Summary
A major obstacle to out understanding of the mechanisms governing the inheritance, recombination and segregation of chloroplast genes in Chlamydomonas is that the majority of antibiotic resistance mutations that have been used to gain insights into such mechanisms have not been physically localized on the chloroplast genome. We report here the physical mapping of two chloroplast antibiotic resistance mutations: one conferring cross-resistance to erythromycin and spiramycin in Chlamydomonas moewusii (er-nM1) and the other conferring resistance to streptomycin in the interfertile species C. eugametos (sr-2). The er-nM1 mutation results from a C to G transversion at a well-known site of macrolide resistance within the peptidyl transferase loop region of the large subunit rRNA gene. This locus, designated rib-2 in yeast mitochondrial DNA, corresponds to residue C-2611 in the 23 S rRNA of Escherichia coli. The sr-2 locus maps within the small subunit (SSU) rRNA gene at a site that has not been described previously. The mutation results from an A to C transversion at a position equivalent to residue A-523 in the E. coli 16 S rRNA. Although this region of the E. coli SSU rRNA has no binding affinity for streptomycin, it binds to ribosomal protein S4, a protein that has long been associated with the response of bacterial cells to this antibiotic. We propose that the sr-2 mutation indirectly affects the nearest streptomycin binding site through an altered interaction between a ribosomal protein and the SSU rRNA.
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References
Biggin MD, Gibson TJ, Hong GF (1983) Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci USA 80:3963–3965
Blanc H, Wright CT, Bibb MJ, Wallace DC, Clayton DA (1981) Mitochondrial DNA of chloromphenicol-resistant mouse cells contains a single nucleotide change in the region encoding the 3′ end of the large ribosomal RNA. Proc Natl Acad Sci USA 78:3789–3793
Cundliffe E (1987) On the nature of antibiotic binding sites in ribosomes. Biochimie 69:863–869
Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395
Dujon B (1980) Sequence of the intron and flanking exons of the mitochondrial 21S rRNA gene of yeast strains having different alleles at the ω and rib-1 loci. Cell 20:185–197
Ettayebi M, Prasad SM, Morgan EA (1985) Chloramphenicol-erythromycin resistance in a 23 S rRNA gene of Escherichia coli. J Bacteriol 162:551–557
Etzold T, Fritz CC, Schell J, Schreier PH (1987) A point mutation in the chloroplast 16 S rRNA gene of a streptomycin resistant Nicotiana tabacum. FEBS Lett 219:343–346
Expert-Bezançon A, Wollenzien PL (1985) Three-dimensional arrangement of the Escherichia coli 16 S ribosomal RNA. J Mol Biol 184:53–66
Fromm H, Edelman M, Aviv D, Galun E (1987) The molecular basis for rRNA-dependent spectinomycin resistance in Nicotiana chloroplasts. EMBO J 6:3233–3237
Gillham NW (1978) Organelle heredity. Raven Press, New York
Gravel M, Melançon P, Brakier-Gingras L (1987) Cross-linking of streptomycin to the 16 S ribosomal RNA of Escherichia coli. Biochemistry 26:6227–6232
Gutell RR, Weiser B, Woese CR, Noller HF (1985) Comparative anatomy of 16-S-like ribosomal RNA. Prog Nucleic Acid Res Mol Biol 32:155–216
Harris EH, Boynton JE, Gillham NW, Tingle CL, Fox SB (1977) Mapping of chloroplast genes involved in chloroplast biogenesis in Chlamydomonas reinhardtii. Mol Gen Genet 155:249–265
Harris EH, Boynton JE, Gillham NW (1987) Interaction of nuclear and chloroplast mutations in biogenesis of chloroplast ribosomes in Chlamydomonas. In: Weissner W, Robinson DG, Starr R (eds) Algal development. Springer, New York Heidelberg Berlin
Hummel H, Böck A (1987) 23 S ribosomal RNA mutations in halobacteria conferring resistance to the anti-80S ribosome targeted antibiotic anisomycin. Nucleic Acids Res 15:2431–2443
Kearsey SE, Craig IW (1981) Altered ribosomal RNA genes in mitochondria from mammalian cells with chloramphenicol resistance. Nature 290:607–608
Lee RW, Lemieux C (1986) Biparental inheritance of non-Mendelian gene markers in Chlamydomonas moewusii. Genetics 113:589–600
Lemieux B, Turmel M, Lemieux C (1988) Unidirectional gene conversions in the chloroplast of Chlamydomonas interspecific hybrids. Mol Gen Genet 212:48–55
Lemieux C, Lee RW (1987) Nonreciprocal recombination between alleles of the chloroplast 23 S rRNA gene in interspecific Chlamydomonas crosses. Proc Natl Acad Sci USA 84:4166–4170
Lemieux C, Turmel M, Lee RW (1980) Characterization of chloroplast DNA in Chlamydomonas eugametos and C. moewusii and its inheritance in hybrid progeny. Curr Genet 2:139–147
Lemieux C, Turmel M, Seligy VL, Lee RW (1984) Chloroplast DNA recombination in interspecific hybrids of Chlamydomonas: Linkage between a nonmendelian locus for streptomycin resistance and restriction fragments coding for 16 S rRNA. Proc Natl Acad Sci USA 81:1164–1168
Lemieux C, Turmel M, Seligy VL, Lee RW (1985) The large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase is encoded in the inverted repeat sequence of the Chlamydomonas eugametos chloroplast genome. Curr Genet 9:139–145
Marko MA, Chipperfield R, Birnboim HC (1982) A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder. Anal Biochem 121:382–387
McBride AC, McBride JC (1975) Uniparental inheritance in Chlamydomonas eugametos (Chlorophyceae). J Phycol 11:343–344
Mets LJ, Geist LJ (1983) Linkage of a known chloroplast gene mutation to the uniparental genome of Chlamydomonas reinhardtii. Genetics 105:559–579
Moazed D, Noller HF (1987) Interaction of antibiotics with functional sites in 16 S ribosomal RNA. Nature 327:389–394
Montandon PE, Nicolas P, Schurmann P, Stutz E (1985) Streptomycin resistance of Euglena gracilis chloroplasts: identification of a point mutation in the 16 S rRNA gene in an invariant position. Nucleic Acids Res 13:4299–4310
Montandon PE, Wagner R, Stutz E (1986) E. coli ribosomes with a C912 to U base change in the 16 S rRNA are streptomycin resistant. EMBO J 5:3705–3708
Noller HF (1984) Structure of ribosomal RNA. Annu Rev Biochem 53:119–162
Ruusala T, Kurland CG (1984) Streptomycin preferentially perturbs ribosomal proofreading. Mol Gen Genet 198:100–104
Sager R (1977) Genetic analysis of chloroplast DNA in Chlamydomonas. Adv Genet 19:287–340
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Sigmund CD, Ettayebi M, Morgan EA (1984) Antibiotic resistance mutations in 16 S and 23 S ribosomal RNA genes of Escherichia coli. Nucleic Acids Res 12:4653–4663
Slott EF, Shade RO, Lansman RA (1983) Sequence analysis of mitochondrial DNA in a mouse cell line resistant to chloramphenicol and oligomycin. Mol Cell Biol 3:1694–1702
Sor F, Fukuhara H (1982) Identification of two erythromycin resistance mutations in the mitochondrial gene coding for the large ribosomal RNA in yeast. Nucleic Acids Res 10:6571–6577
Sor F, Fukuhara H (1984) Erythromycin and spiramycin resistance mutations of yeast mitochondria: nature of the rib2 locus in the large ribosomal RNA gene. Nucleic Acids Res 12:8313–8318
Stern S, Wilson RC, Noller HF (1986) Localization of the binding site for protein S4 on 16 S ribosomal RNA by chemical and enzymatic probing and primer extension. J Mol Biol 192:101–110
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Communicated by R.G. Herrmann
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Gauthier, A., Turmel, M. & Lemieux, C. Mapping of chloroplast mutations conferring resistance to antibiotics in Chlamydomonas: Evidence for a novel site of streptomycin resistance in the small subunit rRNA. Mol Gen Genet 214, 192–197 (1988). https://doi.org/10.1007/BF00337710
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DOI: https://doi.org/10.1007/BF00337710