Isolation and characterization of cell lines of Nicotiana tabacum lacking nitrate reductase

Summary Chlorate-resistant cell lines were established from survivors after plating allodihaploid cells of Nicotiana tabacum into solid medium containing 20 mM chlorate and amino acids as sole nitrogen source. Data characterizing 9 of the most resistant lines are presented. The mutational origin of these lines was inferred on the basis of the enhancement of the variant frequency by mutagen treatment, and of the persistance of the variant phenotype in cell progeny during growth in the absence of selection for more than 3 years and in plants regenerated from two of the lines.Seven lines completely lacked in vivo nitrate reductase (NR) activity and two lines exhibited low (less than 5% of the wild type) NR activity. The abolition of NR activity was found to be not due to an impaired induction by nitrate. Data reported elsewhere show that one of the NR-negative mutants simultaneously lacks xanthine dehydrogenase activity. This pleiotropic mutation is interpreted to affect the synthesis of a molybdenum-containing cofactor, whereas the 8 other lines carry mutations specifically affecting the synthesis of the NR. Both types of NR-negative mutants were unable to grow on minimal medium containing nitrate as sole nitrogen source, but grew well on amino acids. They proved extremely sensitive to the standard medium containing nitrate and ammonium. Differences between the NR-negative mutants with respect to chlorate resistance suggest that chlorate inhibits cultured N tabacum cells not only via its NR-catalysed conversion to chlorite, but also by NR-independent mechanisms.     

Characterization of a new type of molybdenum cofactor-mutant in cell cultures of Nicotiana tabacum

Summary Four allelic putative cnx (molybdenum-cofactor defective) cell lines (O42, P12, P31 and P47) of Nicotiana tabacum var. Xanthi, biochemically and genetically distinct from N. tabacum var. Gatersleben cnxA mutants, were examined further. Their molybdenum-cofactor could efficiently reconstitute NADPH-nitrate reductase activity from Neurospora crassa mutant nit-1 extract only in the presence of exogenous molybdenum unlike that of the wild-type cofactor which could reconstitute NADPH-nitrate reductase activity in either the absence or presence of exogenous molybdenum. Loss of cofactor activity in vivo was not due to a defect in molybdenum uptake into the cells. In vitro nitrate reductase complementation between extracts of each of these four lines and a nia mutant showed that they possessed a functional nitrate reductase haemoflavoprotein subunit. Both constitutive molybdenum cofactor and NADH cytochrome c reductase activity were derepressed in the four cell lines. These results show that the four cell lines are indeed altered at a cnx locus, called cnxB, that the defect is probably in molybdenum processing and that there is a link between synthesis of functional molybdenum cofactor and nitrate reductase aporprotein.     

Nitrate reductase-deficient mutant cell lines of Nicotiana tabacum

Summary The wild-type line and 14 nitrate reductase-deficient mutant cell lines of Nicotiana tabacum were tested for the presence of nitrate reductase partial activities, and for nitrite reductase and xanthine dehydrogenase activity. Data characterizing the electron donor specificity of nitrate reductase (EC 1.6.6.1., NADH:nitrate oxidoreductase) and nitrite reductase (EC 1.7.7.1., ferredoxin:nitrite oxidoreductase) of the wild-type line are presented. Three lines (designated cnx) simultaneously lack NADH-, FADH₂-, red. benzyl viologen-nitrate reductase, and xanthine dehydrogenase activities, but retain the nitrate reductase-associated NADH-cytochrome c reductase activity. These mutants are, therefore, interpreted to be impaired in gene functions essential for the synthesis of an active molybdenum-containing cofactor. For cnx-68 and cnx-101, the sedimentation coefficient of the defective nitrate reductase molecules does not differ from that of the wild-type enzyme (7.6S). In 11 lines (designated nia) xanthine dehydrogenase activity is unaffected, and the loss of NADH-nitrate reductase is accompanied by a loss of all partial activities, including NADH-cytochrome c reductase. However, one line (nia-95) was found to possess a partially active nitrate reductase molecule, retaining its FADH₂- and red. benzyl viologen nitrate reductase activity. It is likely that nia-95 is a mutation in the structural gene for the apoprotein. Both, the nia and cnx mutant lines exhibit nitrite reductase activity, being either nitrate-inducible or constitutive. Evidence is presented that, in Nicotiana tabacum, nitrate, without being reduced to nitrite, is an inducer of the nitrate assimilation pathway.     

Non-coordinate expression of the neighbouring genes rplL and rpoB,C of Escherichia coli.

Summary Two types of nitrate reductase-deficient mutant cell lines (nia and cnx) of Nicotiana tabacum have been used for in vitro reconstitution of NADH-nitrate reductase. The cnx mutants simultaneously lack NADH-,FADH₂-, red benzyl viologen-nitrate reductase, and xanthine dehydrogenase activities, but retain the nitrate reductase-associated NADH-cytochrome c reductase activity. These mutants are interpreted to be defective in the molybdenum-containing cofactor necessary for nitrate reductase activity. In the nia lines xanthine dehydrogenase activity is unaffected, and the loss of NADH-nitrate reductase is accompanied by a loss of all partial activities of nitrate reductase, including NADH-cytochrome c reductase. When cnx cells (induced by nitrate) were homogenized together with nia cells (induced by nitrate or uninduced), NADH-nitrate reductase activity was detectable in the cell extract. No nitrate reductase was observed when the cnx mutants were homogenized together, or after cohomogenization of the nia mutants. Thus, the inactive nitrate reductase molecule formed in the cnx mutants has been complemented in vitro with the molybdenum-containing cofactor supplied by nia extracts, thus giving rise to NADH-nitrate reductase activity. This result gives additional support to the interpretation that the active nitrate reductase of Nicotiana tabacum is composed of at least the NADH-cytochrome c reductase moiety and a molybdenum-containing cofactor which is formed by the action of the cnx gene product(s).     

Biochemical and genetical characterization of nitrate reductase deficient mutants of Petunia

Four NR^– lines were selected by their resistance to 100 mM chlorate from X-ray irradiated protoplasts of haploid Petunia hybrida var. Mitchell. The four cell lines were characterized by the presence of xanthine dehydrogenase activity and by complementation tests via protoplast fusion. One mutant (line 1) was classified as defective in the NR apoprotein (tentatively, nia-type) and the other three (lines 2, 3, 4) in the molybdenum cofactor (tentatively, cnx-type). Some NR activity (15 %) could be restored by adding unphysiologically high concentrations of molybdate to the culture medium in two of the cnx-lines (lines 3 and 4). The third cnx-line (line 2) had no NR activity. A complementation analysis via protoplast fusion confirmed that the mutants comprised 3 non-allelic groups. From these results it can be concluded that these NR^– mutants are recessive and that two of the cnx-mutants (lines 3, 4) are allelic.Abbreviations MS Murashige and Skoog medium (Murashige and Skoog 1962) - MG Müller and Grafe medium (Müller and Grafe 1978), containing amino acids - V47 protoplast medium (Binding 1974) - MS-413-medium (McCormack and Hanson 1980) - IAA indoleacetic acid - BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - AA amino acids - XDH xanthine dehydrogenase - PEG polyethylene glycol - NR nitrate reductase     

Somatic Hybridization of Nitrate Reductase Deficient Mutants of Nicotiana tabacum by Protoplast Fusion

Protoplasts were isolated from two mutant cell lines of Nicotiana tabacum L. cv. Gatersleben and fused with the aid of polyethylene glycol. Both mutants lacked nitrate reductase and were thus auxotrophic for reduced nitrogen. The fusion resulted in a high frequency of hybrid cells which were detected by their regained ability to grow in media containing nitrate as sole nitrogen source. Thus, the two mutants were found to complement each other in the hybrids. In control experiments, back mutation and cross-feeding were excluded as possible explanations for the occurrence of cell lines utilizing nitrate. A total of 1061 hybrid lines capable of sustained proliferation were isolated. Some of them were further characterized with respect to nitrate reductase activity, chlorate sensitivity, chromosome number, and shoot formation. The results demonstrate that protoplast fusion can be used for the genetic analysis of cell variants of higher plants and that nitrate reductase-deficient mutants provide efficient selective systems for hybrid cells.     

Occurrence of xanthine dehydrogenase in Chlamydomonas reinhardii: A common cofactor shared by xanthine dehydrogenase and nitrate reductase

Wild-type Chlamydomonas reinhardii cells have xanthine dehydrogenase activity when grown with nitrate, nitrite, urea, or amino acid media. Mutant strains 102, 104, and 307 of Chlamydomonas, lacking both xanthine dehydrogenase and nitrate reductase activities, were incapable of restoring the NADPH-nitrate reductase activity of the mutant nit-1 of Neurospora crassa, whereas wild type cells and mutants 203 and 305 had xanthine dehydrogenase and were able to reconstitute the nitrate reductase activity of nit-1 of Neurospora. Therefore, it is concluded that in Chlamydomonas a common cofactor is shared by xanthine dehydrogenase and nitrate reductase. Xanthine dehydrogenase is repressed by ammonia and seems to be inessential for growth of Chlamydomonas.     

Biochemical genetics of further chlorate resistant, molybdenum cofactor defective, conditional-lethal mutants of barley

Summary Three plants, R9201 and R11301 (from cv. Maris Mink) and R12202 (from cv. Golden Promise), were selected by screening M₂ populations of barley (Hordeum vulgare L.) seedlings (mutagenised with azide in the M₁) for resistance to 10 mM potassium chlorate. Selections R9201 and R11301 were crossed with the wild-type cv. Maris Mink and analysis of the F₂ progeny showed that one quarter lacked shoot nitrate reductase activity. These F₂ plants also withered and died in the continuous presence of nitrate as sole nitrogen source. Loss of nitrate reductase activity and withering and death were due in each case to a recessive mutation in a single nuclear gene. All F1 progeny derived from selfing selection R12202 lacked shoot nitrate reductase activity and also withered and subsequently died when maintained in the continuous presence of nitrate as sole nitrogen source. All homozygous mutant plants lacked not only shoot nitrate reductase activity but also shoot xanthine dehydrogenase activity. The plants took up nitrate, and possessed wild-type or higher levels of shoot nitrite reductase activity and NADH-cytochrome c reductase activity when treated with nitrate for 18 h. We conclude that loss of shoot nitrate reductase activity, xanthine dehydrogenase activity and withering and death, in the three mutants R9201, R11301 and R12202 is due to a mutation affecting the formation of a functional molybdenum cofactor. The mutants possessed wild-type levels of molybdenum and growth in the presence of unphysiologically high levels of molybdate did not restore shoot nitrate reductase or xanthine dehydrogenase activity. The shoot molybdenum cofactor of R9201 and of R12202 is unable to reconstitute NADPH nitrate reductase activity from extracts of the Neurospora crassa nit-1 mutant and dimerise the nitrate reductase subunits present in the respective barley mutant. The shoot molybdenum cofactor of R11301 is able to effect dimerisation of the R11301 nitrate reductase subunits and can reconstitute NADPH-nitrate reductase activity up to 40% of the wild-type molybdenum cofactor levels. The molybdenum cofactor of the roots of R9201 and R11301 is also defective. Genetic analysis demonstrated that R9201, but not R11301, is allelic to R9401 and Az34 (nar-2a), two mutants previously shown to be defective in synthesis of molybdenum cofactor. The mutations in R9401 and R9201 gave partial complementation of the nar-2a gene such that heterozygotes had higher levels of extractable nitrate reductase activity than the homozygous mutants.We conclude that: (a) the nar-2 gene locus encodes a step in molybdopterin biosynthesis; (b) the mutant R11301 represents a further locus involved in the synthesis of a functional molybdenum cofactor; (c) mutant Rl2202 is also defective in molybdopterin biosynthesis; and (d) the nar-2 gene locus and the gene locus defined by R11301 govern molybdenum cofactor biosynthesis in both shoot and root.     

Correction of nitrate reductase defect in auxotrophic plant cells through protoplast-mediated intergeneric gene transfers

Summary Nitrate reductase-deficient cells of Nicotiana tabacum cv Gatersleben (coded cnx-68) lacking active molybdenum-cofactor were corrected by introducing the genes from Physalis minima and Datura innoxia into NR^- genomes. In these itergeneric reconstruction experiments, X-irradiated inactive mesophyll protoplasts of Physalis and Datura were fused separately with the cultured cell protoplasts of cnx-68 Nicotiana. A total of 45 cell colonies, 37 transformed by Physalis and 8 by Datura, were selected from about 1.7×10³ heteroplasmic fusion products. The selection of transformants was made by their ability to grow on a medium containing nitrate as the sole nitrogen source. Some of these transformants were further characterized with respect to nitrate reductase, xanthine dehydrogenase and glutamate dehydrogenase activities, chlorate sensitivity, and chlorophyll synthesis. The restoration of nitrate reductase and xanthine dehydrogenase activities confirm the presence of an active form of the molybdenum-cofactor by the expression of introduced genes of Physalis and Datura into the genome of cnx-68 Nicotiana. Such stable transformations via fusion of normal and highly irradiated protoplasts may have a considerable application in higher plants for introducing desirable characters from diverse genomes.     

Nitrate reductase deficient cell lines from diploid cell cultures and lethal mutant M2 plants of Arabidopsis thaliana

Summary Cell suspensions of diploid Arabidopsis thaliana were screened for resistance to chlorate on a medium with ammonium nitrate as the nitrogen source, and after plating on filters to increase the plating efficiency. Thirty-nine lines were selected, four of which were still resistant after two years of subculturing on non-selective medium. Of the latter lines three were nitrate reductase deficient but exhibited some residual nitrate reductase activity; the fourth line showed a high level of enzyme activity. Screening M2-seeds for callus production on selective medium with amino acids as the nitrogen source and chlorate revealed resistant calli in 17 out of 483 M2-groups. Nine well-growing lines, all but one (G3) exhibiting no detectable in vivo nitrate reductase activity, were classified as defective in the cofactor. Two lines (G1 and G3) could be analysed genetically at the plant level. Chlorate resistance was monogenic and recessive. Sucrose gradient fractionation of callus extracts of G1 revealed that a complete enzyme molecule can be assembled. Nitrate reductase activity in G1 could partly be restored by excess molybdenum. It is suggested that G1 is disturbed in the catalytic properties of the cofactor. It appeared that G1 is neither allelic with another molybdenum repairable mutant (B73) nor with another cofactor mutant (B25). Wilting of intact G1 plants could be ascribed to non-closing stomata.     

Isolation and characterization of transposon Tn5 mutants of Rhodobacter sphaeroides deficient in both nitrate and chlorate reduction.

Abstract The wild-type strain Rhodobacter sphaeroides DSM 158 is a nitrate-reducing bacterium with a periplasmic nitrate reductase. Addition of chlorate to the culture medium causes a stimulation of the phototrophic growth, indicating that this strain is able to use chlorate as an ancillary oxidant. Several mutant strains of R. sphaeroides deficient in nitrate reductase activity were obtained by transposon Tn5 mutagenesis. Mutant strain NR45 exhibited high constitutive nitrate and chlorate reductase activities and phototrophic growth was also increased by the presence of chlorate. In contrast, the stimulation of growth by chlorate was not observed in mutant strains NR8 and NR13, in which transposon Tn5 insertion causes the simultaneous loss of both nitrate and chlorate reductase activities. Tn5 insertion probably does not affect molybdenum metabolism since NR8 and NR13 mutants exhibit both xanthine dehydrogenase and nitrogenase activities. These results that a single enzyme could reduce both nitrate and chlorate in R. sphaeroides DSM 158.     

Chlorate-resistant variants of Nicotiana tabacum L.

Summary In somatic hybrids of each of four chlorate-resistant cell lines of Nicotiana tabacum L., two of which were also nitrate-nonutilizing, and a common chlorate-sensitive parent, nitrate utilization and/or chlorate resistance were found to be recessive traits. Complementation for nitrate utilization was observed in somatic hybrids of the two nitrate-nonutilizing cell lines. No chlorate-resistant or nitrate-nonutilizing seedlings were detected among a large number of progeny resulting from the self-fertilization of a somatic hybrid plant one of whose parents was one of the nitrate-nonutilizing cell lines.     

Regulation of reductase formation in Proteus mirabilis

Summary Prteus mirabilis can form four reductases after anaerobic growth: nitrate reductase A, chlorate reductase C, thiosulfate reductase and tetrathionate reductase. The last three enzymes are formed constitutively. Nitrate reductase is formed only after growth in the presence of nitrate, which causes repression of the formation of thiosulfate reductase, chlorate reductase C, tetrathionate reductase and hydrogenase. Formic dehydrogenase assayed with methylene blue as hydrogen acceptor is formed under all conditions.Two groups of chlorate resistant mutants were obtained. One group does not form the reductases and formic dehydrogenase. The second group does not form nitrate reductase, chlorate reductase and hydrogenase, but forms formic dehydrogenase and small amounts of formic hydrogenlyase after growth without hydrogen acceptor or after growth in the presence of thiosulfate or tetrathionate. Nitrate prevents the formation of formic dehydrogenase, thiosulfate reductase and tetrathionate reductase in this group of mutants. Only after growth with thiosulfate or tetrathionate the reductases for these compounds are formed. Anaerobic growth of the wild type in complex medium without a fermentable carbon source is strongly stimulated by the presence of nitrate. Tetrathionate and thiosulfate have no effect at all or only a small effect. The results show that in the presence of tetrathionate or thiosulfate the bacterial metabolism is fully anaerobic, as these cells also contain formic hydrogenlyase.     

Nitrate reductase deficient cell lines from haploid protoplast cultures ofNicotiana plumbaginifolia

Summary Nitrate reductase deficient (NR^-) cell lines were selected indirectly by their resistance to 40 mM chlorate in protoplast cultures of haploidNicotiana plumbaginifolia. Frequency of the chlorate resistant clones was 5.8×10^-5 in non-mutagenized cultures, which could be increased up to 25 times by treatment with N-ethyl-N-nitrosourea (NEU) or gamma irradiation.Out of 136 chlorate resistant clones 29 were fully deficient in nitrate reductase. The rest of the clones contained decreased or normal levels of NR activity (91 and 16 clones, respectively).Further characterization was carried out in 9 clones which were fully deficient in NR and in 2 clones containing resisdual (0–5%) NR activity. The clones were tentatively classified as defective in the apoenzyme (7 clones including the 2 with residual NR activity) or the cofactor (4 clones) of NR by the xanthine dehydrogenase activity and in vitro enzyme complementation. The cofactor defectives could be further classified into two groups. In one of these (2 clones) the NR activity could be partially restored by unphysiologically high (0.2–1 mM) molybdate in the culture medium. The other two are new types which have not been described in flowering plants.Plant regeneration was obtained only in the clones which contained residual NR activity.     

Presence of the molybdenum-cofactor in nitrate reductase-deficient mutant cell lines of Nicotiana tabacum

Summary Nicotiana tabacum mutant cell cultures lacking nitrate reductase activity were assayed for the presence of the molybdenum-cofactor using its ability to restore NADPH-nitrate reductase activity in extracts of Neurospora crassa nit-1 mycelia. The molybdenum-cofactor of the tobacco wild-type line was shown to complement efficiently the N. crassa nit-1 mutant in vitro. The molybdenum-cofactor seems to exist in a bound form, as acid-treatment was required for release of cofactor activity. Molybdate (5–10 mM), ascorbic acid, and anaerobic conditions greatly increased the activity of the cofactor, demonstrating its high lability and sensitivity to oxygen. Similar results were obtained with two tobacco nia mutants, which are defective in the apoprotein of nitrate reductase. The four cnx mutants studied were shown to contain exclusively an inactive form of the molybdenum-cofactor. This inactive cofactor could be reactivated in vitro and in vivo by unphysiologically high concentrations of molybdate (1–10 mM), thereby converting the cnx cells into highly active cofactor sources in vitro, and restoring nitrate reductase and xanthine dehydrogenase in vivo to partial acitivity. Thus the defect of the cnx mutants resides in a lack of molybdenum as a catalytically active ligand metal for the cofactor, while the structural moiety of the cofactor seems not to be impaired by the mutation. The subunit assembly of the nitrate reductase was found to be independent of the molybdenum content of the cofactor.     

Induction and Characterization of Chlorate-resistant Strains of Rosa damascena Cultured Cells

The sensitivity of Rosa damascena cultured cells to chlorate was measured by plating samples of suspensions in agar containing NaClO₃. This sensitivity depended on the age of the cultures that were plated. Chlorate-resistant colonies isolated from 5- to 7-day cultures retained their resistance through many generations of growth in medium lacking NaClO₃; they also retained resistance when mixed with sensitive cells. Treating cell aggregates with ultraviolet (UV) light (254 nanometers), or UV light (360 nanometers) in the presence of 4′-methoxymethyltrioxsalen, increased the proportion that was resistant to NaClO₃. However, the amount of increase was low (three times) and required very specific doses of UV light. The UV treatments did not select for chlorate-resistant cells over chlorate-sensitive cells. The data suggested that UV had induced mutations leading to chlorate resistance. Approximately 15% of the resistant strains did not grow on medium containing nitrate as the sole nitrogen source. These strains lacked ability to reduce chlorate to chlorite. This observation supports the current idea that chlorate toxicity depends on the activity of nitrate reductase. Approximately 85% of the resistant strains grew on medium containing nitrate as the sole nitrogen source. These strains lost catalase activity following chlorate treatment, indicating that they took up and reduced chlorate. These strains have a mechanism for tolerating chlorate and its reduction products, rather than avoiding them.     

Somatic hybridization of Nicotiana tabacum and Petunia hybrida

Summary Leaf mesophyll protoplasts of a nitrate reductase deficient streptomycin resistant mutant of Nicotiana tabacum were fused with cell suspension protoplasts of wild type Petunia hybrida. Somatic hybrid cell colonies were selected for streptomycin resistance and nitrate reductase proficiency. Six independent cell lines, capable of growth in selection medium, were analysed by electrophoresis of callus peroxidases and leucine aminopeptidases and also by hybridization with rDNA and a chloroplast encoded gene as molecular probes. The results show that all six lines represented nuclear somatic hybrids, possessing the chloroplast of N. tabacum, at an early stage of development. However, after 6–12 months in culture, genomic incompatibility was observed resulting in the loss of most of the tobacco nuclear genome in the majority of the cell lines. One of the latter cell lines regenerated plants which possessed the chloroplast of N. tabacum in a predominantly P. hybrida nuclear background.     

Complementation analysis of a nitrate reductase deficient Hyoscyamus muticus cell line by somatic hybridisation

Summary Complementation of a nitrate reductase deficient variant of Hyoscyamus muticus (MA-2) and nitrate reductase apoenzyme (nia-115) and cofactor mutants (cnx-68) of Nicotiana tabacum was studied by protoplast fusion. Selection of prototrophic intergeneric somatic hybrids was achieved in combination of MA-2 with the apoenzyme mutant nia-115 of N. tabacum. The H. muticus MA-2 line was therefore classified to be a cnx type variant possessing an altered molybdenum cofactor of the nitrate reductase enzyme complex but unaffected in the apoprotein of nitrate reductase. The nitrate reductase deficient and chlorate resistant characters of MA-2 were functionally coupled recessive traits. Nitrate reductase activity accompanied by chlorate sensitivity could be detected only under inductive conditions in the somatic hybrids. The inductive expression of nitrate reductase in the somatic hybrids arising from the combination of cells harbouring either the inductive or constitutive type nitrate reductase is discussed.Abbreviations DTT 1,4-Dithio-DL-threitol - Mo-co molybdenum containing cofactor - PEG polyethylene glycol     

Nitrate reductase-deficient mutants in barley : immunoelectrophoretic characterization

Nitrate reductase-deficient barley (Hordeum vulgare L.) mutants were assayed for the presence of a functional molybdenum cofactor determined from the activity of the molybdoenzyme, xanthine dehydrogenase, and for nitrate reductase-associated activities. Rocket immunoelectrophoresis was used to detect nitrate reductase cross-reacting material in the mutants. The cross-reacting material levels of the mutants ranged from 8 to 136% of the wild type and were correlated with their nitrate reductase-associated activities, except for nar 1c, which lacked all associated nitrate reductase activities but had 38% of the wild-type cross-reacting material. The cross-reacting material of two nar 1 mutants, as well as nar 2a, Xno 18, Xno 19, and Xno 29, exhibited rocket immunoprecipitates that were similar to the wild-type enzyme indicating structural homology between the mutant and wild-type nitrate reductase proteins. The cross-reacting materials of the seven remaining nar 1 alleles formed rockets only in the presence of purified wild-type nitrate reductase, suggesting structural modifications of the mutant cross-reacting materials. All nar 1 alleles and Xno 29 had xanthine dehydrogenase activity indicating the presence of functional molybdenum cofactors. These results suggest that nar 1 is the structural gene for nitrate reductase. Mutants nar 2a, Xno 18, and Xno 19 lacked xanthine dehydrogenase activity and are considered to be molybdenum cofactor deficient mutants. Cross-reacting material was not detected in uninduced wild-type or mutant extracts, suggesting that nitrate reductase is synthesized de novo in response to nitrate.     

曼陀罗培养细胞的硝酸还原酶突变株

经紫外诱变氯酸钾筛选,得到一个低硝酸还原酶(NADH:硝酸氧化还原酶.EC1.6.6.1.,以下简写为NR)活力的细胞株。其主要特征:NR活力低,约为正常型的1/5;对氯酸钾具有较强的抗性;不适合在单纯以硝酸盐为氮源的培养基上生长,能在以(NH_4)_2SO_4为唯一氮源的培养基上生长。蛋白电泳表明,此细胞株与正常型有不同的蛋白带。这些特征在没有选择压力的培养基上培养二年后,仍保持不变,说明此细胞株是一个遗传型的变异株。