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  • 1
    ISSN: 1432-0983
    Keywords: Neurospora ; Tomato ; Nitrate reductase ; Nitrate regulation ; GATA-binding factor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The nit-2 gene of Neurospora crassa encodes a trans-acting regulatory protein that activates the expression of a number of structural genes which code for nitrogen catabolic enzymes, including nitrate reductase. The NIT2 protein contains a Cys2/Cys2-type zinc-finger DNA-binding domain that recognizes promoter regions of the Neurospora nitrogen-related genes. The NIT2 zincfinger domain/β-Gal fusion protein was shown to recognize and bind in a specific manner to two upstream fragments of the nia gene of Lycopersicon esculentum (tomato) in vitro, whereas two mutant NIT2 proteins failed to bind to the same fragments. The dissociation kinetics of the complexes formed between the NIT2 protein and the Neurospora nit-3 and the tomato nia gene promoters were examined; NIT2 binds more strongly to the nit-3 promoter DNA fragment than it does to fragments derived from the plant nitrate reductase gene itself. The observed specificity of the binding suggests the existence of a NIT2-like homolog which regulates the expression of the nitrate assimilation pathway of higher plants.
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  • 2
    ISSN: 1617-4623
    Keywords: Nicotiana plumbaginifolia ; Nicotiana tabacum ; GATA-binding factor ; Nitrate reductase ; PCR
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In higher plants, the expression of the nitrate assimilation pathway is highly regulated. Although the molecular mechanisms involved in this regulation are currently being elucidated, very little is known about the trans-acting factors that allow expression of the nitrate and nitrite reductase genes which code for the first enzymes in the pathway. In the fungus Neurospora crassa, nit-2, the major nitrogen regulatory gene, activates the expression of unlinked structural genes that specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein containing a single zinc finger motif defined by the C-X2-CX17-C-X2-C sequence. This DNA-binding domain recognizes the promoter region of N. crassa nitrogen-related genes and fragments derived from the tomato nia gene promoter. The observed specificity of the binding suggests the existence of a NIT2-like homolog in higher plants. PCR and cross-hybridization techniques were used to isolate, respectively, a partial cDNA from Nicotiana plumbaginifolia and a full-length cDNA from Nicotiana tabacum. These clones encode a NIT2-like protein (named NTL1 for nit-2-like), characterized by a single zinc finger domain, defined by the C-X2-C-X18-C-X2-C amino acids, and associated with a basic region. The amino acid sequence of NTL1 is 60% homologous to the NIT2 sequence in the zinc finger domain. The Ntl1 gene is present as a unique copy in the diploid N. plumbaginifolia species. The characteristics of Ntl1 gene expression are compatible with those of a regulator of the nitrate assimilation pathway, namely weak nitrate inducibility and regulation by light.
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  • 3
    ISSN: 1617-4623
    Keywords: Key words Methylammonium ; Mutant ; Nicotiana plumbaginifolia ; Nitrate uptake
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  This work reports the isolation and preliminary characterization of Nicotiana plumbaginifolia mutants resistant to methylammonium. Nicotiana plumbaginifolia plants cannot grow on low levels of nitrate in the presence of methylammonium. Methylammonium is not used as a nitrogen source, although it can be efficiently taken up by Nicotiana plumbaginifolia cells and converted into methylglutamine, an analog of glutamine. Glutamine is known to repress the expression of the enzymes that mediate the first two steps in the nitrate assimilatory pathway, nitrate reductase (NR) and nitrite reductase (NiR). Methylammonium has therefore been used, in combination with low concentrations of nitrate, as a selective agent in order to screen for mutants in which the nitrate pathway is de-repressed. Eleven semi-dominant mutants, all belonging to the same complementation group, were identified. The mutant showing the highest resistance to methylammonium was not affected either in the utilization of ammonium, accumulation of methylammonium or in glutamine synthase activity. A series of experiments showed that utilization of nitrite by the wild-type and the mutant was comparable, in the presence or the absence of methylammonium, thus suggesting that the mutation specifically affected nitrate transport or reduction. Although NR mRNA levels were less repressed by methylammonium treatment of the wild-type than the mutant, NR activities of the mutant remained comparable with or without methylammonium, leading to the hypothesis that modified expression of NR is probably not responsible for resistance to methylammonium. Methylammonium inhibited nitrate uptake in the wild-type but had only a limited effect in the mutant. The implications of these results are discussed.
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  • 4
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: A mutant screen was developed to isolate Arabidopsis thaliana mutants affected in the regulation of the nitrate assimilation pathway. A fusion between the tobacco Nii1 gene (that encodes a foliar nitrite reductase involved in nitrate assimilation) and the Gus reporter gene was introduced into A. thaliana, and shown to be properly regulated by nitrate. Moreover, β-glucuronidase (GUS) activity in the transgenic plants was essentially detected in the cotyledons and leaves, showing that the organ-specific expression of the tobacco Nii1 gene was retained in Arabidopsis. M2 plantlets derived from mutagenized seeds homozygous for the Nii-Gus fusion were screened by histochemical staining of whole plates for GUS activity after growth on nitrate or glutamine. About 250 progenies were screened, leading to the isolation of plants showing an enhanced or reduced staining compared to the control non-mutagenized plants. Several mutants were analyzed for the transmission of the phenotype to the M3 generation, as well as for levels of GUS or nitrite reductase activities or mRNA levels. A major problem encountered during the screening was the high background of false positives that reproducibly showed altered GUS histochemical staining compared to control plants and did not, however, display any changes in GUS activity levels. One interesting family of mutants was isolated that overexpressed GUS activity and Nii mRNA in the absence of nitrate. These mutants turned out to be cnx mutants impaired in the molybdenum cofactor biosynthesis that is necessary for nitrate reductase activity. These results may indicate that active nitrate reductase is necessary for a correct regulation of nitrate assimilation genes by nitrate.
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  • 5
    ISSN: 1432-1211
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Mouse embryonal carcinoma (EC) cells do not express the major H-2 class I transplantation antigens. The latter, however, become detectable upon in vitro differentiation of EC cells. Neither class I H-2 genes nor the gene coding for beta-2 microglobulin (β 2m) is transcribed in EC cells. We have constructed two hybrid plasmids containing the 5′ flanking region of an H-2K b gene followed by the coding regions of either the herpes simplex virus thymidine kinase (H-2 tk) or the chloramphenicol acetyl transferase (H-2 CAT) genes. Upon transfer into EC cells, the H-2 tk hybrid gene is expressed in F9 tk− cell lines which thus acquire a stable tk+ phenotype. When such transformed clones are induced to differentiate in vitro, tk activity shows a moderate increase, which reflects an increase in transcription of the hybrid gene. In transient transformation experiments, EC cells were found to express the H-2 CAT hybrid gene as well. We conclude that the 2 kilobase pair region of the H-2K b gene which we used contains an active promoter region, but does not include all the elements required for the correct regulation of the H-2K b gene.
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  • 6
    ISSN: 1432-2048
    Keywords: Key words:Arabidopsis ; Gene expression (differential display) ; Gene regulation (N-source) ; Nitrate transporter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. We used the differential display technique on total RNAs from roots of Arabidopsis thaliana (L.) Heynh. plants which had or had not been induced for 2 h by nitrate. One isolated cDNA clone, designated Nrt2:1At, was found to code for a putative high-affinity nitrate transporter. Two genomic sequences homologous to Nrt2:1At were found to be localized on the same fragment of chromosome 1 in the Arabidopsis genome. Expression analyses of both low- and high-affinity nitrate transporter genes, respectively Nrt1:1At (previously named Chl1) and Nrt2:1At, were carried out on plants grown under different nitrogen regimes. In this paper, we show that both genes are induced by very low levels of nitrate (50 μM KNO3). However, stronger induction was observed with Nrt2:1At than with Nrt1:1At. Moreover, these two genes, although both over-expressed in a nitrate-reductase-deficient mutant, were differently regulated when N-sufficient wild-type or mutant plants were transferred to an N-free medium. Indeed, the steady-state amounts of Nrt1:1At mRNA declined whereas the amount of Nrt2:1At mRNA increased, probably reflecting the de-repression of the high-affinity transport system during N-starvation.
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  • 7
    ISSN: 1573-5028
    Keywords: gene family ; gene expression ; photoreceptor ; phytochrome ; tomato
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Data presented here define five tomato phytochrome genes (PHY) and indicate the existence of additional PHY in the tomato genome. Portions of each gene, encoding amino acids 203 through 315 in a consensus amino acid sequence, were amplified by polymerase chain reaction. Four of these genes, PHYA, PHYB1, PHYB2 and PHYE, are members of previously identified PHY subfamilies, while the fifth, PHYF, is identified as a member of a new PHY subfamily. PHYA, PHYB1, PHYB2 and PHYE fragments encode amino acid sequences that share 88% to 98% sequence identity with their Arabidopsis counterparts. The PHYF fragment, however, encodes a polypeptide that shares only 65% to 74% sequence identity with previously identified Arabidopsis phytochromes. A phylogenetic analysis suggests that PHYF arose soon after, or perhaps prior to, the origin of angiosperms. This analysis leads to the prediction that PHYF might be widespread among angiosperms, including both monocotyledons and dicotyledons. Each of the five tomato PHY is expressed as a transcript of sufficient size to encode a full-length phytochrome apoprotein. Two PHYF transcripts, 4.4 and 4.7 kb in length, have been detected in 9-day-old light-grown seedlings, consistent with either multiple transcription start sites or differential processing. Analyses of genomic Southern blots hybridized with radiolabelled RNA probes derived from the five tomato PHY, as well as Arabidopsis PHYC, indicate that the tomato genome contains as many as 9 to 13 PHY. The tomato PHY family is apparently not only different from, but also larger than, the PHY family presently described for Arabidopsis.
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  • 8
    ISSN: 1573-5028
    Keywords: barley ; Chlamydomonas reinhardtii ; Nicotiana plumbaginifolia ; nitrate transport ; Nrt2 expression ; RT-PCR
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 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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  • 9
    ISSN: 1573-5028
    Keywords: budding yeast ; fission yeast ; nitrate reductase ; transcription initiation ; β-galactosidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A fragment comprising 1 kb of the 5′ region and the 81 first nucleotides of the coding region of the tomato nitrate reductase nia gene was placed in translational fusion with the lacZ reporter gene. This construct was introduced in budding and in fission yeast using a derivative of the Saccharomyces cerevisiae/Schizosaccharomyces pombe autonomously replicating vector pUZL. β-galactosidase activity was detected in S. pombe but not in S. cerevisiae. Primer extension experiments show that in fission yeast transcripts are initiated at the same starting point as in tomato, indicating for the first time that a plant promoter can be correctly recognized in fission yeast.
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  • 10
    ISSN: 1573-5028
    Keywords: nia gene ; regulation ; tomato ; transgenic plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A nitrate reductase (NR) deficient mutant of Nicotiana plumbaginifolia totally impaired in the production of functional nia transcript and protein was restored for NR activity by transformation with a cloned tomato nia gene. The transgenic plants expressed from undetectable to 17% of the control NR activity in their leaves. Restoration of growth rates comparable to the wild type was obtained for transgenic plants expressing as little as 10% of the wild-type activity showing that nitrate reduction is not a growth-limiting factor in the wild-type plant. The analysis of the transgene expression showed that the tomato nia gene transcription was regulated by light, nitrate and a circadian rhythm as in tomato plants. These results suggest that all the cis-acting sequences involved in these regulations are contained in the 3 kb upstream region of the tomato nia gene and are still functional in transgenic N. plumbaginifolia plants. The amount of NR transcript synthesized from the tomato nia gene was reduced when a functional N. plumbaginifolia nia locus was introduced by sexual crosses. These data support the hypothesis that nitrate reduction is regulated by nitrate-derived metabolites as demonstrated in fungi.
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