russia;2. Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012, Bern, Switzerland;1. Department of Genomic Science, Central University of Kerala, Kasaragod, India;2. Department of Biochemistry and Molecular Biology, Central University of Kerala, Kasaragod, India;1. Key Laboratory of Poyang Lake Ecology and Bio-Resource Utilization of Ministry of Education, Nanchang University, Nanchang 330031, China;2. Department of Chemical Engineering, Nanchang University, Nanchang 330031, China;3. School of Foreign Language, Nanchang University, Nanchang 330031, China;1. School of Software, Xiamen University, Xiamen, China;2. School of Information Science and Technology, Xiamen University, Xiamen, China;1. College of Pharmacy, Drug Research and Development Center, Daegu Catholic University, Gyeongbuk 38430, Republic of Korea;2. College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Republic of Korea;3. Division of Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea |
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| Abstract: | 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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