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Nuclear and chloroplast transformation inChlamydomonas reinhardtii: strategies for genetic manipulation and gene expression

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Abstract

Transformation of the nuclear, chloroplast, and mitochondrial genomes can now be accomplished inChlamydomonas reinhardtii. Many biosynthetic pathways are carried out in the chloroplast, and efforts to manipulate these pathways will require that gene products be directed to this compartment. Chloroplast proteins are encoded in either the chloroplast or nuclear genome. In the latter case they are synthesized in the cytoplasm and imported post-translationally into the chloroplast. Thus, strategies for expressing foreign genes or overexpressing endogenous genes whose products reside in the chloroplast could involve either genome. This paper reviews the present status of transformation methodology for the nuclear and chloroplast genomes inChlamydomonas. Considerations for expressing gene products in the chloroplast are discussed. Experimental evidence for homologous recombination during transformation of the nuclear genome is presented.

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References

  • Adam ME, Lentz KE, Loppes R (1993) Insertional mutagenesis to isolate acetate-requiring mutants inChlamydomonas reinhardtii. FEMS microbiol. Lett. 110: 265–258.

    Google Scholar 

  • Bingham SE, Cox JC, Strem MD (1989) Expression of foreign DNA inChlamydomonas reinhardtii. FEMS microbiol. Lett. 65: 77–82.

    Google Scholar 

  • Bingham SE, Xu R, Webber AN (1991) Transformation of chloroplasts with thepsaB gene encoding a polypeptide of the Photosystem I reaction center. FEBS Lett. 292: 137–140.

    Google Scholar 

  • Blankenship JE, Kindle KL (1992) Expression of chimeric genes by the light-regulatedcabII-1 promoter inChlamydomonas reinhardtii. AcabII-1/nit1 gene functions as a dominant selectable marker in anit1 nit2 strain. Mol. cell. Biol. 12: 5268–5279.

    Google Scholar 

  • Blowers AD, Ellmore GS, Klein U, Bogorad L (1990) Transcriptional analysis of endogenous and foreign genes in chloroplast transformants ofChlamydomonas. Plant Cell 2: 1059–1070.

    Google Scholar 

  • Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM, Jones AR, Randolph-Anderson BL, Robertson D, Klein TM, Shark KM, Sanford JC (1988) Chloroplast transformation inChlamydomonas with high velocity microprojectiles. Science 240: 1534–1538.

    Google Scholar 

  • Boynton JE, Gillham NW, Harris EH, Newman SM, Randolph-Anderson BL, Johnson AM, Jones AR (1990) Manipulating the chloroplast genome ofChlamydomonas: Molecular genetics and transformation. In Baltscheffsky M, (ed.), Current Research in Photosynthesis, Vol. 3. Kluwer Academic Publishers, Dordrecht: 509–516.

  • Breyne P, Montagu M, Depicker A, Gheysen G (1992) Characterization of a plant scaffold attachment region in a DNA fragment that normalizes transgene expression in tobacco. Plant Cell 4: 463–471.

    Google Scholar 

  • Brown LE, Sprecher SL, Keller LR (1991) Introduction of exogenous DNA intoChlamydomonas reinhardtii by electroporation. Mol cell. Biol. 11: 2328–2332.

    Google Scholar 

  • Capecchi MR (1989) The new mouse genetics. Altering the genome by gene targeting. Trends Genet. 5: 70–76.

    Google Scholar 

  • Collis P, Antoniou M, Grosveld F (1990) Definition of the minimal requirements within the human β-globin gene and the dominant control region for high level expression. EMBO J. 9: 233–240.

    Google Scholar 

  • Davies JP, Weeks DP, Grossman AR (1992) Expression of the arylsulfatase gene from β2-tubulin promoter inChlamydomonas reinhardtii. Nucl. Acids Res. 20: 2959–2965.

    Google Scholar 

  • Day A, Debuchy R, van Dillewijn J, Purton S, Rochaix JD (1990) Studies on the maintenance and expression of cloned DNA fragments in the nuclear genome of the green algaChlamydomonas reinhardtii. Physiol. Plantarum 78: 254–260.

    Google Scholar 

  • Debuchy R, Purton S, Rochaix J-D (1989) The argininosuccinate lyase gene ofChlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of thearg7 locus. EMBO J. 8: 2803–2809.

    Google Scholar 

  • Diener DR, Curry AM, Johnson KA, Williams BD, Lefebvre PA, Kindle KL, Rosenbaum JL (1990) Rescue of a paralyzed-flagella mutant ofChlamydomonas by transformation. Proc. natl. Acad. Sci. USA 87: 5739–5743.

    Google Scholar 

  • Dunahay TG (1992) Nuclear transformation ofChlamydomonas reinhardtii with silicon carbide fibers. J. Phycol. 28: 11.

    Google Scholar 

  • Fernández E, Schnell R, Ranum LPW, Hussey SC, Silflow CD, Lefebvre PA (1989) Isolation and characterization of the nitrate reductase structural gene ofChlamydomonas reinhardtii. Proc. natl. Acad. Sci USA 86: 6449–6453.

    Google Scholar 

  • Goldschmidt-Clermont M (1991) Transgenic expression of aminoglycoside adenine transferase in the chloroplast — A selectable marker for site-directed transformation ofChlamydomonas. Nucl. Acids Res. 19: 4083–4090.

    Google Scholar 

  • Goldschmidt-Clermont M, Choquet Y, Girard-Bascou J, Michel F, Schirmer-Rahire M, Rochaix J-D (1991) A small chloroplast RNA may be required for trans-splicing inChlamydomonas reinhardtii. Cell 65: 135–143.

    Google Scholar 

  • Grosveld R, Blom van Assendelft G, Greaves D, Kollias G (1987) Position-independent, high-level expression of the human β-globin gene in transgenic mice. Cell 51: 975–985.

    Google Scholar 

  • Hall LM, Taylor KB, Jones DD (1993) Expression of a foreign gene inChlamydomonas reinhardtii. Gene 124: 75–81.

    Google Scholar 

  • Hasnain SE, Manavathu EK, Leung WC (1985) DNA-mediated transformation ofChlamydomonas reinhardtii cells — Use of aminoglycoside 3′ phosphotransferase as a selectable marker. Mol. cell. Biol. 5: 3647–3650.

    Google Scholar 

  • Heiss S, Johanningmeier U (1992) Analysis of a herbicide resistant mutant obtained by transformation of theChlamydomonas chloroplast. Photosynth. Res. 34: 311–317.

    Google Scholar 

  • Hill KL, Li HH, Singer J, Merchant S (1991) Isolation and structural characterization of theChlamydomonas reinhardtii gene for cytochrome c6. J. biol. Chem. 266: 15060–15067.

    Google Scholar 

  • Kindle KL (1990) High-frequency nuclear transformation ofChlamydomonas reinhardtii. Proc. natl. Acad. Sci USA 87: 1228–1232.

    Google Scholar 

  • Kindle KL, Richards KL, Stern DB (1991) Engineering the chloroplast genome — Techniques and capabilities for chloroplast transformation inChlamydomonas reinhardtii. Proc. natl. Acad. Sci. USA 88: 1721–1725.

    Google Scholar 

  • Kindle KL, Schnell RA, Fernández E, Lefebvre PA (1989) Stable nuclear transformation ofChlamydomonas using theChlamydomonas gene for nitrate reductase. J. Cell Biol. 109: 2589–2601.

    Google Scholar 

  • Kindle KL, Suzuki H, Stern DB (1994) Gene amplification can correct a photosynthetic defect caused by mRNA instability inChlamydomonas chloroplasts. Plant Cell (in press).

  • Koller BH, Smithies O (1992) Altering genes in animals by gene targeting. Ann. Rev. Immunol. 10: 705–730.

    Google Scholar 

  • Kozminski KG, Diener DR, Rosenbaum JL (1993) High level expression of non-acetylatable alpha tubulin inChlamydomonas reinhardtii. Cell Motil. Cytoskeleton 25: 158–170.

    Google Scholar 

  • Mayfield SP, Kindle KL (1990) Stable nuclear transformation ofChlamydomonas reinhardtii by using aC. reinhardtii gene as the selectable marker. Proc. natl. Acad. Sci USA 87: 2087–2091.

    Google Scholar 

  • Merchant S, Bogorad L (1986) Regulation by copper of the expression of cytochrome c552inChlamydomonas reinhardtii. Mol. cell. Biol. 6: 462–469.

    Google Scholar 

  • Mitchell DR, Kang Y (1991) Identification ofoda6 as aChlamydomonas dynein mutant by rescue with the wildtype gene. J. Cell Biol. 113: 835–842.

    Google Scholar 

  • Newman SM, Boynton JE, Gillham NW, Randolph-Anderson BL, Johnson AM, Harris EH (1990) Transformation of chloroplast ribosomal RNA genes inChlamydomonas: Molecular and genetic characterization of integration events. Genetics 126: 875–888.

    Google Scholar 

  • Newman SM, Gillham NW, Harris EH, Johnson AM, Boynton JE (1991) Targeted disruption of chloroplast genes inChlamydomonas reinhardtii. Mol. gen. Genet. 230: 65–74.

    Google Scholar 

  • Phi-Van L, von Kries JP, Ostertag W Strätling WH (1989) The chicken lysozyme 5′ matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes. Mol. cell. Biol. 10: 2302–2307.

    Google Scholar 

  • Przibilla E, Heiss S, Johanningmeier J, Trebst A (1991) Site-specific; mutagenesis of the D1 subunit of photosystem II in wild-typeChlamydomonas. Plant Cell 3: 169–174.

    Google Scholar 

  • Quinn J, Li HH, Singer J, Morimoto B, Mets L, Kindle K, Merchant S (1993) The plastocyanin-deficient phenotype ofChlamydomonas reinhardtii ac-208 results from a frame-shift mutation in the nuclear gene encoding preapoplastocyanin. J. biol. Chem. 268: 7832–7841.

    Google Scholar 

  • Randolph-Anderson BL, Boynton JE, Gillham NW, Harris EH, Johnson AM, Dorthu MP, Matagne RF (1993) Further characterization of the respiratory deficientdum-1 mutation ofChlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation. Mol. gen. Genet. 238: 235–244.

    Google Scholar 

  • Rochaix J-D, van Dillewijn J (1982) Transformation of the green algaChlamydomonas reinhardtii with yeast DNA. Nature 296: 70–73.

    Google Scholar 

  • Sakamoto W, Kindle KL, Stern DB (1993)In vivo analysis ofChlamydomonas chloroplast petD gene expression using stable transformation of β-glucuronidase translational fusions. Proc. natl. Acad. Sci. USA 90: 497–501.

    Google Scholar 

  • Smart EJ, Selman BR (1991) Isolation and characterization of aChlamydomonas reinhardtii mutant lacking the γ-subunit of chloroplast coupling factor CF-1. Mol. cell. Biol. 11: 5053–5058.

    Google Scholar 

  • Smart EJ, Selman BR (1993) Complementation of aChlamydomonas reinhardtii mutant defective in the nuclear gene encoding the chloroplast coupling factor 1 (CF1) γ-subunit (atpC). J. Bioenerg. Biomembr. 25: 275–284.

    Google Scholar 

  • Sodeinde OA, Kindle KL (1993) Homologous recombination in the nuclear genome ofChlamydomonas reinhardtii. Proc. natl. Acad. Sci. USA 90: 9199–9203.

    Google Scholar 

  • Stern DB, Radwanski ER, Kindle KL (1991) A 3′ stem/loop structure of theChlamydomonas chloroplastatpB gene regulates mRNA accumulationin vivo. Plant Cell 3: 285–297.

    Google Scholar 

  • Stief A, Winter DM, Stratling WH, Sippel A (1989) A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature 341: 343–345.

    Google Scholar 

  • Takahashi Y, Goldschmidt-Clermont M, Soen SY, Franzen LG, Rochaix J-D (1991) Directed chloroplast transformation inChlamydomonas reinhardtii — Insertional inactivation of thepsaC gene encoding the iron-sulfure protein destabilizes photosystem I. EMBO J. 10: 2033–2040.

    Google Scholar 

  • Tam L-W Lefebvre PA (1993) The use of DNA insertional mutagenesis to clone genes inChlamydomonas. Genetics 135: 375–384.

    Google Scholar 

  • Ye J, Sayre R (1990) Effects of novobiocin on chloroplast DNA steady-state levels and transformation frequency inChlamydomonas. Plant Physiol. 93: 89.

    Google Scholar 

  • Zhang H, Herman P, Weeks DP (1994) Gene isolation through genomic complementation using an indexed library ofChlamydomonas reinhardtii DNA. Plant Mol. Biol. (in press).

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Kindle, K.L., Sodeinde, O.A. Nuclear and chloroplast transformation inChlamydomonas reinhardtii: strategies for genetic manipulation and gene expression. J Appl Phycol 6, 231–238 (1994). https://doi.org/10.1007/BF02186076

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  • DOI: https://doi.org/10.1007/BF02186076

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