Skip to main content
NCBI home page
The Plant Cell logoLink to The Plant Cell
. 1994 Feb;6(2):187–200. doi: 10.1105/tpc.6.2.187

Gene Amplification Can Correct a Photosynthetic Growth Defect Caused by mRNA Instability in Chlamydomonas Chloroplasts.

K L Kindle 1, H Suzuki 1, D B Stern 1
PMCID: PMC160426  PMID: 12244236

Abstract

Chlamydomonas reinhardtii chloroplast transformants that lack an inverted repeat normally found at the 3[prime] end of the chloroplast atpB gene have a slow phototrophic growth phenotype due to reduced accumulation of atpB mRNA and the chloroplast ATPase [beta] subunit. We have recovered transformants exhibiting more robust phototrophic growth at a moderate frequency (~1% relative to slow-growing transformants). Quantitative DNA blot analysis indicated that in one class of these robust photosynthetic transformants, the introduced plasmid DNA is maintained at high copy number-~25 copies per chloroplast genome or 2000 copies per cell. Partial restriction digests resulted in a ladder with at least 15 visible fragments, indicating that most of the transforming DNA is organized as a long head-to-tail tandem repeat. Total atpB transcription and accumulation of atpB mRNA and the ATPase [beta] subunit were increased approximately fivefold relative to transformants that carry a single copy of the truncated atpB gene. The amplified DNA was stably maintained at high copy number under mixotrophic growth conditions. It was inherited uniparentally from the mt+ parent, and its synthesis was sensitive to 5-fluoro-2[prime]-deoxyuridine, an inhibitor of chloroplast DNA synthesis. Therefore, we conclude that the tandem repeat is maintained in the chloroplast. Restriction enzymes that fail to digest the transforming plasmid but have recognition sites in chloroplast DNA did not alter the electrophoretic mobility of the tandem repeat, suggesting that it is not integrated in the chloroplast genome. We conclude that the tandem repeat is probably episomal and hypothesize that its replication is independent of the chloroplast genome.

Full Text

The Full Text of this article is available as a PDF (4.7 MB).

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. Büschlen S., Choquet Y., Kuras R., Wollman F. A. Nucleotide sequences of the continuous and separated petA, petB and petD chloroplast genes in Chlamydomonas reinhardtii. FEBS Lett. 1991 Jun 24;284(2):257–262. doi: 10.1016/0014-5793(91)80698-3. [DOI] [PubMed] [Google Scholar]
  5. Chen X., Kindle K., Stern D. Initiation codon mutations in the Chlamydomonas chloroplast petD gene result in temperature-sensitive photosynthetic growth. EMBO J. 1993 Sep;12(9):3627–3635. doi: 10.1002/j.1460-2075.1993.tb06036.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Drapier D., Girard-Bascou J., Wollman F. A. Evidence for Nuclear Control of the Expression of the atpA and atpB Chloroplast Genes in Chlamydomonas. Plant Cell. 1992 Mar;4(3):283–295. doi: 10.1105/tpc.4.3.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  9. Fox T. D., Sanford J. C., McMullin T. W. Plasmids can stably transform yeast mitochondria lacking endogenous mtDNA. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7288–7292. doi: 10.1073/pnas.85.19.7288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grant D. M., Gillham N. W., Boynton J. E. Inheritance of chloroplast DNA in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 1980 Oct;77(10):6067–6071. doi: 10.1073/pnas.77.10.6067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Guertin M., Bellemare G. Synthesis of chloroplast ribonucleic acid in Chlamydomonas reinhardtii toluene-treated cells. Eur J Biochem. 1979 May 2;96(1):125–129. doi: 10.1111/j.1432-1033.1979.tb13021.x. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Maleszka R., Skelly P. J., Clark-Walker G. D. Rolling circle replication of DNA in yeast mitochondria. EMBO J. 1991 Dec;10(12):3923–3929. doi: 10.1002/j.1460-2075.1991.tb04962.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Myers A. M., Grant D. M., Rabert D. K., Harris E. H., Boynton J. E., Gillham N. W. Mutants of Chlamydomonas reinhardtii with physical alterations in their chloroplast DNA. Plasmid. 1982 Mar;7(2):133–151. doi: 10.1016/0147-619x(82)90073-7. [DOI] [PubMed] [Google Scholar]
  15. Shepherd H. S., Ledoigt G., Howell S. H. Regulation of light-harvesting chlorophyll-binding protein (LHCP) mRNA accumulation during the cell cycle in Chlamydomonas reinhardi. Cell. 1983 Jan;32(1):99–107. doi: 10.1016/0092-8674(83)90500-7. [DOI] [PubMed] [Google Scholar]
  16. Stern D. B., Kindle K. L. 3'end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process. Mol Cell Biol. 1993 Apr;13(4):2277–2285. doi: 10.1128/mcb.13.4.2277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  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. Woessner J. P., Gillham N. W., Boynton J. E. The sequence of the chloroplast atpB gene and its flanking regions in Chlamydomonas reinhardtii. Gene. 1986;44(1):17–28. doi: 10.1016/0378-1119(86)90038-7. [DOI] [PubMed] [Google Scholar]
  20. Wurtz E. A., Boynton J. E., Gillham N. W. Perturbation of chloroplast DNA amounts and chloroplast gene transmission in Chlamydomonas reinhardtii by 5-fluorodeoxyuridine. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4552–4556. doi: 10.1073/pnas.74.10.4552. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Plant Cell are provided here courtesy of Oxford University Press

RESOURCES