Expression of the arylsulphatase reporter gene under the control of the nit1 promoter in Chlamydomonas reinhardtii

Ohresser, Marc; Matagne, René-Fernand; Loppes, Roland

doi:10.1007/s002940050204

Cited by 103 publications

(82 citation statements)

References 27 publications

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“…Hereafter in the text, mutant 20.73 will therefore be referred to as cyg56. AI phenotype of this mutant was determined by ARS activity assays (Ohresser et al, 1997) after growth on solid medium containing different sources of nitrogen as previously described (Gonzá lezBallester et al, 2005a).…”

Section: Resultsmentioning

confidence: 99%

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A Soluble Guanylate Cyclase Mediates Negative Signaling by Ammonium on Expression of Nitrate Reductase in Chlamydomonas

et al. 2010

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Nitrate assimilation in plants and related organisms is a highly regulated and conserved pathway in which the enzyme nitrate reductase (NR) occupies a central position. Although some progress has been made in understanding the regulation of the protein, transcriptional regulation of the NR gene (NIA1) is poorly understood. This work describes a mechanism for the ammonium-mediated repression of NIA1. We report the characterization of a mutant defective in the repression of NIA1 and NR in response to ammonium and show that a gene (CYG56) coding for a nitric oxide (NO)-dependent guanylate cyclase (GC) was interrupted in this mutant. NO donors, cGMP analogs, a phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), and a calcium ionophore (A23187) repress the expression of NIA1 in Chlamydomonas reinhardtii wild-type cells and also repress the expression of other ammonium-sensitive genes. In addition, the GC inhibitors LY83,583 (6-anilino-5,8-quinolinedione) and ODQ (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one) release cells from ammonium repression. Intracellular NO and cGMP levels were increased in the presence of ammonium in wild-type cells. In the cyg56 mutant, NIA1 transcription was less sensitive to NO donors and A23187, but responded like the wild type to IBMX. Results presented here suggest that CYG56 participates in ammonium-mediated NIA1 repression through a pathway that involves NO, cGMP, and calcium and that similar mechanisms might be occurring in plants.

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Section: Resultsmentioning

confidence: 99%

“…ARS activity was determined directly on the agar plates after removing cells from the agar surface with a razor blade and following the procedure previously reported (Ohresser et al, 1997). The reaction was stopped after 15 min.…”

Section: Ars Activitymentioning

confidence: 99%

A Soluble Guanylate Cyclase Mediates Negative Signaling by Ammonium on Expression of Nitrate Reductase in Chlamydomonas

et al. 2010

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“…The use of a reporter gene fused to a promoter of interest is a useful tool for studying dynamic changes in gene expression and to characterize cis-acting factors, like promoter or enhancer elements. Such a system utilizing the Ars gene as the reporter has been employed widely in C. reinhdartii to investigate transcriptional regulation of genes involved in, for example, nitrate metabolism (Ohresser et al 1997), carbon-concentrating mechanisms (Kucho et al 2003), copper sensing (Quinn et al 2000), flagella regeneration (Kang and Mitchell 1998) and reactive oxygen signalling (Leisinger et al 2001). The usual underlying assumption in all these studies is that the promoter region used in making the 'positive control' construct contains all the elements required for transcriptional activation of the gene under investigation.…”

Section: Discussionmentioning

confidence: 99%

“…Aliquots (1 ml) of cells were collect and stored at À20 C. Thawed samples were pelleted and the supernatant used to assay Ars activity according to Ohresser et al (1997). In a total volume of 500 ml, the reaction mixture contained 400 ml supernatant, 0.4 M glycine-NaOH pH 9.0, 10 mM imidazole and 0.8 mM a-naphthylsulfate.…”

Section: Measurements Of Ars Activitymentioning

confidence: 99%

Characterization of the alternative oxidase of Chlamydomonas reinhardtii in response to oxidative stress and a shift in nitrogen source

Molen

Rosso

Piercy

et al. 2006

Physiologia Plantarum

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To study the transcriptional regulation of Aox1, the major alternative oxidase (AOX) gene, we fused 1072 bp of its promoter to the promoterless arylsulfatase reporter gene. We find that the reporter is strongly activated when cells are shifted from a medium containing ammonium to one containing nitrate but is unresponsive to treatments known to induce Aox1 in higher plants -H 2 O 2 , antimycin A and cold stress. However, induction of Aox1 by all these factors was found when changes in gene expression were monitored using RNA blot analysis. Our data suggest that transcriptional upregulation of Aox1 by H 2 O 2 , antimycin A and cold-stress requires one or more enhancers which are not found in the proximal promoter region of the gene. Interestingly, while nitrate, H 2 O 2 and cold stress result in increased AOX abundance and increased alternative pathway respiration, these changes are not seen when cells are treated with antimycin A. We find that H 2 O 2 , antimycin A and cold-stress result in an increase in intracellular reactive oxygen species (ROS) but that a significant change in ROS does not occur when cells are shifted into a medium-containing nitrate. Overall, our data are consistent with there being two distinct pathways regulating Aox1 transcription and AOX abundance in Chlamydomonas reinhardtii: one in response to oxidative stress and a second due to metabolic changes brought about by a shift in nitrogen source from ammonium to nitrate.

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“…The promoter of NIT1 is negatively regulated by ammonium; in the presence of ammonium, NIT1 transcripts do not accumulate (16). NIT1-regulated transcripts are induced in ammonium-free medium and enhanced by nitrate (40,49). The NIT1 promoter and genomic and cDNA fragments of GSK3 were obtained by using PCR.…”

Section: Methodsmentioning

confidence: 99%

Regulation of Flagellar Assembly by Glycogen Synthase Kinase 3 in Chlamydomonas reinhardtii

2004

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Chlamydomonas reinhardtii controls flagellar assembly such that flagella are of an equal and predetermined length. Previous studies demonstrated that lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), induced flagellar elongation, suggesting that a lithium-sensitive signal transduction pathway regulated flagellar length (S. Nakamura, H. Takino, and M. K. Kojima, Cell Struct. Funct. 12: [369][370][371][372][373][374] 1987). Here, we demonstrate that lithium treatment depletes the pool of flagellar proteins from the cell body and that the heterotrimeric kinesin Fla10p accumulates in flagella. We identify GSK3 in Chlamydomonas and demonstrate that its kinase activity is inhibited by lithium in vitro. The tyrosine-phosphorylated, active form of GSK3 was enriched in flagella and GSK3 associated with the axoneme in a phosphorylation-dependent manner. The level of active GSK3 correlated with flagellar length; early during flagellar regeneration, active GSK3 increased over basal levels. This increase in active GSK3 was rapidly lost within 30 min of regeneration as the level of active GSK3 decreased relative to the predeflagellation level. Taken together, these results suggest a possible role for GSK3 in regulating the assembly and length of flagella.How cells regulate the size of their organelles is poorly understood. Cilia and flagella are of particular interest, as the precise regulation of their length is critical to their function. The importance of the correct assembly of these organelles, including maintenance of the appropriate length, is illustrated by studies involving intraflagellar transport (IFT). IFT is required for the assembly and maintenance of cilia and flagella in organisms ranging from Chlamydomonas reinhardtii (where it was first identified) to sea urchins, Tetrahymena thermophila, Caenorhabditis elegans, and mice (10). In the absence of IFT, cilia and flagella fail to form correctly, resulting in various human diseases, including polycystic kidney disease and retinal degeneration, and anomalous determination of the left-right axis during development (51). Ultrastructural studies of children suffering from a particular form of primary ciliary dyskinesia revealed the presence of abnormally long cilia within their respiratory tracts, resulting in recurrent respiratory infections (1, 39). This observation suggests that not only do cilia and flagella need to assemble properly but that they also need to be of appropriate and defined lengths.The green alga Chlamydomonas reinhardtii makes an ideal model organism for studies involving ciliary and flagellar assembly and length control. Chlamydomonas has two apically localized flagella that are maintained at equal lengths. Moreover, within a population of wild-type cells, flagellar lengths fall within a very narrow range. The maintenance of flagella of equal lengths as an active process was first demonstrated in the "long-zero" experiments of Rosenbaum et al. (52). In these experiments, cells were deflagellated under conditions that amputated only one...

show abstract

Expression of the arylsulphatase reporter gene under the control of the nit1 promoter in Chlamydomonas reinhardtii

Cited by 103 publications

References 27 publications

A Soluble Guanylate Cyclase Mediates Negative Signaling by Ammonium on Expression of Nitrate Reductase in Chlamydomonas

A Soluble Guanylate Cyclase Mediates Negative Signaling by Ammonium on Expression of Nitrate Reductase in Chlamydomonas

Characterization of the alternative oxidase of Chlamydomonas reinhardtii in response to oxidative stress and a shift in nitrogen source

Regulation of Flagellar Assembly by Glycogen Synthase Kinase 3 in Chlamydomonas reinhardtii

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