Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1991 Aug 11;19(15):4083–4089. doi: 10.1093/nar/19.15.4083

Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of chlamydomonas.

M Goldschmidt-Clermont 1
PMCID: PMC328544  PMID: 1651475

Abstract

Expression vectors for Chlamydomonas reinhardtii chloroplast transformation have been constructed with transcription and translation signals from chloroplast genes. The bacterial aadA sequence, coding for aminoglycoside 3" adenyl transferase, was inserted in these vectors and introduced into the C. reinhardtii chloroplast by particle gun transformation. The stable transgenic expression of this foreign protein in the chloroplast confers spectinomycin and streptomycin resistance to the transformed cells. This new marker can be used as a reporter of gene expression, and as a portable selectable cassette for chloroplast reverse genetics. Targetted gene disruption mutants of loci required for photosynthesis, tscA and psaC, were thus obtained. A gene disruption of an unidentified open reading frame, ORF472, remained heteroplasmic, suggesting that it has a vital function.

Full text

PDF
4083

Images in this article

4085Image
on p.4085
4087Image
on p.4087
4087Image
on p.4087

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blowers A. D., Bogorad L., Shark K. B., Sanford J. C. Studies on Chlamydomonas chloroplast transformation: foreign DNA can be stably maintained in the chromosome. Plant Cell. 1989 Jan;1(1):123–132. doi: 10.1105/tpc.1.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Choquet Y., Goldschmidt-Clermont M., Girard-Bascou J., Kück U., Bennoun P., Rochaix J. D. Mutant phenotypes support a trans-splicing mechanism for the expression of the tripartite psaA gene in the C. reinhardtii chloroplast. Cell. 1988 Mar 25;52(6):903–913. doi: 10.1016/0092-8674(88)90432-1. [DOI] [PubMed] [Google Scholar]
  5. Daniell H., Vivekananda J., Nielsen B. L., Ye G. N., Tewari K. K., Sanford J. C. Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors. Proc Natl Acad Sci U S A. 1990 Jan;87(1):88–92. doi: 10.1073/pnas.87.1.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Debuchy R., Purton S., Rochaix J. D. The argininosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus. EMBO J. 1989 Oct;8(10):2803–2809. doi: 10.1002/j.1460-2075.1989.tb08426.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dron M., Rahire M., Rochaix J. D. Sequence of the chloroplast DNA region of Chlamydomonas reinhardii containing the gene of the large subunit of ribulose bisphosphate carboxylase and parts of its flanking genes. J Mol Biol. 1982 Dec 25;162(4):775–793. doi: 10.1016/0022-2836(82)90547-2. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Goldschmidt-Clermont M., Girard-Bascou J., Choquet Y., Rochaix J. D. Trans-splicing mutants of Chlamydomonas reinhardtii. Mol Gen Genet. 1990 Sep;223(3):417–425. doi: 10.1007/BF00264448. [DOI] [PubMed] [Google Scholar]
  10. Haas M. J., Dowding J. E. Aminoglycoside-modifying enzymes. Methods Enzymol. 1975;43:611–628. doi: 10.1016/0076-6879(75)43124-x. [DOI] [PubMed] [Google Scholar]
  11. Hollingshead S., Vapnek D. Nucleotide sequence analysis of a gene encoding a streptomycin/spectinomycin adenylyltransferase. Plasmid. 1985 Jan;13(1):17–30. doi: 10.1016/0147-619x(85)90052-6. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Kindle K. L., Schnell R. A., Fernández E., Lefebvre P. A. Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J Cell Biol. 1989 Dec;109(6 Pt 1):2589–2601. doi: 10.1083/jcb.109.6.2589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Rochaix J. D. Restriction endonuclease map of the chloroplast DNA of Chlamydomonas reinhardii. J Mol Biol. 1978 Dec 25;126(4):597–617. doi: 10.1016/0022-2836(78)90011-6. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Thompson R. J., Mosig G. Stimulation of a Chlamydomonas chloroplast promoter by novobiocin in situ and in E. coli implies regulation by torsional stress in the chloroplast DNA. Cell. 1987 Jan 30;48(2):281–287. doi: 10.1016/0092-8674(87)90431-4. [DOI] [PubMed] [Google Scholar]
  19. Vallet J. M., Rahire M., Rochaix J. D. Localization and sequence analysis of chloroplast DNA sequences of Chlamydomonas reinhardii that promote autonomous replication in yeast. EMBO J. 1984 Feb;3(2):415–421. doi: 10.1002/j.1460-2075.1984.tb01822.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ye G. N., Daniell H., Sanford J. C. Optimization of delivery of foreign DNA into higher-plant chloroplasts. Plant Mol Biol. 1990 Dec;15(6):809–819. doi: 10.1007/BF00039421. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES