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.     

Molecular approaches to the analysis of nitrate assimilation

Abstract. The application of molecular approaches such as mutant analysis and recombinant DNA technology, in conjunction with immunology, are set to revolutionize our understanding of the nitrate assimilation pathway. Mutant analysis has already led to the identification of genetic loci encoding a functional nitrate reduction step and is expected to lead ultimately to the identification of genes encoding nitrate uptake and nitrite reduction. Of particular significance would be identification of genes whose products contribute to regulatory networks controlling nitrogen metabolism. Recombinant DNA techniques are particularly powerful and have already allowed the molecular cloning of the genes encoding the apoprotein of nitrate reductase and nitrite reductase. These successes allow for the first lime the possibility to study directly the role of environmental factors such as type of nitrogen source (NO₃⁻ or NH₄⁺) available to the plant, light, temperature water potential and CO₂ and O₂ tensions on nitrate assimilation gene expression and its regulation at the molecular level. This is an important advance since our current understanding of the regulation of nitrate assimilation is based largely on changes of activity of the component steps. The availability of mutants, cloned genes, and gene transfer systems will permit attempts to manipulate the nitrate assimilation pathway.     

Biochemical and genetic comparison of two nitrate reductase-deficient pea mutants disturbed in the cofactor

Two nitrate reductase (NaR)-deficient mutants of pea (Pisum sativum L.), E₁ and A300, both disturbed in the molybdenum cofactor function and isolated, respectively, from cv Rondo and cv Juneau, were tested for allelism and were compared in biochemical and growth characteristics. The F₁ plants of the cross E₁ × A300 possessed NaR and xanthine dehydrogenase (XDH) activities comparable to those of the wild types, indicating that these mutants belong to different complementation groups, representing two different loci. Therefore, mutant E₁ represents, besides mutant A300 and the allelic mutants A317 and A334, a third locus governing NaR and is assigned the gene destignation nar 3. In comparison with the wild types, cytochrome c reductase activity was increased in both mutants. The mutants had different cytochrome c reductase distribution patterns, indicating that mutant A300 could be disturbed in the ability to dimerize NaR apoprotein monomers, and mutant E₁ in the catalytic function of the molybdenum cofactor. In growth characteristics studied, A300 did not differ from the wild types, whereas fully grown leaves of mutant E₁ became necrotic in soil and in liquid media containing nitrate.     

Regulation of pteridine biosynthesis and aromatic amino acid hydroxylation inDrosophila melanogaster

The relationship between high dietary levels of aromatic amino acid and regulation of pteridines inDrosophila eyes was examined by measuring changes in pool levels of six pterins in the wild type and mutants and amino acid pool levels in flies that carry mutations for pteridine biosynthesis. The effect upon relative viability and developmental times was also analyzed; relative viability was affected byl-phenylalanine,l-tryptophan, andl-tyrosine in decreasing order and thed-amino acids had little or no effect. The changes in concentration of biopterin, dihydrobiopterin, pterin, sepiapterin, drosopterins, and isoxanthopterin showed a characteristic pattern of increased and/or decreased amounts in response to each of the threel-amino acids. Pterin was regularly increased, and isoxanthopterin decreased.l-Tyrosine caused a 2.1-fold increase in dihydrobiopterin, the largest increase found in this study;l-tryptophan also caused dihydrobiopterin to increase butl-phenylalanine did not. Of 18 eye-color mutants examined, 2 were found to contain high levels of phenylalanine and/or tyrosine,Pu ² andHn ^r3. These two mutants, along withpr ^c4 cn/pr ^m2b cn, were shown to be very sensitive to dietaryl-phenylalanine, indicating that having low levels of certain pteridines makes them susceptible to toxic effects of these amino acids. Therefore, high levels of aromatic amino acids can perturb the balance among pteridine pools, and low levels of some pteridines in mutants are correlated with the inability to withstand the toxic effects of phenylalanine. From the patterns of change in the pteridines we suggest that tetrahydropterin may also be a cofactor for hydroxylation of phenylalanine, along with tetrahydrobiopterin.This work was sponsored in part by a grant from the U.S.-Spain Joint Committee for Scientific and Technological Cooperation.     

Characterization of a methyl-specific restriction system in Clostridium acetobutylicum strain N1-4081

A type II restriction endonuclease, named CacI, was detected in Clostridium acetobutylicum strain N1-4081. CacI cleaved the tetranucleotide sequence [5' decreases -GATC-3']. The modification system consisted of the methylation of the adenine present in this sequence. CacI, an isoschizomer of MboI, is inactive on dam methylated substrates.     

Release of molybdenum co-factor from nitrate reductase and xanthine oxidase by heat treatment

Heat treatment (90 sec at 70°) is shown to convert the bound molybdenum co-factor of tobacco cell-free extracts and bovine milk xanthine oxidase into a form capable of complementing the Neurospora crassa mutant nit-1.In the presence of 1 mM ascorbic acid, 25 mM molybdate and, for plant extracts, sulphydryl group protecting agents, the molybdenum co-factor can survive incubations up to 100° whilst maintaining its biological activity. Especially with plant extracts, the efficiency of heat treatment is considerably higher than that of the acidification procedure which is often utilized for releasing molybdenum co-factor.     

谷氨酸棒杆菌生产异亮氨酸辅因子策略及其基因组整合研究进展

L?异亮氨酸属于三大支链氨基酸,是人体8种必需氨基酸之一,广泛应用于食品、药品、保健品、化妆品等领域。目前,微生物发酵法是工业生产L?异亮氨酸的主要方法,其中谷氨酸棒杆菌（Corynebacterium glutamicum）是发酵生产L?异亮氨酸的优势菌株,然而随机诱变会使产量的提高能力达到饱和,难以得到更加高产的菌株,因此针对诱变菌株进行理性改造已成为进一步提高产量的主要方式;且随着遗传操作技术在谷氨酸棒杆菌中的应用与优化,代谢工程育种已逐渐取代传统的诱变育种。综述了谷氨酸棒杆菌中L?异亮氨酸的生物合成途径、代谢调控机制和理性改造L?异亮氨酸生产菌株的策略,并对辅助因子工程应用于理性改造及对谷氨酸棒杆菌基因组整合策略进行了系统阐述,以期为工业水平稳定生产L?异亮氨酸高产菌株的基因组整合策略提供参考依据。     

The ammonia-lyases: enzymes that use a wide range of approaches to catalyze the same type of reaction

Abstract

The paradigm that protein structure determines protein function has been clearly established. What is less clear is whether a specific protein structure is always required to carry out a specific function. Numerous cases are now known where there is no apparent connection between the biological function of a protein and the other members of its structural class, and where functionally related proteins can have quite diverse structures. A set of enzymes with these diverse properties, the ammonia-lyases, will be examined in this review. These are a class of enzymes that catalyze a relatively straightforward deamination reaction. However, the individual enzymes of this class possess a wide variety of different structures, utilize a diverse set of cofactors, and appear to catalyze this related reaction through a range of different mechanisms. This review aims to address a basic question: if there is not a specific protein structure and active site architecture that is both required and sufficient to define a catalyst for a given chemical reaction, then what factor(s) determine the structure and the mechanism that is selected to catalyze a particular reaction?     

An essential quality control mechanism at the eukaryotic basal body prior to intraflagellar transport

Constructing a eukaryotic cilium/flagellum is a demanding task requiring the transport of proteins from their cytoplasmic synthesis site into a spatially and environmentally distinct cellular compartment. The clear potential hazard is that import of aberrant proteins could seriously disable cilia/flagella assembly or turnover processes. Here, we reveal that tubulin protein destined for incorporation into axonemal microtubules interacts with a tubulin cofactor C (TBCC) domain-containing protein that is specifically located at the mature basal body transitional fibres. RNA interference-mediated ablation of this protein results in axonemal microtubule defects but no effect on other microtubule populations within the cell. Bioinformatics analysis indicates that this protein belongs to a clade of flagellum-specific TBCC-like proteins that includes the human protein, XRP2, mutations which lead to certain forms of the hereditary eye disease retinitis pigmentosa. Taken with other observations regarding the role of transitional fibres in cilium/flagellum assembly, we suggest that a localized protein processing capacity embedded at transitional fibres ensures the 'quality' of tubulin imported into the cilium/flagellum, and further, that loss of a ciliary/flagellar quality control capability may underpin a number of human genetic disorders.     

Electrical conduction behavior of organic light‐emitting diodes using fluorinated self‐assembled monolayer with molybdenum oxide‐doped hole transporting layer

The electrical conductivity behavior of a fluorinated self‐assembled monolayer (FSAM) of a molybdenum oxide (MoOx)‐doped α‐naphthyl diamine derivative (α‐NPD) in organic light‐emitting diodes (OLEDs) was investigated. The current density of the MoOx‐doped α‐NPD/FSAM device was proportional to its voltage owing to smooth carrier injection through the FSAM and the high carrier density of its bulk. The temperature‐dependent characteristics of this device were investigated. The current density–voltage characteristics at different temperatures were almost the same owing to its very low activation energy. The activation energy of the device was estimated to be 1.056 × 10^?2 [eV] and was very low due to the inelastic electron tunneling of FSAM molecules. Copyright © 2014 John Wiley & Sons, Ltd.