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

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-2promoter and activity was measured under different light conditions. Chop-2promoter 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.

[1]  Peter Berthold,et al.  An engineered Streptomyces hygroscopicus aph 7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. , 2002, Protist.

[2]  W. D. Mcelroy,et al.  The function of coenzyme A in luminescence. , 1958, Biochimica et biophysica acta.

[3]  P. Hegemann,et al.  A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. , 2001, Gene.

[4]  M. Schroda,et al.  Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas. , 2002, The Plant journal : for cell and molecular biology.

[5]  M. Melkonian,et al.  In vivo localization of centrin in the green alga Chlamydomonas reinhardtii. , 2002, Cell motility and the cytoskeleton.

[6]  P. Hegemann,et al.  A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. , 1999, The Plant journal : for cell and molecular biology.

[7]  U. Kück,et al.  Identification of DNA sequences controlling light- and chloroplast-dependent expression of the lhcb1 gene from Chlamydomonas reinhardtii , 1999, Current Genetics.

[8]  C. Johnson,et al.  Transcription ofCABII is regulated by the biological clock inChlamydomonas reinhardtii , 1996, Plant Molecular Biology.

[9]  Yoshihiro Ugawa,et al.  Plant cis-acting regulatory DNA elements (PLACE) database: 1999 , 1999, Nucleic Acids Res..

[10]  Dan S. Prestridge,et al.  SIGNAL SCAN: a computer program that scans DNA sequences for eukaryotic transcriptional elements , 1991, Comput. Appl. Biosci..

[11]  H. Cerutti,et al.  A eubacterial gene conferring spectinomycin resistance on Chlamydomonas reinhardtii: integration into the nuclear genome and gene expression. , 1997, Genetics.

[12]  A. Hallmann,et al.  An inducible arylsulfatase of Volvox carteri with properties suitable for a reporter-gene system. Purification, characterization and molecular cloning. , 1994, European journal of biochemistry.

[13]  K. Kindle.  High-frequency nuclear transformation of Chlamydomonas reinhardtii. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[14]  U. Goodenough.  THE EFFECTS OF INHIBITORS OF RNA AND PROTEIN SYNTHESIS ON CHLOROPLAST STRUCTURE AND FUNCTION IN WILD-TYPE CHLAMYDOMONAS REINHARDI , 1971, The Journal of cell biology.

[15]  M. J. Cormier,et al.  Isolation and expression of a cDNA encoding Renilla reniformis luciferase. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[16]  C. Johnson,et al.  Transcription of CABII is regulated by the biological clock in Chlamydomonas reinhardtii. , 1996, Plant molecular biology.

[17]  M. J. Cormier,et al.  Purification and properties of Renilla reniformis luciferase. , 1977, Biochemistry.

[18]  K. Kindle.  Expression of a gene for a light-harvesting chlorophyll a/b-binding protein in Chlamydomonas reinhardtii: effect of light and acetate , 1987, Plant Molecular Biology.

[19]  Kwang-Hwan Jung,et al.  Chlamydomonas sensory rhodopsins A and B: cellular content and role in photophobic responses. , 2004, Biophysical journal.

[20]  H. Cerutti,et al.  Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas. , 1997, The Plant cell.

[21]  E. Bamberg,et al.  Channelrhodopsin-2, a directly light-gated cation-selective membrane channel , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[22]  M. Rahire,et al.  Sequence, evolution and differential expression of the two genes encoding variant small subunits of ribulose bisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii. , 1986, Journal of molecular biology.

[23]  P. Dumas,et al.  Crystal structure and site‐directed mutagenesis of a bleomycin resistance protein and their significance for drug sequestering. , 1994, The EMBO journal.

[24]  K. Killham,et al.  Toxicity of the bacterial luciferase substrate, n‐decyl aldehyde, to Saccharomyces cerevisiae and Caenorhabditis elegans , 2001, FEBS letters.

[25]  S. P. Holloway,et al.  Renilla luciferase as a vital reporter for chloroplast gene expression in Chlamydomonas , 1999, Molecular and General Genetics MGG.

[26]  J Stables,et al.  Development of a dual glow-signal firefly and Renilla luciferase assay reagent for the analysis of G-protein coupled receptor signalling. , 1999, Journal of receptor and signal transduction research.

[27]  J. Rochaix,et al.  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. , 1989, The EMBO journal.

[28]  B. Piechulla,et al.  Identification of tomato Lhc promoter regions necessary for circadian expression , 1998, Plant Molecular Biology.

[29]  W. Terzaghi,et al.  Light-Regulated Transcription , 1995 .

[30]  S. Mayfield,et al.  Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast. , 2004, The Plant journal : for cell and molecular biology.

[31]  E. Tobin,et al.  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. , 1996, The Plant cell.

[32]  V. Lumbreras,et al.  Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron , 1998 .

[33]  K. Lechtreck,et al.  Analysis of Chlamydomonas SF-assemblin by GFP tagging and expression of antisense constructs. , 2002, Journal of cell science.

[34]  M. Sumper,et al.  Evidence for Autocatalytic Cross-Linking of Hydroxyproline-Rich Glycoproteins during Extracellular Matrix Assembly in Volvox Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.000711. , 2002, The Plant Cell Online.

[35]  S. Howell,et al.  Regulation of light-harvesting chlorophyll-binding protein mRNA accumulation in Chlamydomonas reinhardi. Possible involvement of chlorophyll synthesis precursors. , 1984, The Journal of biological chemistry.

[36]  J. Rochaix,et al.  The bacterial phleomycin resistance gene , 1996 .

[37]  C. Johnson,et al.  Circadian rhythms of gene expression in Chlamydomonas reinhardtii: circadian cycling of mRNA abundances of cab II, and possibly of beta-tubulin and cytochrome c. , 1994, European journal of cell biology.