Summary
A novel and efficient genetic procedure is described for generating mitochondrial mutants of the green alga Chlamydomonas reinhardtii. The development of a mutagenesis procedure using manganese cations and the application of cytoduction techniques resulted in a combined approach for the generation and analysis of mitochondrial mutants. Although mitochondrial mutations are inherited in sexual crosses from the minus mating type parent, the cytoduction technique can be used to transfer mitochondrial mutations into recipient strains with different genetic backgrounds, irrespective of their mating type. Cytoduction allows the transfer of mitochondrial markers from diploid to haploid cells also, which is of great benefit since diploid cells do not germinate in C. reinhardtii. We report here the isolation and characterisation of eight mutants, which are resistant to the antibiotics myxothiazol and mucidin. The mutants all have point mutations in the mitochondrial gene for apocytochrome b. Using in vitro-amplified cytb gene fragments as probes for direct DNA sequencing, three different types of single base pair substitutions were revealed in all mutants tested. In particular, amino acid substitutions in the mutant apocytochrome b polypeptide have been identified at residues 129, 132 and 137, which have been implicated in forming part of an antibiotic-binding niche. The amino acid substitution at position 132 has not been so far described for mutant apocytochrome b in any other organism, prokaryotic or eukaryotic. The genetic approach presented here confirms C. reinhardtii as a model system that is unique among plant cells.
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References
Bennoun P, Chua NH (1976) Methods for detection and characterization of photosynthetic mutants in Chlamydomonas reinhardtii. In: Bücher T, Neupert W, Sebald W, Werner S (eds) Genetics and biogenesis of chloroplasts and mitochondria. Elsevier/North-Holland, Amsterdam, pp 33–39
Bennoun P, Delepelaire P (1982) Isolation of photosynthesis mutants in Chlamydomonas. In: Edelman M, Chua N-H, Hallick RB (eds) Methods in chloroplast molecular biology. Elsevier/North-Holland, Amsterdam, pp 25–38
Bennoun P, Levine RP (1969) Detecting mutants that have impaired photosynthesis by their increased level of fluorescence. Plant Physiol 42:1284–1287
Bennoun P, Masson A, Piccioni R, Chua NH (1978) Uniparental mutants of Chlamydomonas reinhardtii defective in photosynthesis. In: Akoyunoglou G, Argyroudi-Akoyunoglou J (eds) Chloroplast development. Elsevier/North-Holland, Amsterdam, pp 721–726
Bennoun P, Delosme M, Kück U (1991) Mitochondrial genetics of Chlamydomonas reinhardtii: Resistance mutations marking the cytochrome b gene. Genetics 127:335–343
Boer PH, Gray MW (1988) Scrambled ribosomal RNA gene pieces in Chlamydomonas reinhardtii mitochondrial DNA. Cell 59:399–411
Coen D, Deutsch J, Netter P, Petrochili E, Slonimski PP (1970) Mitochondrial genetics. I. Methodology and phenomenology. In: Miller HP (ed) Control of organelle development. Soc Exp Biol Symp vol 24. Cambridge University Press, London, pp 449–496
Colson AM, Trumpower BL, Brasseur R (1990) Insight into cytochrome b structure-function relationships by the combined approaches of genetics and computer modeling. In: Brasseur R (ed) Molecular description of biological membrane components by computer aided conformational analysis, vol II. CRC Press, Boca Raton, pp 147–159
Conde J, Fink GR (1976) A mutant of Saccharomyces cerevisiae defective for nuclear fusion. Proc Natl Acad Sci USA 73:3651–3655
Daldal F, Tokito MK, Davidson E, Faham M (1989) Mutations conferring resistance to quinol oxidation (Qz) inhibitors of the cytbc 1 complex of Rhodobacter capsulatus. EMBO J 8:3951–3961
Girard J, Chua NH, Bennoun P, Schmidt G, Delosme M (1980) Studies on mutants deficient in the photosystem I reaction centers in Chlamydomonas reinhardtii. Curr Genet 2:215–221
Gorman DS, Levine RP (1965) Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 54:1665–1669
Harris E (1989) The Chlamydomonas sourcebook. Academic Press, New York
Howell N, Gilbert K (1988) Mutational analysis of the mouse mitochondrial cytochrome b gene. J Mol Biol 203:607–618
Innis MA, Gelfand DH (1990) Optimization of PCRs. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols. A guide to methods and applications. Academic Press, San Diego New York Boston London Sydney Tokyo Toronto, pp 3–12
Kück U (1989) The intron of a plastid gene from a green alga contains an open reading frame for a reverse transcriptase-like enzyme. Mol Gen Genet 218:257–266
Loppes R (1969) A new class of arginine-requiring mutants in Chlamydomonas reinhardtii. Mol Gen Genet 104:172–177
Matagne R, Remacle C, Dinant M (1991) Cytoduction in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 88:7447–7450
McCabe PC (1990) Production of single-stranded DNA by asymmetric PCR. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols. A guide to methods and applications. Academic Press, San Diego New York Boston London Sydney Tokyo Toronto, pp 76–83
Mets LJ, Bogorad L (1971) Mendelian and uniparental alterations in erythromycin binding by plastid ribosomes. Science 174:707–709
Meyerowitz EM (1989) Arabidopsis, a useful weed. Cell 56:263–269
Olive J, Wellman F-A, Bennoun P, Recouvreur M (1981) Ultrastructure of thylakoid membranes in Chlamydomonas reinhardtii. evidence for variations in the partition coefficient of the light-harvesting complex-containing particles upon membrane fracture. Arch Biochem Biophys 208:456–467
Putrement A, Baranowska H, Prazmo W (1973) Induction of manganese of mitochondrial antibiotic resistant mutations in yeast. Mol Gen Genet 126:357–366
Sanger F, Nicklen S, Coulsen AR (1977) DNA sequencing with chain-termination inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Subik J, Takacsova G (1978) Genetic determination of ubiquinolcytochrome creductase. Mol Gen Genet 161:99–108
Sueoka N (1960) Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardtii Proc Natl Acad Sci USA 46:83–91
Thierbach G, Michaelis G (1982) Mitochondrial and nuclear myxothiazol resistance in Saccharomyces cerevisiae. Mol Gen Genet 186:501–506
Tron T, Lemesle-Meunier D (1990) Two substitutions at the same position in the mitochondrial cytochrome b gene of S. cerevisiae induce a mitochondrial myxothiazol resistance and impair the respiratory growth of the mutated strains albeit maintaining a good electron transfer activity. Curr Genet 18:413–419
Tron T, Infossi P, Coppee JY, Colson AM (1991) Molecular analysis of revertants from a respiratory-deficient mutant affecting the center o domain of cytochrome b in Saccharomyces cerevisiae induce a mitochondrial myxothiazol resistance and impair the respiratory growth of the mutated strains albeit maintaining a good electron transfer activity. Curr Genet 18:413–419
Tzagoloff A, Akai A, Needleman RB (1975a) Properties of cytoplasmic mutants of Saccharomyces cerevisiae with specific lesions in cytochrome oxidase. Proc Natl Acad sci USA 72:2054–2058
Tzagoloff A, Akai A, Needleman RB (1975b) Assembly of the mitochondrial membrane system: Isolation of nuclear and cytoplasmic mutants of Saccharomyces cerevisiae with specific defects in mitochondrial functions. J Bacteriol 122:826–831
Von Jagow G, Link TA (1986) Use of specific inhibitors on mitochondrial bel complex. Methods Enzymol 126, 253–271
Yannisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13 mpl8 and pUC19 vectors. Gene 33:103–119
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Communicated by R.G. Herrmann
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Bennoun, P., Delosme, M., Godehardt, I. et al. New tools for mitochondrial genetics of Chlamydomonas reinhardtii: manganese mutagenesis and cytoduction. Molec. Gen. Genet. 234, 147–154 (1992). https://doi.org/10.1007/BF00272356
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DOI: https://doi.org/10.1007/BF00272356