Skip to main content
SpringerLink
Log in

Gaussia-luciferase as a sensitive reporter gene for monitoring promoter activity in the nucleus of the green alga Chlamydomonas reinhardtii

  • Original Paper
  • Published:
Molecular Genetics and Genomics Aims and scope Submit manuscript

Abstract

For the model organism Chlamydomonas reinhardtii, a codon-adapted gene variant of the extracellular luciferase of Gaussia princeps was generated as a sensitive molecular tool to study gene expression from the nuclear genome. In the past, monitoring promoter activity in Chlamydomonas employing the commonly used luciferase encoded by Renilla reniformis was hampered due to the detection limit of the reporter assay, especially if analyzing weak promoters. In this work, the expression of Gaussia-luciferase from such promoters resulted in an average luminescent activity at least 500 times higher than that detected for the Renilla enzyme. The wildtype signal peptide of Gaussia princeps efficiently mediated the export of the luciferase into the culture medium of Chlamydomonas strain cw15arg −, and the characterization of the secreted protein showed an unexpected temperature instability, probably arising from post-translational modifications made by the algae. To further test the utility of Gaussia-luciferase, promoter sequences originating from different viral genomes were analyzed for their ability to drive transgene expression in Chlamydomonas. Solely, the 35S-promoter of the Cauliflower mosaic virus (CaMV) displayed a significant transcriptional activity and this happened only when the shunting region of the 5′-untranslated region of the 35S-sequence was omitted from the luciferase expression cassette. Gaussia-luciferase proved to be a superior quantifiable reporter gene for the analysis of constitutive promoter sequences in Chlamydomonas reinhardtii.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Babinger K, Hallmann A, Schmitt R (2006) Translational control of regA, a key gene controlling cell differentiation in Volvox carteri. Development 133:4045–4051

    Article  PubMed  CAS  Google Scholar 

  • Battraw MJ, Hall TC (1990) Histochemical analysis of CaMV 35S promoter-beta-glucuronidase gene expression in transgenic rice plants. Plant Mol Biol 15:527–538

    Article  PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  • Croft MT, Moulin M, Webb ME, Smith AG (2007) Thiamine biosynthesis in algae is regulated by riboswitches. Proc Natl Acad Sci USA 104:20770–20775

    Article  PubMed  CAS  Google Scholar 

  • Davies JP, Weeks DP, Grossman AR (1992) Expression of the arylsulfatase gene from the beta 2-tubulin promoter in Chlamydomonas reinhardtii. Nucleic Acids Res 20:2959–2965

    Article  PubMed  CAS  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 alga Chlamydomonas reinhardtii. Physiol Plant 78:254–260

    Article  CAS  Google Scholar 

  • de Hostos EL, Togasaki RK, Grossman A (1988) Purification and biosynthesis of a derepressible periplasmic arylsulfatase from Chlamydomonas reinhardtii. J Cell Biol 106:29–37

    Article  PubMed  Google Scholar 

  • Fuhrmann M (2002) Expanding the molecular toolkit for Chlamydomonas reinhardtii—from history to new frontiers. Protist 153:357–364

    Article  PubMed  Google Scholar 

  • Fuhrmann M, Hausherr A, Ferbitz L, Schodl T, Heitzer M, Hegemann P (2004) Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene. Plant Mol Biol 55:869–881

    PubMed  CAS  Google Scholar 

  • Fuhrmann M, Oertel W, 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

    Article  PubMed  CAS  Google Scholar 

  • Griffiths CM, Page MJ (1997) Production of heterologous proteins using the baculovirus/insect expression system. Methods Mol Biol 75:427–440

    PubMed  CAS  Google Scholar 

  • Harris EH (1989) The Chlamydomonas sourcebook: a comprehensive guide to biology and laboratory use. Academic Press, San Diego

    Google Scholar 

  • Harris EH (2001) Chlamydomonas as a model organism. Annu Rev Plant Physiol Plant Mol Biol 52:363–406

    Article  PubMed  CAS  Google Scholar 

  • Hasnain SE, Manavathu EK, Leung WC (1985) DNA-mediated transformation of Chlamydomonas reinhardi cells: use of aminoglycoside 3′-phosphotransferase as a selectable marker. Mol Cell Biol 5:3647–3650

    PubMed  CAS  Google Scholar 

  • Heitzer M, Zschoernig B (2007) Construction of modular tandem expression vectors for the green alga Chlamydomonas reinhardtii using the Cre/lox-system. Biotechniques 43:324, 326, 328 passim

  • Inouye S, Sahara Y (2008) Identification of two catalytic domains in a luciferase secreted by the copepod Gaussia princeps. Biochem Biophys Res Commun 365:96–101

    Article  PubMed  CAS  Google Scholar 

  • Julenius K, Molgaard A, Gupta R, Brunak S (2005) Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites. Glycobiology 15:153–164

    Article  PubMed  CAS  Google Scholar 

  • Kang M, Duncan DD, van Etten JL (2005) Chlorovirus: a genus of Phycodnaviridae that infects certain chlorella-like green algae. Mol Plant Pathol 6:213–224

    Article  CAS  Google Scholar 

  • Kang M, Han JG, Liu PF, Ye Y, Tien P (2000) [The regulation activity of Chlorella virus gene 5′ upstream sequence in Escherichia coli and eucaryotic alage]. Sheng Wu Gong Cheng Xue Bao 16:443–446

    PubMed  CAS  Google Scholar 

  • Kiaulehn S, Voytsekh O, Fuhrmann M, Mittag M (2007) The presence of UG-repeat sequences in the 3′-UTRs of reporter luciferase mRNAs mediates circadian expression and can determine acrophase in Chlamydomonas reinhardtii. J Biol Rhythms 22:275–277

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Kobayashi K, Tsuge S, Stavolone L, Hohn T (2002) The Cauliflower mosaic virus virion-associated protein is dispensable for viral replication in single cells. J Virol 76:9457–9464

    Article  PubMed  CAS  Google Scholar 

  • Kutish GF, Li Y, Lu Z, Furuta M, Rock DL, Van Etten JL (1996) Analysis of 76 kb of the chlorella virus PBCV-1 330-kb genome: map positions 182 to 258. Virology 223:303–317

    Article  PubMed  CAS  Google Scholar 

  • Matsuo T, Onai K, Okamoto K, Minagawa J, Ishiura M (2006) Real-time monitoring of chloroplast gene expression by a luciferase reporter: evidence for nuclear regulation of chloroplast circadian period. Mol Cell Biol 26:863–870

    Article  PubMed  CAS  Google Scholar 

  • Mayfield SP, Kindle KL (1990) Stable nuclear transformation of Chlamydomonas reinhardtii by using a C. reinhardtii gene as the selectable marker. Proc Natl Acad Sci USA 87:2087–2091

    Article  PubMed  CAS  Google Scholar 

  • Mayfield SP, Schultz J (2004) Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast. Plant J 37:449–458

    Article  PubMed  CAS  Google Scholar 

  • Minko I, Holloway SP, Nikaido S, Carter M, Odom OW, Johnson CH, Herrin DL (1999) Renilla luciferase as a vital reporter for chloroplast gene expression in Chlamydomonas. Mol Gen Genet 262:421–425

    Article  PubMed  CAS  Google Scholar 

  • Narva KE, Wendell DL, Skrdla MP, Van Etten JL (1987) Molecular cloning and characterization of the gene encoding the DNA methyltransferase, M.CviBIII, from Chlorella virus NC-1A. Nucleic Acids Res 15:9807–9823

    Article  PubMed  CAS  Google Scholar 

  • Ow DW, Jacobs JD, Howell SH (1987) Functional regions of the Cauliflower mosaic virus 35S RNA promoter determined by use of the firefly luciferase gene as a reporter of promoter activity. Proc Natl Acad Sci USA 84:4870–4874

    Article  PubMed  CAS  Google Scholar 

  • Pooggin MM, Futterer J, Skryabin KG, Hohn T (2001) Ribosome shunt is essential for infectivity of Cauliflower mosaic virus. Proc Natl Acad Sci USA 98:886–891

    Article  PubMed  CAS  Google Scholar 

  • Rajam MV, Kumar SV (2006) Green Alga (Chlamydomonas reinhardtii). Methods Mol Biol 344:421–433

    PubMed  CAS  Google Scholar 

  • Rochaix JD (1995) Chlamydomonas reinhardtii as the photosynthetic yeast. Annu Rev Genet 29:209–230

    Article  PubMed  CAS  Google Scholar 

  • Ryabova LA, Hohn T (2000) Ribosome shunting in the Cauliflower mosaic virus 35S RNA leader is a special case of reinitiation of translation functioning in plant and animal systems. Genes Dev 14:817–829

    PubMed  CAS  Google Scholar 

  • Schroda M (2006) RNA silencing in Chlamydomonas: mechanisms and tools. Curr Genet 49:69–84

    Article  PubMed  CAS  Google Scholar 

  • Shao N, Krieger-Liszkay A, Schroda M, Beck CF (2007) A reporter system for the individual detection of hydrogen peroxide and singlet oxygen: its use for the assay of reactive oxygen species produced in vivo. Plant J 50:475–487

    Article  PubMed  CAS  Google Scholar 

  • Southern PJ, Berg P (1982) Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet 1:327–341

    PubMed  CAS  Google Scholar 

  • Stevens DR, Rochaix JD, Purton S (1996) The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas. Mol Gen Genet 251:23–30

    PubMed  CAS  Google Scholar 

  • Tabor S, Richardson CC (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 82:1074–1078

    Article  PubMed  CAS  Google Scholar 

  • Tang DK, Qiao SY, Wu M (1995) Insertion mutagenesis of Chlamydomonas reinhardtii by electroporation and heterologous DNA. Biochem Mol Biol Int 36:1025–1035

    PubMed  CAS  Google Scholar 

  • Tannous BA, Kim DE, Fernandez JL, Weissleder R, Breakefield XO (2005) Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol Ther 11:435–443

    Article  PubMed  CAS  Google Scholar 

  • Vallon O, Wollman FA (1995) Mutations affecting O-glycosylation in Chlamydomonas reinhardtii cause delayed cell wall degradation and sex-limited sterility. Plant Physiol 108:703–712

    PubMed  CAS  Google Scholar 

  • Verhaegent M, Christopoulos TK (2002) Recombinant Gaussia luciferase. Overexpression, purification, and analytical application of a bioluminescent reporter for DNA hybridization. Anal Chem 74:4378–4385

    Article  PubMed  CAS  Google Scholar 

  • Wiles S, Ferguson K, Stefanidou M, Young DB, Robertson BD (2005) Alternative luciferase for monitoring bacterial cells under adverse conditions. Appl Environ Microbiol 71:3427–3432

    Article  PubMed  CAS  Google Scholar 

  • Wurm F, Bernard A (1999) Large-scale transient expression in mammalian cells for recombinant protein production. Curr Opin Biotechnol 10:156–159

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We would like to thank Markus Fuhrmann for designing the cgluc gene and Meike Priesmeier-Gradl for perfect technical assistance. This work was supported by the German Federal Ministry of Education and Research and the Bavarian Ministry of Economic Affairs, Infrastructure, Transport and Technology.

Author information

Author notes

  1. Markus Heitzer

    Present address: Geneart AG, Josef-Engert-Str. 11, 93053, Regensburg, Germany

Authors and Affiliations

  1. Center of Excellence for Fluorescent Bioanalysis, University of Regensburg, Josef-Engert-Str. 9, 93053, Regensburg, Germany

    Ovidiu Ruecker, Karina Zillner, Regina Groebner-Ferreira & Markus Heitzer

Authors
  1. Ovidiu Ruecker
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Karina Zillner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Regina Groebner-Ferreira
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Markus Heitzer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Markus Heitzer.

Additional information

Communicated by C. Dieckmann.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ruecker, O., Zillner, K., Groebner-Ferreira, R. et al. Gaussia-luciferase as a sensitive reporter gene for monitoring promoter activity in the nucleus of the green alga Chlamydomonas reinhardtii. Mol Genet Genomics 280, 153–162 (2008). https://doi.org/10.1007/s00438-008-0352-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-008-0352-3

Keywords

Search

Navigation