Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.7.1.2 (nitrate reductase)
3,861 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Spontaneous chlorate-resistant (CR) mutants have been isolated from Chlamydomonas reinhardtii wild-type strains. Most of them, 244, were able to grow on nitrate minimal medium, but 23 were not. Genetic and in vivo complementation analyses of this latter group of mutants indicated that they were defective either at the regulatory locus nit-2, or at the nitrate reductase (NR) locus nit-1, or at very closely linked loci. Some of these nit-1 or nit-2 mutants were also defective in pathways not directly related to nitrate assimilation, such as those of amino acids and purines. Chlorate treatment of wild-type cells resulted in both a decrease in cell survival and an increase in mutant cells resistant to a number of different chemicals (chlorate, methylammonium, sulphanilamide, arsenate, and streptomycin). The toxic and mutagenic effects of chlorate in minimal medium were not found when cells were grown either in darkness or in the presence of ammonium, conditions under which nitrate uptake is drastically inhibited. Chlorate was also able to induce reversion of nit- mutants of C. reinhardtii, but failed to produce His+ revertants or Arar mutants in the BA-13 strain of Salmonella typhimurium. In contrast, chlorate treatment induced mutagenesis in strain E1F1 of the phototrophic bacterium Rhodobacter capsulatus. Genetic analyses of nitrate reductase-deficient CR mutants of C. reinhardtii revealed two types of CR, to low (1.5 mM) and high (15 mM) chlorate concentrations. These two traits were recessive in heterozygous diploids and segregated in genetic crosses independently of each other and of the nit-1 and nit-2 loci. Three hcr loci and four lcr loci mediating resistance to high (HC) and low (LC) concentrations of chlorate were identified. Mutations at the nit-2 locus, and deletions of a putative locus for nitrate transport were always epistatic to mutations responsible for resistance to either LC or HC. In both nit+ and nit- chlorate-sensitive (CS) strains, nitrate and nitrite gave protection from the toxic effect of chlorate. Our data indicate that in C. reinhardtii chlorate toxicity is primarily dependent on the nitrate transport system and independent of the existence of an active NR enzyme. At least seven loci unrelated to the nitrate assimilation pathway and mediating CR are thought to control indirectly the efficiency of the nitrate transporter for chlorate transport. In addition, chlorate appears to be a mutagen capable of inducing a wide range of mutations unrelated to the nitrate assimilation pathway.
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PMID:Toxicity of and mutagenesis by chlorate are independent of nitrate reductase activity in Chlamydomonas reinhardtii. 848 58

During the course of this study a novel family of Chlamydomonas mobile elements has been identified in natural isolate strain 224. The first member of this class to be characterized, a 2.8-kb element named Pioneer1, was trapped in an intron of the nitrate reductase structural gene, NIT1. This element has been cloned and completely sequenced and found to be unusual in structure. Pioneer elements are present in a very low-copy number of three per genome in strain 224. The copy number increased by one upon transposition of Pioneer1. Hybridization of Pioneer1 to a variety of Chlamydomonas strains confirmed that this element differed from previously described Chlamydomonas transposons. It also indicated that related elements are present in low-copy number in natural isolate strains 356 and S1D2, but not in the most commonly used laboratory strains 137c and 21 gr. For these reasons, members of the Pioneer family might prove useful as insertional mutagens.
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PMID:Isolation and characterization of Pioneer1, a novel Chlamydomonas transposable element. 857 15

Plasmid DNA carrying either the nitrate reductase (NR) gene or the argininosuccinate lyase gene as selectable markers and the corresponding Chlamydomonas reinhardtii mutants as recipient strains have been used to isolate regulatory mutants for nitrate assimilation by insertional mutagenesis. Identification of putative regulatory mutants was based on their chlorate sensitivity in the presence of ammonium. Among 8975 transformants, two mutants, N1 and T1, were obtained. Genetic characterization of these mutants indicated that they carry recessive mutations at two different loci, named Nrg1 and Nrg2. The mutation in N1 was shown to be linked to the plasmid insertion. Two copies of the nitrate reductase plasmid, one of them truncated, were inserted in the N1 genome in inverse orientation. In addition to the chlorate sensitivity phenotype in the presence of ammonium, these mutants expressed NR, nitrite reductase and nitrate transport activities in ammonium-nitrate media. Kinetic constants for ammonium (I4C-methylammonium) transport, as well as enzymatic activities related to the ammonium-regulated metabolic pathway for xanthine utilization, were not affected in these strains. The data strongly suggest that Nrg1 and Nrg2 are regulatory genes which specifically mediate the negative control exerted by ammonium on the nitrate assimilation pathway in C. reinhardtii.
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PMID:Isolation and characterization of two new negative regulatory mutants for nitrate assimilation in Chlamydomonas reinhardtii obtained by insertional mutagenesis. 870 50

The small gene family encoding the chlorophyll a/b-binding proteins of photosystem II (CABII or lhcb) is known to exhibit circadian rhythms of mRNA abundance in Chlamydomonas reinhardtii. In this study we investigated the role of transcription in the phenomenon. We used as reporters Chlamydomonas genes that encode nitrate reductase (NITI) and arylsulfatase (ARS2) transcriptionally fused to sequences upstream of one of the CABII genes (called CABII-1). We found that both reporters exhibited the same circadian rhythm of mRNA abundance in phase, period, and amplitude as does the endogenous CABII-1 gene. We also evaluated the efficacy of arylsulfatase enzymatic activity as a reporter and found that its half-life is too long to make it a useful reporter of rhythmic transcription during a circadian or diurnal cycle. The amount of mRNA synthesis from the CABII-1 gene was examined by in vivo labeling experiments and a circadian rhythm in transcription rate was demonstrated. In vivo labeling also revealed a circadian rhythm of mRNA synthesis for the CABII gene family as a whole. The results from the transcriptional reporter assays together with the in vivo labeling experiments strongly support the conclusion that the biological clock regulates the transcriptional activity of the CABII-I gene, and moreover that regulation at the transcriptional level is the predominant mode by which the clock regulates this gene.
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PMID:Transcription of CABII is regulated by the biological clock in Chlamydomonas reinhardtii. 891 33

DNA sequencing in the phage lambda JA13 isolated from a lambda EMBL3 Hansenula polymorpha genomic DNA library containing the nitrate reductase-(YNR1) and nitrite reductase-(YNI1) encoding genes revealed an open reading frame (YNT1) of 1524 nucleotides encoding a putative protein of 508 amino acids with great similarity to the nitrate transporters from Aspergillus nidulans and Chlamydomonas reinhardtii. Disruption of the chromosomal YNT1 copy resulted in incapacity to grow in nitrate and a significant reduction in rate of nitrate uptake. The disrupted strain is still sensitive to chlorate, and, in the presence of 0.1 mM nitrate, the expression of YNR1 and YNI1 and the activity of nitrate reductase and nitrite reductase are significantly reduced compared with the wild-type. Northern-blot analysis showed that YNT1 is expressed when the yeast is grown in nitrate and nitrite but not in ammonium solution.
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PMID:The YNT1 gene encoding the nitrate transporter in the yeast Hansenula polymorpha is clustered with genes YNI1 and YNR1 encoding nitrite reductase and nitrate reductase, and its disruption causes inability to grow in nitrate. 902 Aug 72

In Chlamydomonas reinhardtii, the expression of the nit1 gene encoding nitrate reductase is dependent on the nature of the nitrogen source and on other environmental factors. We have fused the nit1 promoter region to the arylsulphatase (ars) reporter gene lacking its own promoter and introduced this chimeric construction (nit1/ars) into a wall-less strain of C. reinhardtii. A new and sensitive method, based on the use of alpha-naphthylsulphate as a substrate and a diazonium salt as a chromogenic post-coupling agent, was developed to detect the activity of arylsulphatase (an enzyme which is almost completely secreted in the culture medium) both in vitro and in agar plates. The transformants carrying nit1/ars did not express arylsulphatase when grown in ammonium-sufficient medium but readily accumulated the enzyme in ammonium-free medium either supplemented, or not supplemented, with nitrate or nitrite. The nit1/ars construct, however, was not expressed in the nit2 mutant lacking a specific transcription regulator controlling the expression of nit1. These results, together with the observation that the transcription of nit1/ars is initiated at the same sites as the nit1 endogenous gene, confirms the hypothesis that the regulation of nit1 expression takes place mainly at the transcriptional level. The expression of the ars gene from the nit1 promoter was high enough to allow direct measurements of arylsulphatase activities in pools of transformants without prior isolation of nit1/ars clones. This original procedure has permitted the analysis of the effects of nested deletions in the nit1 promoter region on the expression of the reporter gene. The results indicate that the -282 to -198 sequence is required for transcription to occur and that the -751 to -282 region contains several elements mediating nit1 expression.
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PMID:Expression of the arylsulphatase reporter gene under the control of the nit1 promoter in Chlamydomonas reinhardtii. 906 90

In Chlamydomonas reinhardtii, the genes required for nitrate assimilation, including the gene encoding nitrate reductase (NIT1), are subject to repression by ammonia. To study the mechanism of ammonia repression, we employed two approaches to search for mutants with defective repression of NIT1 gene expression. (1) PF14, a gene required for flagellar function, was used as a reporter gene for expression from the NIT1 promoter. When introduced into a pf14 mutant host, the NIT1;PF14 chimeric construct produced a transformant (T10-10B) with a conditional swimming phenotype. Spontaneous mutants with defective ammonia repression of the NIT1 promoter were screened for by isolating cells that gained constitutive motility. (2) Insertional mutagenesis was performed, followed by screening for chlorate sensitivity in the presence of ammonia ion. One insertional mutant and six spontaneous mutants were allelic and defined a new gene, FAR1 (free from ammonia repression). FAR1 was mapped to Linkage Group I, 7.7 cM to the right of the centromere. The far1-1 mutant strain was used to clone DNA adjacent to the site of plasmid insertion, which was then used as a hybridization probe to clone the FAR1 gene from wild type.
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PMID:FAR1, a negative regulatory locus required for the repression of the nitrate reductase gene in Chlamydomonas reinhardtii. 913 6

A family of high-affinity nitrate transporters has been identified in Aspergillus nidulans and Chlamydomonas reinhardtii, and recently homologues of this family have been cloned from a higher plant (barley). Based on six of the peptide sequences most strongly conserved between the barley and C. reinhardtii polypeptides, a set of degenerate primers was designed to permit amplification of the corresponding genes from other plant species. The utility of these primers was demonstrated by RT-PCR with cDNA made from poly(A)+ RNA from barley, C. reinhardtii and Nicotiana plumbaginifolia. A PCR fragment amplified from N. plumbaginifolia was used as probe to isolate a full-length cDNA clone which encodes a protein, NRT2;1Np, that is closely related to the previously isolated crnA homologue from barley. Genomic Southern blots indicated that there are only 1 or 2 members of the Nrt2 gene family in N. plumbaginifolia. Northern blotting showed that the Nrt2 transcripts are most strongly expressed in roots. The effects of external treatments with different N sources showed that the regulation of the Nrt2 gene(s) is very similar to that reported for nitrate reductase and nitrite reductase genes: their expression was strongly induced by nitrate but was repressed when reduced forms of N were supplied to the roots.
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PMID:PCR-identification of a Nicotiana plumbaginifolia cDNA homologous to the high-affinity nitrate transporters of the crnA family. 920 42

In Chlamydomonas reinhardtii, molybdopterin cofactor (MoCo) able to reconstitute active nitrate reductase (NR) with apoenzyme from the Neurospora crassa mutant nit-1 was found mostly bound to a carrier protein (CP). This protein is scarce in the algal free extracts and has been purified 520-fold. MoCoCP is a protein of 64 kDa with subunits of 16.5 kDa and an isoelectric point of 4.5. In contrast to free MoCo, MoCo bound to CP was remarkably protected against inactivation under both aerobic conditions and basic pH. MocoCP transferred active MoCo to apoNR in vitro without addition of molybdate, though reconstituted activity was 20% higher in the presence of molybdate. Incubation with tungstate specifically inhibited MoCoCP activity but had no effect on the activity of free MoCo released from milk xanthine oxidase. MoCoCP did not charge molybdate unless in the presence of N. crassa extracts. Our data support that MoCoCP stabilizes MoCo in an active form charged with molybdate to provide MoCo to apomolybdoenzymes.
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PMID:The Chlamydomonas reinhardtii MoCo carrier protein is multimeric and stabilizes molybdopterin cofactor in a molybdate charged form. 970 3

The NAD(P)H nitrate reductase (NR) from Chlamydomonas reinhardtii is encoded by the structural gene Nia1. Numerous data from the literature indicate that this enzyme is submitted to complex regulation mechanisms involving multiple controls at transcriptional and post-transcriptional levels. To specifically investigate the regulation of the Nia1 gene at the transcriptional level, NR+ and NR- transformed cells harbouring the Nia1:Ars construct (Nia1 promoter fused to the arylsulfatase (ARS)-encoding Ars reporter gene) were cultivated under various experimental conditions and the ARS activities were recorded. ARS levels were very low in cells grown in the presence of NH4Cl and dramatically increased on agar medium deprived of any nitrogen source or containing nitrate, nitrite, urea, arginine or glutamine. Compared to nitrogen-free medium, a slight positive effect of nitrate in the NR+ strain and a significant negative effect of nitrite in both NR+ and NR- strains were observed. The ARS activities were high in the light and very low in the dark or in the light in the presence of DCMU, indicating that Nia1 transcription is strikingly dependent on photosynthetic activity. Acetate used as a carbon source in the dark did not substitute for light in stimulating Nia1:Ars expression. Inactivation of NR by tungstate treatment of the NR+ strain resulted in a dramatic increase of ARS level suggesting that in Chlamydomonas, like in higher plants, active NR negatively regulates the transcription of the NR structural gene. Deleting the major part of the Nia1 leader sequence still present in the chimeric gene resulted in a decrease of ARS level but did not modify the regulation pattern.
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PMID:Transcriptional regulation of the Nia1 gene encoding nitrate reductase in Chlamydomonas reinhardtii: effects of various environmental factors on the expression of a reporter gene under the control of the Nia1 promoter. 1064 29


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