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

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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.

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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

References

  • 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 

  • 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 

  • 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 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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 

  • 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 

  • 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 

  • 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 

  • 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 

  • 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 

  • 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 

  • 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 

  • 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 

  • 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 

  • Harris, E.H. 1989. The Chlamydomonas Sourcebook. Academic Press Inc., San Diego, CA.

    Google Scholar 

  • 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 

  • 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 

  • 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 

  • 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 

  • 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 

  • 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 

  • Kindle, K.L. 1990. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 87: 1228–1232.

    Google Scholar 

  • 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 

  • 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 

  • 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 

  • Matthews, J.C., Hori, K. and Cormier, M.J. 1977. Purification and properties of Renilla reniformis luciferase. Biochemistry 16: 85–91.

    Google Scholar 

  • 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 

  • 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 

  • 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 

  • 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 

  • Prestridge, D.S. 1991. SIGNAL SCAN: a computer program that scans DNA sequences for eukaryotic transcriptional elements. CABIOS 7: 203–206.

    Google Scholar 

  • 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 

  • 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 

  • 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 

  • 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 

  • 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 

  • Terzaghi, W.B. and Cashmore, A.R. 1995. Light-regulated transcription. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46: 445–474.

    Google Scholar 

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Author notes

  1. Amparo Hausherr

    Present address: , Matthias-Mayer-Str. 4, 81379, München, Germany

  2. Lars Ferbitz

    Present address: Institute for Molecular Biology and Biophysics ETHZ, ETH Hoenggerberg, HPK building, 8093, Zuerich, Switzerland

  3. Thomas Schödl

    Present address: GeneArt GmbH, Josef-Engert-Str. 9, 93053, Regensburg, Germany

Authors and Affiliations

  1. Institut für Biochemie I, Universität Regensburg, 93040, Regensburg, Germany

    Markus Fuhrmann, Amparo Hausherr, Lars Ferbitz, Thomas Schödl & Peter Hegemann

  2. Center of excellence for fluorescent bioanalysis, Biotechnology Division, Universität Regensburg, Josef-Engert-Str. 9, 93053, Regensburg, Germany

    Markus Fuhrmann & Markus Heitzer

Authors
  1. Markus Fuhrmann
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  2. Amparo Hausherr
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Correspondence to Markus Fuhrmann.

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Fuhrmann, M., Hausherr, A., Ferbitz, L. et al. Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene. Plant Mol Biol 55, 869–881 (2004). https://doi.org/10.1007/s11103-005-2150-1

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  • DOI: https://doi.org/10.1007/s11103-005-2150-1

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