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

---

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

Six mutant strains (301, 102, 203, 104, 305, and 307) affected in their nitrate assimilation capability and their corresponding parental wild-type strains (6145c and 21gr) from Chlamydomonas reinhardii have been studied on different nitrogen sources with respect to NAD(P)H-nitrate reductase and its associated activities (NAD(P)H-cytochrome c reductase and reduced benzyl viologen-nitrate reductase) and to nitrite reductase activity. The mutant strains lack NAD(P)H-nitrate reductase activity in all the nitrogen sources. Mutants 301, 102, 104, and 307 have only NAD(P)H-cytochrome c reductase activity whereas mutant 305 solely has reduced benzyl viologen-nitrate reductase activity. Both activities are repressible by ammonia but, in contrast to the nitrate reductase complex of wild-type strains, require neither nitrate nor nitrite for their induction. Moreover, the enzyme from mutant 305 is always obtained in active form whereas nitrate reductase from wild-types needs to be reactivated previously with ferricyanide to be fully detected. Wild-type strains and mutants 301, 102, 104, and 307, when properly induced, exhibit an NAD(P)H-cytochrome c reductase distinguishable electrophoretically from constitutive diaphorases as a rapidly migrating band. Nitrite reductase from wild-type and mutant strains is also repressible by ammonia and does not require nitrate or nitrite for its synthesis. These facts are explained in terms of a regulation of nitrate reductase synthesis by the enzyme itself.
...
PMID:Regulation of the nitrate-reducing system enzymes in wild-type and mutant strains of Chlamydomonas reinhardii. 681 63

The mRNA accumulation pattern of the Chlamydomonas reinhardtii nitrate assimilation-related gene cluster has been elucidated. In ammonium-grown wild-type cells, nit-1 (nitrate reductase, NR), nar-1, nar-2 and nar-3 (nitrate transporter) genes showed very similar kinetics of expression when transferred to nitrate medium. Transcripts of all these genes accumulated transiently in ammonium-grown wild-type cells after a one-hour incubation in nitrogen-free medium, and practically disappeared at about 2 hours. Mutant strains lacking functional nitrate reductase showed similar accumulation kinetics of these transcripts during both nitrate induction and derepression in nitrogen-free media. In contrast to the other nar transcripts, that nar-4, a gene sharing similar sequences with nar-3, accumulated in small amounts in wild-type cells, and only increased after a long nitrate induction period. Nitrate and light showed a strong positive effect on the accumulation of nit-1 gene transcripts. Acetate as a carbon source allowed accumulation of nit-1 mRNA in the dark, indicating the existence of interactions between light and carbon metabolism in nit-1 gene expression. Our data strongly suggest that NR negatively autoregulates its own expression and that of nar genes.
...
PMID:Expression of nitrate assimilation related genes in Chlamydomonas reinhardtii. 811 Oct 16

The Chlamydomonas reinhardtii nar-2, nar-3, and nar-4 genes, which are within a nitrate-regulated gene cluster containing the nitrate reductase structural gene nit-1, have been related to nitrate transport. Mutant strains defective in nitrate transport and having an active nitrate reductase have been genetically constructed. Their nitrate non-utilizing phenotype has been directly complemented by transformation using the pCO-5 plasmid which carries the nar-2, nar-3, and nar-4 clustered genes. Integration of pCO-5 DNA in the genome of nitrate transport mutants resulted in the expression of these nar transcripts and the recovery of a high affinity nitrate transport activity. Complementation of the nitrate non-utilizing phenotype of the constructed strains was also achieved by co-transformation with plasmids containing nar-2 and nar-3 genes or nar-2 and nar-4, but not with single plasmids containing each individual gene. In addition, DNA sequences of a practically complete cDNA of nar-3 and a partial one of nar-4 have been generated and the deduced amino acid sequences showed a very significant identity with that of the nitrate transporter gene (crnA) from Aspergillus nidulans. These data strongly support the hypothesis that the nitrate transport system in C. reinhardtii contains at least two protein components encoded by the nar-2 and nar-3 genes. The nar-4 gene would produce a protein with a high identity to that of nar-3.
...
PMID:Identification of nitrate transporter genes in Chlamydomonas reinhardtii. 818 Jun 24

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.
...
PMID:The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation. 819 40

Nuclear transformation of intact (walled) cells of the green alga Chlamydomonas reinhardtii was achieved by agitating the cells in the presence of plasmid DNA and silicon carbide (SiC) whiskers. The protocol was used to introduce the wild-type nitrate reductase structural gene into the nitrate reductase-deficient mutant strain nit1-305. Using SiC whiskers, 10-100 transformants per 10(7) cells were routinely produced, which is comparable to transformation rates achieved by agitating the cells with glass beads. In contrast to the glass bead protocol, cell viability was very high following treatment with SiC, with greater than 80% cell survival after agitation for 10 min. Agitation with SiC whiskers appears to be an efficient method for introducing DNA into intact C. reinhardtii cells and may prove to be applicable to other algal species for which cell wall mutants or protoplasting procedures are unavailable.
...
PMID:Transformation of Chlamydomonas reinhardtii with silicon carbide whiskers. 821 58

Chlamydomonas is a popular genetic model system for studying many cellular processes. In this report, we describe a new approach to isolate Chlamydomonas genes using the cloned nitrate reductase gene (NIT1) as an insertional mutagen. A linearized plasmid containing the NIT1 gene was introduced into nit1 mutant cells by glass-bead transformation. Of 3000 Nit+ transformants examined, 74 showed motility defects of a wide range of phenotypes, suggesting that DNA transformation is an effective method for mutagenizing cells. For 13 of 15 such motility mutants backcrossed to nit- mutant strains, the motility phenotype cosegregated with the Nit+ phenotype, indicating that the motility defects of these 13 mutants may be caused by integration of the plasmid. Further genetic analysis indicated that three of these mutants contained alleles of previously identified loci: mbo2 (move backward only), pf13 (paralyzed flagella) and vfl1 (variable flagellar number). Three other abnormal-flagellar-number mutants did not map to any previously described loci at which mutations produce similar phenotypes. Genomic sequences flanking the integrated plasmid in the mbo2 and vfl1 mutants were isolated and used as probes to obtain wild-type genomic clones, which complemented the motility defects upon transformation into cells. Our results demonstrate the potential of this new approach for cloning genes identified by mutation in Chlamydomonas.
...
PMID:Cloning of flagellar genes in Chlamydomonas reinhardtii by DNA insertional mutagenesis. 824 2

Genomic transformation of Chlamydomonas reinhardtii exposed to glass-bead abrasion was accomplished with a chimeric neomycin phosphotransferaseII (NPTII)-encoding gene (nos::npt) flanked by the nopaline synthase promoter and polyadenylation sequences obtained from the Ti plasmid of Agrobacterium tumefaciens. These sequences were in a plasmid (pGA482) which also contained gene nit1 encoding nitrate reductase of C. reinhardtii. Transformants were selected by their ability to grow on medium containing nitrate, and 52% of these was also resistant to kanamycin. Evidence for nos::npt expression includes: (1) hybridization with probes specific for npt, (2) demonstration of NPTII activity after electrophoresis of extracts, and (3) chromatographic identification of the reaction product of NPTII, kanamycin phosphate. The highly biased codon usage in Chlamydomonas does not preclude expression.
...
PMID:Expression of a foreign gene in Chlamydomonas reinhardtii. 838 57

Three overlapping clones covering a Chlamydomonas reinhardtii genomic region of about 32 kb appear to contain five genes potentially involved in nitrate assimilation in addition to the nitrate reductase structural locus nit-1. These new loci produced transcripts of 2.8, 2.2, 1.8 and 1.7 kb in nitrate-induced wild-type cells that, like the 3.4 kb transcript of nit-1, were undetectable in cells grown in ammonium. In addition, in a mutant defective at the regulatory locus, nit-2 for nitrate assimilation, which does not express the nit-1 gene transcript, accumulation of the four other transcripts was also blocked. They have been named nar (nitrate assimilation related) genes. The nar-1 and nar-2 loci are transcribed in the same orientation as nit-1. The nar-3 and nar-4 loci are transcribed divergently from nit-1. DNA and RNA sequences from both nar-3 and nar-4 cross-hybridized with each other indicating that they share similar sequences. Four nitrate assimilation-deficient mutants (C2, D2, F6 and G1) were characterized. These mutants lack nar transcripts and have major deletions and/or rearrangements in the nar gene cluster. In contrast to other nitrate reductase-deficient mutants and to wild type, deletion mutants and the regulatory mutant nit-2 were incapable of accumulating intracellular nitrate. Two of the mutants in which expression of all the nar loci did not occur, C2 and D2, grew in nitrite medium and showed wild-type levels of both nitrite uptake and nitrite reductase activities.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Five nitrate assimilation-related loci are clustered in Chlamydomonas reinhardtii. 841 88

Nuclear transformation of the unicellular green alga Chlamydomonas reinhardtii has thus far been characterized by integration of the introduced DNA into nonhomologous sites. In this study, the occurrence of homologous recombination events during transformation was investigated with the intent of developing strategies for gene targeting and gene disruption. Homologous recombination was monitored by using nonfunctional 5' and 3' deletion derivatives of the wild-type C. reinhardtii nit1 gene (encodes nitrate reductase) as selectable markers (p5' delta and p3' delta respectively) and the low reverting nit1-305 strain as the transformation recipient. After introduction of the DNA into the cell, intermolecular recombination between p5' delta and p3' delta occurs at a high frequency to restore a functional nit1 gene, indicating the presence of homologous recombination machinery in mitotic cells. Gene-targeting events at the nit1 locus were selected by restoring nit1-305 cells to prototrophy after transformation with only p5' delta and were confirmed by analysis of genomic DNA. By comparing the number of transformants obtained after transformation with p5' delta to the number obtained after transformation with a functional nit1 gene, the frequency of homologous-to-random integration events ranged between 1:1000 after glass bead-mediated transformation and 1:24 after bombardment with DNA-coated tungsten microprojectiles.
...
PMID:Homologous recombination in the nuclear genome of Chlamydomonas reinhardtii. 841 77

Ac-208 is a plastocyanin-deficient mutant of Chlamydomonas reinhardtii that contains only 2-3% of the wild-type level of plastocyanin-encoding mRNA and no detectable plastocyanin. Sequence analysis of the ac-208 plastocyanin-encoding gene reveals a single nucleotide insertion in the first exon compared with the wild-type gene; this alters the reading frame and results in a premature nonsense codon. We have introduced the genomic sequence encoding plastocyanin from a wild-type strain into ac-208 by cotransformation with a selectable marker encoding nitrate reductase. Of 22 nit+ transformants characterized, nine contained additional plastocyanin-encoding sequences (compared with untransformed cells) and each of these nine transformants was found to accumulate the protein. Transformants that do not contain newly introduced plastocyanin sequences retain the plastocyanin-deficient phenotype. The introduced plastocyanin-encoding sequences are stable during mitotic growth in liquid culture over a period of several months, as is expression from the introduced sequences. We suggest that the decreased steady state level of plastocyanin-encoding messages is a consequence of the frame-shift mutation in the structural gene. The ability to complement ac-208 with plastocyanin-encoding sequences will allow the introduction and analysis of in vitro mutagenized plastocyanin sequences in vivo in transgenic C. reinhardtii cells.
...
PMID:The plastocyanin-deficient phenotype of Chlamydomonas reinhardtii Ac-208 results from a frame-shift mutation in the nuclear gene encoding preapoplastocyanin. 846 10


<< Previous 1 2 3 4 5 6 7 8 Next >>

---