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. 1992 Jan;4(1):39–45. doi: 10.1105/tpc.4.1.39

Long regions of homologous DNA are incorporated into the tobacco plastid genome by transformation.

J M Staub 1, P Maliga 1
PMCID: PMC160104  PMID: 1356049

Abstract

We investigated the size of flanking DNA incorporated into the tobacco plastid genome alongside a selectable antibiotic resistance mutation. The results showed that integration of a long uninterrupted region of homologous DNA, rather than of small fragments as previously thought, is the more likely event in plastid transformation of land plants. Transforming plasmid pJS75 contains a 6.2-kb DNA fragment from the inverted repeat region of the tobacco plastid genome. A spectinomycin resistance mutation is encoded in the gene of the 16S rRNA and, 3.2 kb away, a streptomycin resistance mutation is encoded in exon II of the ribosomal protein gene rps12. Transplastomic lines were obtained after introduction of pJS75 DNA into leaf cells by the biolistic process and selection for the spectinomycin resistance marker. Homologous replacement of resident wild-type sequences resulted in integration of all, or almost all, of the 6.2-kb plastid DNA sequence from pJS75. Plasmid pJS75, which contains engineered cloning sites between two selectable markers, can be used as a plastid insertion vector.

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Selected References

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  1. Blowers A. D., Ellmore G. S., Klein U., Bogorad L. Transcriptional analysis of endogenous and foreign genes in chloroplast transformants of Chlamydomonas. Plant Cell. 1990 Nov;2(11):1059–1070. doi: 10.1105/tpc.2.11.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boynton J. E., Gillham N. W., Harris E. H., Hosler J. P., Johnson A. M., Jones A. R., Randolph-Anderson B. L., Robertson D., Klein T. M., Shark K. B. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science. 1988 Jun 10;240(4858):1534–1538. doi: 10.1126/science.2897716. [DOI] [PubMed] [Google Scholar]
  3. Dahlberg A. E. The functional role of ribosomal RNA in protein synthesis. Cell. 1989 May 19;57(4):525–529. doi: 10.1016/0092-8674(89)90122-0. [DOI] [PubMed] [Google Scholar]
  4. Galili S., Fromm H., Aviv D., Edelman M., Galun E. Ribosomal protein S12 as a site for streptomycin resistance in Nicotiana chloroplasts. Mol Gen Genet. 1989 Aug;218(2):289–292. doi: 10.1007/BF00331280. [DOI] [PubMed] [Google Scholar]
  5. Goldschmidt-Clermont M., Choquet Y., Girard-Bascou J., Michel F., Schirmer-Rahire M., Rochaix J. D. A small chloroplast RNA may be required for trans-splicing in Chlamydomonas reinhardtii. Cell. 1991 Apr 5;65(1):135–143. doi: 10.1016/0092-8674(91)90415-u. [DOI] [PubMed] [Google Scholar]
  6. Goldschmidt-Clermont M. Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of chlamydomonas. Nucleic Acids Res. 1991 Aug 11;19(15):4083–4089. doi: 10.1093/nar/19.15.4083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gordon-Kamm W. J., Spencer T. M., Mangano M. L., Adams T. R., Daines R. J., Start W. G., O'Brien J. V., Chambers S. A., Adams W. R., Jr, Willetts N. G. Transformation of Maize Cells and Regeneration of Fertile Transgenic Plants. Plant Cell. 1990 Jul;2(7):603–618. doi: 10.1105/tpc.2.7.603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hildebrand M., Hallick R. B., Passavant C. W., Bourque D. P. Trans-splicing in chloroplasts: the rps 12 loci of Nicotiana tabacum. Proc Natl Acad Sci U S A. 1988 Jan;85(2):372–376. doi: 10.1073/pnas.85.2.372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kindle K. L., Richards K. L., Stern D. B. Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1721–1725. doi: 10.1073/pnas.88.5.1721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Newman S. M., Boynton J. E., Gillham N. W., Randolph-Anderson B. L., Johnson A. M., Harris E. H. Transformation of chloroplast ribosomal RNA genes in Chlamydomonas: molecular and genetic characterization of integration events. Genetics. 1990 Dec;126(4):875–888. doi: 10.1093/genetics/126.4.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Przibilla E., Heiss S., Johanningmeier U., Trebst A. Site-specific mutagenesis of the D1 subunit of photosystem II in wild-type Chlamydomonas. Plant Cell. 1991 Feb;3(2):169–174. doi: 10.1105/tpc.3.2.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Roffey R. A., Golbeck J. H., Hille C. R., Sayre R. T. Photosynthetic electron transport in genetically altered photosystem II reaction centers of chloroplasts. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9122–9126. doi: 10.1073/pnas.88.20.9122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Shimada H., Sugiura M. Fine structural features of the chloroplast genome: comparison of the sequenced chloroplast genomes. Nucleic Acids Res. 1991 Mar 11;19(5):983–995. doi: 10.1093/nar/19.5.983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Stern D. B., Radwanski E. R., Kindle K. L. A 3' stem/loop structure of the Chlamydomonas chloroplast atpB gene regulates mRNA accumulation in vivo. Plant Cell. 1991 Mar;3(3):285–297. doi: 10.1105/tpc.3.3.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sun E., Wu B. W., Tewari K. K. In vitro analysis of the pea chloroplast 16S rRNA gene promoter. Mol Cell Biol. 1989 Dec;9(12):5650–5659. doi: 10.1128/mcb.9.12.5650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Svab Z., Hajdukiewicz P., Maliga P. Stable transformation of plastids in higher plants. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8526–8530. doi: 10.1073/pnas.87.21.8526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Svab Z., Maliga P. Mutation proximal to the tRNA binding region of the Nicotiana plastid 16S rRNA confers resistance to spectinomycin. Mol Gen Genet. 1991 Aug;228(1-2):316–319. doi: 10.1007/BF00282483. [DOI] [PubMed] [Google Scholar]
  18. Takaiwa F., Sugiura M. Nucleotide sequence of the 16S - 23S spacer region in an rRNA gene cluster from tobacco chloroplast DNA. Nucleic Acids Res. 1982 Apr 24;10(8):2665–2676. doi: 10.1093/nar/10.8.2665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tohdoh N., Sugiura M. The complete nucleotide sequence of 16S ribosomal RNA gene from tobacco chloroplasts. Gene. 1982 Feb;17(2):213–218. doi: 10.1016/0378-1119(82)90074-9. [DOI] [PubMed] [Google Scholar]
  20. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]

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