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.     

Development of aminoacyl tRNA synthetases in cultured Nicotiana tabacum cells

Changes in the activity of aminoacyl tRNA synthetases during growth of tobacco XD cells in suspension culture have been determined by the pyrophosphate exchange assay. Alanyl, arginyl, glutamyl, glutaminyl and seryl tRNA synthetases showed the lowest activity, whilst lysyl, histidyl, leucyl, isoleucyl, phenylalanyl threonyl and valyl tRNA synthetases were most active. Most synthetases, and total protein, increased to a maximum, at around 7 days, just before mid-exponential phase, and then fell.     

ATP binding and crossbridge structure in muscle

Thick filaments extracted from insect flight muscle were used in examining whether the dependence of actin-myosin crossbridge structure on nucleotide, generally presumed to underlie the power-stroke, is exhibited by myosin alone. The strongly periodic crossbridge arrangement seen in the presence of ATP (corresponding to relaxed muscle) is reversibly lost in conditions that induce rigor in intact muscle fibres. These observations suggest that the power-stroke may involve changes in the steric relation of the myosin head to the thick as well as to the thin filament.     

Correlated changes of some enzyme activities and cofactor and substrate contents of pea cotyledon tissue during germination

1. The activities of six enzymes (hexokinase, phosphoglucose isomerase, phosphofructokinase, aldolase, glucose 6-phosphate dehydrogenase and amylase) in extracts of pea cotyledons were determined. The activities during the first 10 days after germination showed individual and characteristic changes that indicate a specific control of both synthesis and destruction of enzymes. 2. Tissue contents of glucose, inorganic phosphate, glucose 6-phosphate, fructose 6-phosphate, ATP, ADP, AMP, NAD and NADP were also determined, and a correlation is reported between the substrate concentrations at day 1 and the subsequent enzymic activity. 3. The initial NAD(+)/NADH ratio value of 1 changed to about 3 by day 4; the NADP content was lower and changes in the oxidation state were less striking. The ratio of ATP to ADP and AMP remained virtually constant.     

Accumulation of phenolic acid conjugates and betacyanins,and changes in the activities of enzymes involved in feruloylglucose metabolism in cell-suspension cultures ofChenopodium rubrum L.

Cell-suspension cultures ofChenopodium rubrum accumulate various soluble secondary phenolic metabolites such as the hydroxybenzoic acid glycosides 4-hydroxybenzoic acid--glucoside, vanillic acid--glucoside, the hydroxycinnamic acid acylglycosides 1-O-(4-coumaroyl)--glucose, 1-O-feruloyl--glucose, 1-O-sinapoyl--glucose and 1-O-feruloyl-(-1,2-glucuronosyl)--glucose, the hydroxycinnamic acid amide N-feruloylaspartate, and the betacyanins betanin, amaranthin and celosianin II. In addition, accumulation of the insoluble cell wall-bound hydroxycinnamic acids with ferulic acid as the major component occurs parallel to culture growth. The changes of three pivotal enzymatic activities, all O-transferases which are involved in the formation of the dominant ferulic acid conjugates, were determined. These are (i) uridine 5-diphosphate(UDP)glucose-hydroxycinnamic acid O-glucosyltransferase (EC 2.4.1), (ii) UDP-glucuronic acid:1-O-hydroxycin-namoyl--glucose O-glucuronosyltransferase (EC 2.4.1) and (iii) 1-O-hydroxycinnamoyl--glucose:amaranthin O-hydroxycinnamoyltransferase (EC 2.3.1). The patterns of metabolite accumulation associated with these enzyme activities show that the hydroxycinnamic acid-glucose esters play a central role as metabolically active intermediates in the secondary metabolism ofCh. rubrum. Two cell lines of this culture (CH, CHN), differing in their betacyanin content, were compared with respect to this metabolism. A markedly higher total betacyanin content in the CHN line might possibly be the consequence of an increased supply of the key precursor for betalain biosynthesis, i.e. 3,4-dihydroxyphenylalanine (DOPA). In addition, the enhanced accumulation of celosianin II in the CHN line correlates well with a higher activity of the enzyme catalyzing the transfer of ferulic acid from 1-O-feruloyl--glucose to amaranthin.Abbreviations CH line red-coloured betalain-producing cell-suspension cultures ofChenopodium rubrum (lower betacyanin content) - CHN line deep-red-coloured betalain-producing cell-suspension cultures ofCh. rubrum (higher betacyanin content), selected from CH line - DOPA 3,4-dihydroxyphenylalanine - glucosyltransferase uridine 5-diphosphate-glucose hydroxycinnamic acid O-glucosyltransferase (EC 2.4.1) - glucuronosyltransferase uridine 5-diphosphate-glucuronic acid: 1-O-hydroxycinnamoyl--glucose O-glucuronosyltransferase (EC 2.4.1) - HPLC high-performance liquid chromatography - hydroxycinnamoyltransferase 1-O-hydroxycinnamoyl--glucose:amaranthin O-hydroxycinnamoyltransferase (EC 2.3.1) - NMR nuclear magnetic resonance Support by the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie to D.S. is gratefully acknowledged. We thank Sabine Fehling for help in cell wall analyses and Heike Steingaß for optimization of enzyme assays. Our special thanks are due to Dr H. Harms (FAL, Braunschweig, FRG) and Dr J. Berlin (BBA, Braunschweig) for establishing and providing the CH and CHN lines, respectively, of theChenopodium rubrum cell culture. We are grateful to Christel Kokoschka, H. Dirks and Inge Schweer (GBF, Braunschweig) for recording the NMR, FAB MS and EI MS data, respectively.     

Slipped-strand mispairing: a major mechanism for DNA sequence evolution

Simple repetitive DNA sequences are a widespread and abundant feature of genomic DNA. The following several features characterize such sequences: (1) they typically consist of a variety of repeated motifs of 1-10 bases--but may include much larger repeats as well; (2) larger repeat units often include shorter ones within them; (3) long polypyrimidine and poly-CA tracts are often found; and (4) tandem arrangements of closely related motifs are often found. We propose that slipped-strand mispairing events, in concert with unequal crossing- over, can readily account for all of these features. The frequent occurrence of long tandem repeats of particular motifs (polypyrimidine and poly-CA tracts) appears to result from nonrandom patterns of nucleotide substitution. We argue that the intrahelical process of slipped-strand mispairing is much more likely to be the major factor in the initial expansion of short repeated motifs and that, after initial expansion, simple tandem repeats may be predisposed to further expansion by unequal crossing-over or other interhelical events because of their propensity to mispair. Evidence is presented that single-base repeats (the shortest possible motifs) are represented by longer runs in mammalian introns than would be expected on a random basis, supporting the idea that SSM may be a ubiquitous force in the evolution of the eukaryotic genome. Simple repetitive sequences may therefore represent a natural ground state of DNA unselected for coding functions.      

Complete sequences of the rRNA genes of Drosophila melanogaster

In this, the first of three papers, we present the sequence of the ribosomal RNA (rRNA) genes of Drosophila melanogaster. The gene regions of D. melanogaster rDNA encode four individual rRNAs: 18S (1,995 nt), 5.8S (123 nt), 2S (30 nt), and 28S (3,945 nt). The ribosomal DNA (rDNA) repeat of D. melanogaster is AT rich (65.9% overall), with the spacers being particularly AT rich. Analysis of DNA simplicity reveals that, in contrast to the intergenic spacer (IGS) and the external transcribed spacer (ETS), most of the rRNA gene regions have been refractory to the action of slippage-like events, with the exception of the 28S rRNA gene expansion segments. It would seem that the 28S rRNA can accommodate the products of slippage-like events without loss of activity. In the following two papers we analyze the effects of sequence divergence on the evolution of (1) the 28S gene "expansion segments" and (2) the 28S and 18S rRNA secondary structures among eukaryotic species, respectively. Our detailed analyses reveal, in addition to unequal crossing-over, (1) the involvement of slippage and biased mutation in the evolution of the rDNA multigene family and (2) the molecular coevolution of both expansion segments and the nucleotides involved with compensatory changes required to maintain secondary structures of RNA.      

Characterization of the near infrared absorption spectra of cytochrome aa3 and haemoglobin for the non-invasive monitoring of cerebral oxygenation

Near infrared (IR) spectroscopy can give continuous, direct information about cerebral oxygenation in vivo by providing signals from oxygenated and deoxygenated haemoglobin and cytochrome aa3. Due to a lack of precise spectral information and uncertainties about optical path length it has previously been impossible to quantify the data. We have therefore obtained the cytochrome aa3 spectrum in vivo from the brains of rats after replacing the blood with a fluorocarbon substitute. Near infrared haemoglobin spectra were also obtained, at various oxygenation levels, from cuvette studies of lysed human red blood cells. Estimates of optical path length have been obtained. The data were used to construct an algorithm for calculating the changes in oxygenated and deoxygenated haemoglobin and oxygenated cytochrome aa3 in tissue from changes in near IR absorption.