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Plant Molecular Biology

, Volume 55, Issue 6, pp 869–881 | Cite as

Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene

  • Markus Fuhrmann
  • Amparo Hausherr
  • Lars Ferbitz
  • Thomas Schödl
  • Markus Heitzer
  • Peter Hegemann
Article

Abstract

For monitoring the expression profile of selected nuclear genes in Chlamydomonas reinhardtii in response to altered environmental parameters or during cell cycle, in the past many RNA or protein samples had to be taken and analyzed by RNA hybridization or protein immunoblotting. Here we report the synthesis of a gene that codes for the luciferase of Renilla reniformis (RLuc) and is adapted to the nuclear codon usage of C. reinhardtii. This crluc gene was expressed alone or as a fusion to the zeocin resistance gene ble under control of different promoter variants. Luciferase activity was monitored in living cells, increased with the promoter strength and paralleled the amount of expressed protein. Under control of the Lhcb-1 promoter the Luc-activity in synchronized cultures was dependent on the dark-light cycle. Additionally, crluc was placed under control of the Chop-2 promoter and activity was measured under different light conditions. Chop-2 promoter activity was found to be most pronouced under low-light and dark conditions, further supporting that channelrhodopsin-2 is most active in dark-adapted cells. We conclude that crluc is a reliable tool for convenient monitoring of nuclear gene expression in C. reinhardtii.

Key words

bioluminescence channelrhodopsin-2 Chop-2 gene expression synthetic gene 

Abbreviations

Chop-1/2

channelopsin-1/2

ChR1/2

channelrhodopsin-1/2

HSM

high salt medium

Lhc

light-harvesting complex

Luc

luciferase

mAb

monoclonal antibody

RbcS2

small subunit of ribulosebisphosphate-carboxylase/oxygenase

RCF

relative codon frequency

RLU

relative luminescence units

TAP

Tris/acetate/phosphate medium

UTR

untranslated region

wt

wild type

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References

  1. Airth, R.L., Rhodes, W.C. and McElroy, W.D. 1958. The function of CoA in luminescence. Biochem. Biophy. Acta 27: 519–532.Google Scholar
  2. Berthold, P., Schmitt, R. and Mages, W. 2002. An engineered Streptomyces hygroscopicus aph 7″ gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. Protist 153: 401–412.Google Scholar
  3. Cerutti, H., Johnson, A.M., Gillham, N.W. and Boynton, J.E. 1997a. A eubacterial gene conferring spectinomycin resistance on Chlamydomonas reinhardtii: integration into the nuclear genome and gene expression. Genetics 145: 97–110.Google Scholar
  4. Cerutti, H., Johnson, A.M., Gillham, N.W. and Boynton, J.E. 1997b. Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas. Plant Cell 9: 925–945.CrossRefPubMedGoogle Scholar
  5. Debuchy, R., Purton, S. and Rochaix, J.D. 1989. 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. 8: 2803–2809.Google Scholar
  6. Degenhardt, J. and Tobin, E.M. 1996. A DNA binding activity for one of two closely defined phytochrome regulatory elements in an Lhcb promoter is more abundant in etiolated than in green plants. Plant Cell 8: 31–41.Google Scholar
  7. Dumas, P., Bergdoll, M., Cagnon, C. and Masson, J.-M. 1994. Crystal structure and site directed mutagenesis of a bleomycine resistance protein and their significance for drug sequestering. EMBO J. 13: 2492–2499.Google Scholar
  8. Ender, F., Godl, K., Wenzl, S. and Sumper, M. 2002. Evidence for autocatalytic cross-linking of hydroproline-rich glycoproteins during extracellular matrix assembly in Volvox. Plant Cell 14: 1147–1180.Google Scholar
  9. Fuhrmann, M., Oertel, W. and Hegemann, P. 1999. A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant J. 19: 353–361.Google Scholar
  10. Goldschmidt-Clermont, M. and Rahire M. 1986. Sequence, evolution and differential expression of the two genes encoding variant small subunits of ribulose bisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii. J. Mol. Biol. 191: 421–432.Google Scholar
  11. Goodenough, U.W. 1971. The effects of inhibitors of RNA and protein synthesis on chloroplast structure and function in wild-type Chlamydomonas reinhardi. J. Cell Biol. 50: 35–49.Google Scholar
  12. Govorunova, E.G., Jung, K.H., Sineshchekov, O.A. and Spudich, J.L. 2004. Chlamydomonas sensory rhodopsins A and B: cellular content and role in photophobic responses. Biophys. J. 86: 2342–2349.Google Scholar
  13. Hahn, D. and Kück, U. 1999. Identification of DNA sequences controlling light and chloroplast dependent expression of the lhcb1 gene from Chlamydomonas reinhardtii. Curr. Genet. 34: 459–466.Google Scholar
  14. Hallmann, A. and Sumper, M. 1994. An arylsulfatase of Volvox carteri with properties suitable for a reporter gene system. Eur. J. Biochem. 221: 143–150.Google Scholar
  15. Harris, E.H. 1989. The Chlamydomonas Sourcebook. Academic Press Inc., San Diego, CA.Google Scholar
  16. Higo, K., Ugawa, Y., Iwamoto, M. and Korenaga, T. 1999. Plant cis-acting regulatory DNA elements (PLACE) database. Nucl. Acids Res. 27: 297–300.Google Scholar
  17. Hollis, R.P., Lagido, C., Pettitt, J., Porter, A.J., Killham, K., Paton, G.I. and Glover L.A. 2001. Toxicity of the bacterial luciferase substrate, n-decyl aldehyde, to Saccharomyces cerevisiae and Caenorhabditis elegans. FEBS Lett. 506: 140–142.Google Scholar
  18. Jakobshagen, S. and Johnson, C.H. 1994. Circadian rhythm of gene expression in Chlamydomonas reinhardtii: circadian cycling of mRNA abundance of lhcB and possibly of beta tubulin and cytochrome c. Eur. J. Cell Biol. 64: 142–152.Google Scholar
  19. Jakobshagen, S., Kindle, K.L. and Johnson, C.H. 1996. Transcription of cabII is regulated by the biological clock in Chlamydomonas reinhardtii. Plant Mol. Biol. 31: 1173–1184.Google Scholar
  20. Johanningmeier, U. and Howell, S.H. 1984. Regulation of light-harvesting chlorophyll-binding protein mRNA accumulation in Chlamydomonas reinhardi. Possible involvement of chlorophyll synthesis precursors. J. Biol. Chem. 259: 13541–13549.Google Scholar
  21. Kindle, K.L. 1987. Expression of a gene for a light-harvesting chlorophyll a/b-binding protein in Chlamydomonas reinhardtii: effect of light and acetate. Plant Mol. Biol. 9: 547–563.Google Scholar
  22. Kindle, K.L. 1990. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 87: 1228–1232.Google Scholar
  23. Lechtreck, K.F., Rostmann, J. and Grunow, A. 2002. Analysis of Chlamydomonas SF-assemblin by GFP tagging and expression of antisense constructs. J. Cell Sci. 115: 1511–1522.Google Scholar
  24. Lorenz, W.W., McCann, R.O., Longiaru, M. and Cormier, M.J. 1991. Isolation and expression of a cDNA encoding Renilla reniformis luciferase. Proc. Natl. Acad. Sci. USA 88: 4438–4442.Google Scholar
  25. Lumbreras, V., Stevens, D.R. and Purton, S. 1998. Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. Plant J. 14: 441–447.Google Scholar
  26. Matthews, J.C., Hori, K. and Cormier, M.J. 1977. Purification and properties of Renilla reniformis luciferase. Biochemistry 16: 85–91.Google Scholar
  27. Mayfield, S.P. and Schultz, J. 2004. Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast. Plant J. 37: 449–458.Google Scholar
  28. Minko, I., Holloway, S.P., Nikaido, S., Carter, M., Odom, O.W., Johnson, C.H. and Herrin, D.L. 1999. Renilla luciferase as a vital reporter for chloroplast gene expression in Chlamydomonas. Mol. Gen. Genet. 262: 421–425.Google Scholar
  29. Nagel, G., Szellas, T., Huhn, W., Kateriya, S., Adeishvili, N., Berthold, P., Ollig, D., Hegemann, P. and Bamberg, E. 2003. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl. Acad. Sci. USA 100: 13940–13945.Google Scholar
  30. Piechulla, B., Merforth, N. and Rudolph, B. 1998. Identification of tomato Lhc promoter regions necessary for circadian expression. Plant Mol. Biol. 38: 655–662.Google Scholar
  31. Prestridge, D.S. 1991. SIGNAL SCAN: a computer program that scans DNA sequences for eukaryotic transcriptional elements. CABIOS 7: 203–206.Google Scholar
  32. Ruiz-Binder, N.E., Geimer, S. and Melkonian, M. 2002. In vivo localization of centrin in the green alga Chlamydomonas reinhardtii. Cell Motil. Cytoskel. 52: 43–55.Google Scholar
  33. Schroda, M., Beck, C. and Vallon, O. 2002. Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas. Plant J. 31: 445–455.Google Scholar
  34. Sizova, I., Fuhrmann, M. and Hegemann, P. 2001. A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to the green alga C. reinhardtii. Gene 277: 221–229.Google Scholar
  35. Stables, J., Scott, S., Brown, S., Roelant, C., Burns, D., Lee, M.G. and Rees, S. 1999. Development of a dual glow-signal firefly and Renilla luciferase assay reagent for the analysis of G-protein coupled receptor signalling. J. Recept. Signal Transduct. Res. 19: 395–410.Google Scholar
  36. Stevens, D.R., Rochaix, J.-D. and Purton, S. 1996. The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas. Mol. Gen. Genet. 251: 23–30.Google Scholar
  37. Terzaghi, W.B. and Cashmore, A.R. 1995. Light-regulated transcription. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46: 445–474.Google Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Markus Fuhrmann
    • 1
    • 2
  • Amparo Hausherr
    • 1
  • Lars Ferbitz
    • 1
  • Thomas Schödl
    • 1
  • Markus Heitzer
    • 2
  • Peter Hegemann
    • 1
  1. 1.Institut für Biochemie IUniversität RegensburgRegensburgGermany
  2. 2.Center of excellence for fluorescent bioanalysis, Biotechnology DivisionUniversität RegensburgRegensburgGermany

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