Restriction endonuclease analysis of chloroplast DNA from streptomycin-resistant mutants of Nicotiana plumbaginifolia KIN-YINGTO Department of Botany, National Taiwan University, Taipei, Taiwan, Republic of China

YIU-KAYLAI Institute of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China

TENG-YUNG FENG Institute of Botany, Academia Sinica, Taipei, Taiwan, Republic of China AND,

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CHI-CHANG CHEN' Department of Botany, National Taiwan University, Taipei, Taiwan, Republic of China Corresponding Editor: R. Kemble Received August 12, 1991 Accepted October 16, 1991 To, K.-Y ., LAI, Y .-K., FENG,T.-Y ., and CHEN,C.-C. 1992. Restriction endonuclease analysis of chloroplast DNA from streptomycin-resistant mutants of Nicotiana plumbaginifolia. Genome, 35: 220-224. Chloroplast DNA isolated from wild-type Nicotiana plumbaginifolia and 12 maternally inherited streptomycin-resistant mutants was digested with various restriction enzymes and the resultant patterns were compared. No gross structural alterations of the chloroplast genome were detected in any mutants; however, variant patterns owing to the gain or loss of a restriction site were found in three mutants, SR1007, SR1019, and SR1022. The variant patterns in SR1019 and SR1022 are identical and are the results of mutation in the psbG gene coding for a chloroplast membrane protein G, and that in SR1007 is due to mutation in the 16s rRNA gene. Inheritance of the variant patterns in mutants SR1007 and SR1019 was studied. The results showed that the variant patterns and streptomycin resistance were co-transmitted in reciprocal crosses. Key words: Nicotiana plumbaginifolia, streptomycin resistance, chloroplast DNA, restriction endonuclease analysis, 16s rRNA. To, K.-Y ., LAI, Y .-K., FENG,T.-Y ., et CHEN,C.-C. 1992. Restriction endonuclease analysis of chloroplast DNA from streptomycin-resistant mutants of Nicotiana plumbaginifolia. Genome, 35 : 220-224. L'ADN chloroplastique isole d'un type indigene de Nicotiana plumbaginifolia ainsi que de 12 mutants, dont l'heredite de la resistance a la streptomycine etait d'origine maternelle, a etC digere avec diverses enzymes de restriction et les profils obtenus ont Cte compares. Chez les mutants, aucune alteration structurale importante du genome des chloroplastes n'a ete decelee; toutefois, les profils ont variC en relation avec le gain ou la perte d'un site de restriction chez trois des mutants, les SR1007, SR1019 et SR1022. Les profils des deux derniers mutants ont Cte identiques et sont resultes d'une mutation dans le gene psbG, lequel encode la proteine G des membranes chloroplastiques, alors que le profil du SR1007 est le resultat d'une mutation dans le gene de 1'ARNr 16s. L'heredite des variations de profils chez les mutants SR1007 et SR1019 a etC Ctudiee. Les resultats montrent que les variations de profils des mutants et la resistance a streptomycine ont Cte co-transmis dans des croisements rkciproques. Mots elks : Nicotiana plumbaginifolia, resistance a la streptomycine, ADN chloroplastique, analyse des endonucleases de restriction, ARNr 16s. [Traduit par la redaction]

Introduction In a previous paper, we reported the successful isolation of maternally inherited streptomycin-resistant mutants from protoplast cultures of Nicotiana plumbaginifolia and presented cytological and biochemical evidence for chloroplast control of the resistance trait (To et al. 1989). The molecular basis of streptomycin resistance in these mutants was not investigated. However, it has been shown in other plant species that the resistance is due to a single nucleotide change either in the chloroplast 16s rRNA gene (Montandon et al. 1985; Etzold et al. 1987; Gauthier et al. 1988; Fromm et al. 1989; Harris et al. 1989) or in the gene coding for ribosomal protein S12 (Galili et al. 1989; Liu et al. 1989). To check for possible DNA duplication, deletion, or rearrangement induced by culture treatments during mutant isolation and to find new mutation sites conferring streptomycin resis' ~ u t h o rfor correspondence. Printed in Canada / lrnprime au Canada

tance, we screened chloroplast DNA (cpDNA) from our mutants with lo'restriction endonucleases. Using the cpDNA sequence of N. tabacum (Shinozaki et al. 1986) as a reference, we also analyzed the putative mutation sites in mutants showing variant restriction patterns. Finally, we studied the linkage between streptomycin resistance and the variant restriction patterns. The results of these studies are presented in this paper. Materials and methods Plant material Seeds of wild-type Nicotiana plumbaginifolia Viviani (2n = 2~ = 20) were provided by the U.S. Department of Agriculture. The 12 streptomycin-resistant mutants, SRO(l02, SRO(l04, SR1007, SR1009, SR1012, SR1018, SR1019, SR1021, SR1022, SR1036, SR1037, and SR1046, were isolated from protoplast culture of N. plumbaginifolia (To et al. 1989). Seeds of N. tabacum L. cv.

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Wisconsin 38 (2n = 4x = 48) were supplied by Taiwan Agricultural Research Institute. All plants were grown in pots in the greenhouse.

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Restriction endonuclease analysis of cpDNA Chloroplasts were isolated from leaves of potted plants according to the procedure of Palmer (1986) with modifications. The chloroplast fraction was collected at the 30/60% sucrose interface and lysed in buffer containing 20 mM Tris-HC1 (pH 8.0), 0.5 M NaC1,0.2% Triton X-100, and 1% sodium dodecylsulfate (SDS). . . The lysate was extracted three times with an equal volume of phenol - chloroform - isoamyl alcohol (24:24:1 by volume) and once with diethylether. Chloroplast DNA was precipitated in 70% ethanol overnight at - 20°C. After centrifugation at 13 000 rpm (Hitachi RPR-20-7 rotor) for 30 min, the pellets were treated with RNase A (10 mg/mL) at 37°C for 15 min, then the DNA was purified as in the extraction steps, and finally dissolved in TE buffer containing 10 mM Tris-HC1 (pH 8.0) and 1 mM Na2EDTA. Chloroplast DNA was digested with restriction endonucleases AvaI, BamHI, Bgn, ClaI, HindIII, PstI, PvuII, San, SmaI, and XhoI, respectively. Digestions were carried out as recommended by the manufacturer (Boehringer Mannheim). The restriction fragments were separated by 0.8% agarose gel electrophoresis in TAE buffer containing 40 mM Tris-base, 20 mM sodium acetate, and 2 mM Na2EDTA (pH 8.0).

Preparation of oligonucleotide probe An oligonucleotide (5 ' -CCGGAGAAGAAGCAATGA-3 ' ) corresponding to the sequence in positions 412-429 of the N. tabacum chloroplast 16s rRNA gene (Shinozaki et al. 1986) was synthesized by Gene Assembler (Pharmacia Piscapaway, NJ) and further purified with Mono Q column (Liquid Chromatography Controller LCC-500, Pharmacia). The oligonucleotide was labelled by ~ ~ ~ phosphorylation of the 5 ' terminus with [ y - 3 2 ~as ]described by Maniatis et al. (1982). The labelled probe was purified by passing it through a Sephadex G-25 mini-column. Southern hybridization After agarose gel electrophoresis, restriction fragments were transferred to nitrocellulose filters (Schleicher and Schull BA85) as described by Maniatis et al. (1982). The filter was incubated at 42°C for 2 h with prehybridization solution containing 6 x SSC (1 x SSC is 0.15 M NaCl and 0.015 M sodium citrate), 5 x Denhardt's (50 x Denhardt's is 1% Ficoll, 1% polyvinylpyrrolidone, and 1% bovine serum albumin), 20 mM NaH2P04, and 500 pg/mL salmon sperm DNA. Hybridization was carried out in a solution containing 6 x SSC, 0.4% SDS, 20 mM NaH2P04, 500 pg/mL salmon sperm DNA and labelled oligonucleotide probe at 42°C overnight. After hybridization, the filter was washed three times at 5-min intervals in a solution containing 2 x SSC and 0.1 % SDS at room temperature. Final wash was in the same solution at 44°C for 10 min. The filter was blotted dry and exposed to X-ray film using two intensifying screens at - 70°C.

Results Restriction endonuclease analysis Chloroplast DNA isolated from each of the 12 mutants was digested with restriction enzymes AvaI, BamHI, Bgn, ClaI, HindIII, PstI, PvuII, Sari, SmaI, and XhoI, respectively, and the restriction patterns were compared with those of the wild type. No obvious size alterations of the chloroplast genome were detected in any digestions. Mutants SR1019 and SR 1022 showed identical variant patterns when cpDNA was digested with either AvaI or XhoI. Mutant SR1007 differed from the wild type in PvuII digestion (Fig. 1). The mutants and wild type showed identical patterns for all other digestions (data not shown). AvaI recognizes four different sequences (C 1PyCGPuG), and one

FIG. 1. Restriction fragment patterns of AvaI, PvuII, and XhoI digests of cpDNA from wild-type N. plumbaginifolia (Np, wt) and mutants SR1007, SR1019, and SR1022. Marker: HindIII digest of phage lambda DNA.

of these is also recognized by XhoI (CITCGAG). Because only one restriction site change was detected in the cpDNA of SR1019 and SR1022 when digested with AvaI or XhoI, we surmised that both mutants were mutated at site(s) within the recognition sequence of XhoI. Since the complete cpDNA sequence of N. tabacum has been determined (Shinozaki et al. 1986), it is possible to search for the putative mutation site(s) in mutants with variant restriction patterns using the cpDNA sequence of N. tabacum as a reference. As shown in Fig. 2A, the 8.8-kb (X8) and 5.5-kb (X5) .XhoI fragments present in wild-type N. tabacum and N. plumbaginifolia were replaced by a 14-kb (X12a) fragment in mutants SR1019 and SR1022. Apparently, the XhoI restriction site at the junction of fragments X8 and X5 of the wild type was mutated in both mutants, so that the enzyme no longer recognized this sequence. According to the nucleotide sequence of N. tabacum cpDNA, the mutation site is probably in positions 201-206 in the psbG gene coding for a chloroplast membrane protein G. The cpDNA of N. tabacum and N. plumbaginifolia displayed identical restriction patterns when they were digested with PvuII. In mutant SR1007, the 2.4-kb P1 fragment detected in the wild type was replaced by a smaller fragment of 2.1 kb (Plb in Fig. 2B). The result suggests that a mutation in SR1007 might create a new PvuII recognition

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FIG. 2. Comparison of the restriction fragments of wild-type N. tabacum (Nt, wt) and N. plumbaginifolia (Np, wt), and mutants SR1007, SR1019, and SR1022. ( A ) XhoI digests. ( B ) PvuII digests. Designation of the fragments is in accordance with that of Fluhr and Edelman (1981).

sequence in fragment P1. Upon digestion with PvuII, two fragments, PI b and P l a , were generated from the P1 fragment; fragment P l b is 2.1 kb in length, while P l a is only 0.3 kb in length and was not detected in the gel shown in Fig. 2B. In N. tabacum cpDNA, the P1 fragment contains 1351 bp of the 3' end of the 16s rRNA gene (Fig. 3A). To determine whether the mutation site in SR1007 is located in this region (Figs. 3B and 3B1), cpDNA from wild-type N. plumbaginifolia and the mutant was digested with PvuII and used for Southern blot analysis. The synthetic oligonucleotide was used as probe. As shown in Fig. 3C, the probe hybridized to the 2.4-kb P1 fragment of the wild type and to the 0.3-kb P l a fragment of the mutant. The oligonucleotide is in positions 412-429 of the N. tabacum 16s rRNA gene. The result from Southern blot analysis therefore suggests that the mutation site in SR1007 is around position 470. Inheritance of the variant restriction patterns Mutants SR 1007 and SR 1019 were crossed reciprocally to the wild type, and seeds from the crosses were germinated on medium without streptomycin according to the method of To et al. (1989). When in vitro grown plants reached the stage of 8-10 leaves, 2 leaves were cut off from each plant for assay of streptomycin resistance (To et al. 1989); meanwhile, the plants were transplanted to pots for further growth. Chloroplast DNA was isolated from leaves of potted plants, digested with the relevant restriction enzymes, and the restriction patterns were analyzed. The results revealed that when mutants were the female parent all progeny plants showed streptomycin resistance and the variant restriction patterns, and that when mutants were the male parent all progeny plants showed streptomycin sensitivity and the wildtype restriction patterns (Fig. 4).

Discussion Grossly altered cpDNA molecules have been detected in plants regenerated from pollen (Day and Ellis 1984) but not in plants regenerated from leaf protoplasts (Kemble and Shepard 1984; Rose et al. 1986). It is likely that the cpDNA alterations in pollen plants are the result of pollen development and not a consequence of in vitro culture. Grossly altered forms of cpDNA have also been found in Chlamydomonas reinhardtii treated with 5-fluorodeoxyuridine (Myers et al. 1982) and in Euglena gracilis treated with 500 pg/mL streptomycin (Heizmann et al. 1982). Most streptomycin-resistant mutants used in this study were isolated from N-methyl-N' -nitro-N-nitrosoguanidine treated protoplast cultures in the presence of 1000 pg/mL streptomycin (To et al. 1989). Failure in detection of gross alterations of cpDNA in these mutants suggests that the mutagen, selective agent, and culture method used in isolating the mutants do not appear to induce structural changes of the chloroplast genome of N. plumbaginifolia. The only changes detected from restriction endonuclease analysis of cpDNA were the elimination of a XhoI site in mutants SR1019 and SR1022, and the appearance of a new PvuII site in mutant SR1007. The elimination of a XhoI site in mutants SR1019 and SR1022 is probably due to a point mutation in the psbG gene coding for a chloroplast membrane protein G (Shinozaki et al. 1986). However, we do not know whether this mutation is responsible for streptomycin resistance. The results from Southern hybridization indicate that the appearance of a new PvuII site in cpDNA of mutant SR1007 is caused by a point mutation around nucleotide 470 in the chloroplast 16s rRNA gene. A comparison of the nucleotide sequence of the chloroplast 16s rRNA gene of N. tabacum (Tohdoh and Sugiura 1982; Shinozaki et al. 1986) and that

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FIG. 3. (A) Schematic diagram of the PvuII fragments containing 16s rRNA gene. 5' and 3 ' denote the transcriptional orientation of the 16s rRNA gene. The asterisk indicates the approximate location of the synthetic oligonucleotide in the 16s rRNA gene. (B) and (B') The two possible mutation sites (arrows) in P1 fragment in mutant SR1007 deduced from restriction fragment pattern. Depending on the site of mutation, either P l a or P l b will hybridize t o the synthetic probe. ( C ) Restriction fragment patterns of PvuII digests of cpDNA from wild-type N. plumbaginijiolia (Np, wt) and mutant SR1007, and corresponding autoradiogram after Southern hybridization with the oligonucleotide probe. Fragments P1 and P l a are indicated by arrows. Marker: HindIII digest of phage lambda DNA.

of the 16s rRNA gene of Escherichia coli (Brosius et al. 1981) suggests that the point mutation falls in a phylogenetically conserved region (Stiegler et al. 1981) known as the "530 loop" (Moazed and Noller 1987). The nucleotide sequence of this region (5 ' -GCCAGCAGCCGCGGTAAT-3' ) is identical in E. coli (nucleotides 5 17-534) and N. tabacum (nucleotides 464-48 1). Thus, according to the numbering system in E. coli, either an A to T transversion at position 523 or a C to T transition at position 526 can create a new PvuII recognition sequence (CAG I CTG) in this region. The involvement of position 523 with streptomycin resistance has been shown in E. coli (Melanqon et al. 1988) and Chlamydomonas (Gauthier et al. 1988; Harris et al. 1989). In N. tabacum, a C to T transition at position 525 has also been suggested to confer streptomycin resistance (Fromm et al. 1989). The "530 loop" of the 16s rRNA is known to be associated with ribosomal proofreading (Stern et al. 1986; Moazed and Noller 1987). As one effect of streptomycin is to induce misreading of the genetic code (Ruusala and Kurland 1984), it is possible that mutations at several positions within this loop may diminish ribosomal sensitivity and thus confer resistance to the antibiotic. Analysis of F, plants from reciprocal crosses between streptomycin-resistant mutants SR1007 and SR1019 and the wild type shows that streptomycin resistance and the variant restriction patterns of cpDNA are linked and transmitted to the progeny only through the female parent. The strict co-transmission of the two traits suggests the chloro~last localization of streptomycin resistance. Having one selective marker (streptomycin resistance) and one identification marker (mutant-specific restriction pattern) in their chloroplasts, mutants SR1007, SR1019, and SR1022 are useful

FIG. 4. Inheritance of the variant restriction patterns. AvaI digests of CPDNA from wild-type N.plumbaginijiolia (wt), mutant ~ ~ 1 0 1 and 9 , five progeny plants from each of the crosses of wt Q x SR1019w and SR1019 Q x wtw. Marker: HindIII digest of phage lambda DNA.

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material for the study of chloroplast genetics and chloroplast transfer. Acknowledgements We are grateful to M. F. Tang for synthesizing the oligonucleotide, M. Wu for advice and aid in molecular biology techniques, and S. Y. Tung and D. I. Yang for technical assistance. This work was supported by National Science Council grants NSC78-02 11-B002-24 to C.C.C. and NSC780203-B007-09 to Y .K. L. Brosius, J., Dull, T. J., and Sleeter, D.D. 1981. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J. Mol. Biol. 148: 107-127. Day, A., and Ellis, T.H.N. 1984. Chloroplast DNA deletions associated with wheat plants regenerated from pollen: possible basis for maternal inheritance of chloroplasts. Cell, 39: 359-368. Etzold, T., Fritz, C.C., Schell, J., and Schreier, P.H. 1987. A point mutation in the chloroplast 16s rRNA gene of a streptomycin resistant Nicotiana tabacum. FEBS Lett. 219: 343-346. Fluhr, R., and Edelman, M. 1981. Physical mapping of Nicotiana tabacum chloroplast DNA. Mol. Gen. Genet. 181: 484-490. Fromm, H., Galun, E., and Edelman, M. 1989. A novel site for streptomycin resistance in the "530 loop" of chloroplast 16s ribosomal RNA. Plant Mol. Biol. 12: 499-505. Galili, S., Fromm, H., Aviv, D., et al. 1989. Ribosomal protein S12 as a site for streptomycin resistance in Nicotiana chloroplast. Mol. Gen. Genet. 218: 289-292. Gauthier, A., Turmel, M., and Lemieux, C. 1988. 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. Harris, E.H., Burkhart, B.D., Gillham, H.W., and Boynton, J.E. 1989. Antibiotic resistance mutations in the chloroplast 16s and 23s rRNA genes of Chlamydomonas reinhardtii: correlation of genetic and physical maps of the chloroplast genome. Genetics, 123: 281-292. Heizmann, P., Hussein, Y., Nicolas, P., and Nigon, V. 1982. Modification of chloroplast DNA during streptomycin induced mutagenesis in Euglena gracilis. Curr. Genet. 5 : 9-1 5. Kemble, R. J., and Shepard, J.F. 1984. Cytoplasmic DNA variation in a potato protoclonal population. Theor. Appl. Genet. 69: 211-216. Liu, X.Q., Gillham, N.W., and Boynton, J.E. 1989. Chloroplast

ribosomal protein gene rpsl2 of Chlamydomonas reinhardtii: wild-type sequence, mutation to streptomycin resistance and dependence, and function in Escherichia coli. J. Biol. Chem. 264: 16100-16108. Maniatis, T., Fritsch, E.F., and Sambrook, J. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor, New York. Melan~on,P., Lemieux, C., and Brakier-Gingras, L. 1988. A mutation in the 530 loop of Escherichia coli 16s ribosomal RNA causes resistance to streptomycin. Nucleic Acids Res. 16: 9631-9639. Moazed, D., and Noller, H.F. 1987. Interaction of antibiotics with functional sites in 16s ribosomal RNA. Nature (London), 327: 389-394. Montandon, P.E., Nicolas, P., Schurmann, P., and Stutz, E. 1985. Streptomycin resistance of Euglena gracilis chloroplast: identification of a point mutation in the 16s rRNA gene in an invariant position. Nucleic Acids Res. 13: 4299-4310. Myers, A.M., Grant, D.M., Rabert, D.K., et al. 1982. Mutants of Chlamydomonas reinhardtii with physical alterations in their chloroplast DNA. Plasmid, 7 : 133-1 5 1. Palmer, J.D. 1986. Isolation and structural analysis of chloroplast DNA. Methods Enzymol. 118: 167-186. Rose, R.J., Johnson, L.B., and Kemble, R.J. 1986. Restriction endonuclease studies on the chloroplast and mitochondria1 DNAs of alfalfa (Medicago sativa L.) protoclones. Plant Mol. Biol. 6: 331-338. Ruusala, T., and Kurland, C.G. 1984. Streptomycin preferentially perturbs ribosomal proofreading. Mol. Gen. Genet. 198: 100-104. Shinozaki, K., Ohme, M., Tanaka, M., et al. 1986. The complete nucleotide sequence of the tobacco chloroplast genome. Plant Mol. Biol. Rep. 4: 110-147. Stern, S., Wilson, R.C., and Noller, H.F. 1986. Localization of the binding site for protein S4 on the 16s ribosomal RNA by chemical and enzymatic probing and primer extension. J. Mol. Biol. 192: 101-110. Stiegler, P., Carbon, P., Ebel, J.P., and Ehresmann, C. 1981. A general secondary structure model for procaryotic and eucaryotic RNAs of the small ribosomal subunits. Eur. J. Biochem. 120: 487-4.95. To, K.Y., Chen, C.C., and Lai, Y.K. 1989. Isolation and characterization of streptomycin-resistant mutants in Nicotiana plumbaginifolia. Theor. Appl. Genet. 78: 81-86. Tohdoh, N., and Sugiura, M. 1982. The complete nucleotide sequence of a 16s ribosomal RNA gene from tobacco chloroplasts. Gene, 17: 213-218.

Restriction endonuclease analysis of chloroplast DNA from streptomycin-resistant mutants of Nicotiana plumbaginifolia.

Chloroplast DNA isolated from wild-type Nicotiana plumbaginifolia and 12 maternally inherited streptomycin-resistant mutants was digested with various...
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