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)

In Chlamydomonas reinhardtii, expression of the cabII-1 gene increases dramatically in response to light (cabII-1 encodes one of the light-harvesting chlorophyll a/b-binding proteins of photosystem II). We have used a region upstream of the cabII-1 gene in translational fusions to the bacterial uidA gene (encodes beta-glucuronidase) and transcriptional fusions to the Chlamydomonas nitrate reductase gene (nit1). Chlamydomonas transformants carrying intact copies of the chimeric uidA gene do not express beta-glucuronidase at the level of enzyme activity or mRNA accumulation. Methylation in the cabII-1 promoter region of the introduced gene is extensive in these strains, suggesting that newly introduced foreign genes may be recognized and silenced by a cellular mechanism that is correlated with increased methylation. Transformants that express the chimeric cabII-1/nit1 gene have been recovered. In contrast to the endogenous nit1 gene, the chimeric cabII-1/nit1 gene is expressed in ammonium-containing medium. Moreover, nit1 mRNA accumulation is dramatically stimulated by light, with a time course that is indistinguishable from that of the endogenous cabII-1 gene. The cabII-1/nit1 gene has been used to select transformants in a nit1- nit2- Chlamydomonas strain (CC400G) and should be useful for transformation of the large number of mutants in the Ebersold-Levine lineage, which carry the same mutations.
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PMID: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. 140 96

A Chlamydomonas reinhardtii molybdenum cofactor (MoCo)-carrier protein (CP), capable of reconstituting nitrate reductase activity with apoprotein from the Neurospora crassa mutant nit-1, was subjected to experiments of diffusion through a dialysis membrane and gel filtration. CP bonded firmly MoCo and did not release it efficiently unless aponitrate reductase was present in the incubation mixture. Stability of MoCo bound to CP against air and heat was very similar to that of free-MoCo released from milk xanthine oxidase. Our data strongly suggest that MoCo is directly transferred from CP to aponitrate reductase to form an active enzyme.
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PMID:Direct transfer of molybdopterin cofactor to aponitrate reductase from a carrier protein in Chlamydomonas reinhardtii. 164 69

Molybdenum cofactor (MoCo) of molybdoenzymes is constitutively produced in cells of the green alga Chlamydomonas reinhardtii grown in ammonium media, under which conditions certain molybdoenzymes are not synthesized. In soluble form, MoCo was found to be present in several forms: (i) as a low Mr free species; (ii) bound to a MoCo-carrier protein of about 50 kDa that could release MoCo to directly reconstitute in vitro nitrate reductase activity in the nit-1 mutant of Neurospora crassa, but not to Thiol-Sepharose which, in contrast, bonded free MoCo; and (iii) bound to other proteins, putatively constitutive molybdoenzymes, which only released MoCo after a denaturing treatment. The amount of total MoCo (free, carrier-bound and heat releasable forms) was dependent on the growth phase of cell cultures. Constitutive levels of total MoCo in ammonium-grown cells markedly increased when cells were transferred to media lacking ammonium (nitrate, urea or nitrogen-free media). This increase did not require de novo protein synthesis and was stimulated by light. Levels of both total MoCo and free plus carrier-bound MoCo seemed to be unrelated to either nitrate reductase synthesis or functioning of nit-1 and nit-2 genes responsible for nitrate reductase structure and regulation, respectively. Results suggest that MoCo is continuously synthesized in C. reinhardtii and that its levels are regulated by ammonium in a way independent of nitrate reductase synthesis.
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PMID:Regulation of molybdenum cofactor species in the green alga Chlamydomonas reinhardtii. 182 14

By using a method in which cell-wall-deficient Chlamydomonas reinhardtii cells were agitated in the presence of DNA, glass beads, and polyethylene glycol, nuclear transformation rates of approximately 10(3) transformants per micrograms of plasmid DNA were achieved. The nitrate reductase gene from wild-type Chlamydomonas was used to complement a mutation in the corresponding gene of a strain containing nit1-305. Transformants were selected by growth with nitrate as sole source of nitrogen. The transforming DNA integrated into the genome at a low-copy number in nit+ transformants. When cells carrying nit1-305 were agitated in the presence of two plasmids, one with the gene for nitrate reductase and the second with an unselected gene, the unselected gene was present in 10-50% of nit+ transformants. This high frequency of cotransformation will allow any cloned gene to be introduced into Chlamydomonas. Moreover, the overall efficiency of transformation should be high enough to permit isolation of genes from genomic libraries by complementation of stable nuclear mutants. The availability of efficient nuclear and chloroplast transformation in Chlamydomonas provides specific advantages for the study of chloroplast biogenesis, photosynthesis, and nuclear-chloroplast genome interactions.
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PMID:High-frequency nuclear transformation of Chlamydomonas reinhardtii. 210 99

The biflagellate alga Chlamydomonas has been used extensively in the genetic and biochemical analysis of flagellar assembly and motility. We have restored motility to a paralyzed-flagella mutant of Chlamydomonas by transforming with the corresponding wild-type gene. A nitrate reductase-deficient paralyzed-flagella strain, nit1-305 pf-14, carrying mutations in the genes for nitrate reductase and radial spoke protein 3, was transformed with wild-type copies of both genes. Two-thirds of the cells that survived nitrate selection also regained motility, indicating that they had been transformed with both the nitrate reductase and radial spoke protein 3 genes. Transformants typically contained multiple copies of both genes, genetically linked to each other, but not linked to the original mutant loci. Complementation of paralyzed-flagella mutants by transformation is a powerful tool for investigating flagellar assembly and function.
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PMID:Rescue of a paralyzed-flagella mutant of Chlamydomonas by transformation. 237 11

The nitrate reductase structural gene of Chlamydomonas reinhardtii has been isolated from a genomic library by using a nitrate reductase cDNA probe from barley. Restriction fragment length polymorphism analyses mapped the Chlamydomonas clone (B6a) to the nitrate reductase structural gene locus nit-1. Overlapping inserts cover a region of the genome of about 24 kilobases containing the entire gene, which spans approximately 5-8 kilobases. Sequence analysis of DNA fragments from the B6a clone demonstrated a high degree of sequence similarity at the amino acid level with regions corresponding to portions of the heme and FAD/NADH-binding domains of tobacco and Arabidopsis thaliana nitrate reductases and human NADH cytochrome b5 reductase. The identity of the cloned gene as nitrate reductase was confirmed by its ability to complement a nit-1 mutation upon transformation. The nitrate reductase gene produced a 3.4-kilobase transcript in cells derepressed with nitrate; the transcript was undetectable in cells grown in the presence of ammonium. In cells that contain a mutation in the putative regulatory gene nit-2, significantly lower levels of the 3.4-kilobase transcript were found, indicating that the wild-type nit-2 gene is involved in the control of nitrate reductase transcript levels.
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PMID:Isolation and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii. 247 71

We have developed a nuclear transformation system for Chlamydomonas reinhardtii, using micro-projectile bombardment to introduce the gene encoding nitrate reductase into a nit1 mutant strain which lacks nitrate reductase activity. By using either supercoiled or linear plasmid DNA, transformants were recovered consistently at a low efficiency, on the order of 15 transformants per microgram of plasmid DNA. In all cases the transforming DNA was integrated into the nuclear genome, usually in multiple copies. Most of the introduced copies were genetically linked to each other, and they were unlinked to the original nit1 locus. The transforming DNA and nit+ phenotype were stable through mitosis and meiosis, even in the absence of selection. nit1 transcripts of various sizes were expressed at levels equal to or greater than those in wild-type nit+ strains. In most transformants, nitrate reductase enzyme activity was expressed at approximately wild-type levels. In all transformants, nit1 mRNA and nitrate reductase enzyme activity were repressed in cells grown on ammonium medium, showing that expression of the integrated nit1 genes was regulated normally. When a second plasmid with a nonselectable gene was bombarded into the cells along with the nit1 gene, transformants carrying DNA from both plasmids were recovered. In some cases, expression of the unselected gene could be detected. With the advent of nuclear transformation in Chlamydomonas, it becomes the first photosynthetic organism in which both the nuclear and chloroplast compartments can be transformed.
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PMID:Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. 259 99

A new methylammonium-resistant mutant strain from Chlamydomonas reinhardtii, henceforth termed 2172 (ma-2), has been isolated. This mutant is affected in a single mendelian gene different from and linked to the ma-1 locus which is defective in the methylammonium-resistant mutant 2170. Both mutations in ma-1 (2170) and ma-2 (2172) are linked to the nit-1 gene coding for the nitrate reductase apoenzyme. Mutant 2172 is affected in methylammonium but not in ammonium uptake capacity and shows derepressed nitrate and nitrite reductase activities in media containing nitrate plus methylammonium but not in nitrate plus ammonium media. The following two enzymatic components for the transport of both ammonium and methylammonium in wild-type cells have been identified: component 1, with high Vmax and K values, which is constitutive, and component 2, with low Vmax and K values, which is ammonium-repressible. Mutant 2170 lacks component 1 whereas mutant 2172 lacks component 2 for both methylammonium and ammonium transport. From genetic and kinetic evidences we conclude that in C. reinhardtii two different carriers are responsible for the transport of both ammonium and methylammonium and that methylammonium (ammonium) transport is a reversible process probably inhibited by the intracellular ammonium which, in turn, regulates nitrate and nitrite reductase levels.
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PMID:Two different carriers transport both ammonium and methylammonium in Chlamydomonas reinhardtii. 317 May 37

The inhibitor of mRNA synthesis, 6-methylpurine, inhibited nitrate reductase derepression in either ammonium-grown or methylammonium-treated wild-type cells of Chlamydomonas reinhardtii, but not in nitrogen-starved cells. In contrast, 6-methylpurine did not inhibit nitrate reductase synthesis in the methylammonium-resistant mutant 2170 (ma-1) either grown on ammonium, treated with methylammonium or nitrogen starved, but did inhibit the continuous synthesis of nitrate reductase, which required the presence of nitrate in the media. In both wild-type and mutant 2170 grown on ammonium and transferred to nitrate media, cycloheximide immediately prevented nitrate reductase derepression when added either at the beginning or at different times of induction treatment. Unlike wild-type cells, mutant 2170 was able to take up either nitrate or nitrite simultaneously with ammonium in whose presence nitrate and nitrite reductases were synthesized. However, synthesis of nitrate reductase was progressively inhibited in the mutant cells when the intracellular ammonium levels were raised as a result of an increase in either the external pH or the extracellular ammonium concentrations. The results rule out the existence of maturase-like proteins in Chlamydomonas and indicate that ammonium has a double effect on the regulation of nitrate reductase synthesis: (a) it prevents nitrate reductase mRNA production; and (b) it controls negatively the expression of this mRNA.
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PMID:Regulation by ammonium of nitrate and nitrite assimilation in Chlamydomonas reinhardtii. 319 Nov 35

In vitro complementation of the soluble assimilatory NAD(P)H-nitrate reductase (NAD(P)H:nitrate oxidoreductase, EC 1.6.6.2) was attained by mixing cell-free preparations of Chlamydomonas reinhardii mutant 104, uniquely possessing nitrate-inducible NAD(P)H-cytochrome c reductase, and mutant 305 which possesses solely the nitrate-inducible FMNH2- and reduced benzyl viologen-nitrate reductase activities. Full activity and integrity of NAD(P)H-cytochrome c reductase from mutant 104 and reduced benzyl viologen-nitrate reductase from mutant 305 are needed for the complementation to take place. A constitutive and heat-labile molybdenum-containing cofactor, that reconstitutes the NAD(P)H-nitrate reductase activity of nit-1 Neurospora crassa but is incapable of complementing with 104 from C. reinhardii, is present in the wild type and 305 algal strains. The complemented NAD(P)H-nitrate reductase has been purified 100-fold and was found to be similar to the wild enzyme in sucrose density sedimentation, molecular size, pH optimum, kinetic parameters, substrate affinity and sensitivity to inhibitors and temperature. From previous data and data presented in this article on 104 and 305 mutant activities, it is concluded that C. reinhardii NAD(P)H-nitrate reductase is a heteromultimeric complex consisting of, at least, two types of subunits separately responsible for the NAD(P)H-cytochrome c reductase and the reduced benzyl viologen-nitrate reductase activities.
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PMID:In vitro complementation of assimilatory NAD(P)H-nitrate reductase from mutants of Chlamydomonas reinhardii. 645 69


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