Synchronous loss of quasispecies memory in parallel viral lineages: a deterministic feature of viral quasispecies

Viral quasispecies are endowed with a memory of their past evolutionary history in the form of minority genomes of their mutant spectra. To determine the fate of memory genomes in evolving viral quasispecies, we have measured memory levels of antigenic variant of foot-and-mouth disease virus (FMDV) RED, which includes an Arg-Glu-Asp (RED) at a surface antigenic loop of the viral capsid. The RED reverted to the standard Arg-Gly-Asp (RGD), and the RED remained as memory in the evolving quasispecies. In four parallel evolutionary lineages, memory reduction followed a strikingly similar pattern, and at passage 60 memory levels were indistinguishable from those of control populations (devoid of memory). Nucleotide sequence analyses indicated that memory loss occurred synchronously despite its ultimate molecular basis being the stochastic occurrence of mutations in the evolving quasispecies. These results on the kinetics of memory levels have unveiled a deterministic feature of viral quasispecies. Molecular mechanisms that may underlie synchronous memory loss are the averaging of noise signals derived from mutational input, and constraints to genome diversification imposed by a nucleotide sequence context in the viral genome. Possible implications of the behaviour of complex, adaptive viral systems as experimental models to address primary mechanisms of neurological memory are discussed.     

Comparative analysis of the RED1 and RED2 A-to-I RNA editing genes from mammals, pufferfish and zebrafish

One type of RNA editing involves the deamination of adenosine (A) residues to inosines (I) at specific sites in specific pre-mRNAs. These inosines are subsequently read as guanosines by the ribosome, with potentially significant consequences for protein sequence. In mammals, two such A-to-I RNA editases are RED1, which edits some serotonin and glutamate receptors, and RED2, with unidentified substrates. To study the evolutionary conservation among these editases, we have isolated homologous genes from the Japanese pufferfish, Fugu rubripes. Fugu has two genes homologous to Red1 that are similar in size and organization and that show a fivefold compaction relative to the human gene; they differ, however, in their base compositional features. The Fugu gene for RED2 is unusually large, spanning more than 50 kb; within the largest intron, there is evidence for a novel gene on the opposite strand. Because of these unusual features, the partial genomic structure was determined for the mouse RED2 gene. A partial cDNA for RED1 was also isolated from zebrafish. Comparisons between fish and between fish and mammals of the protein sequences show that the catalytic domains are highly conserved for each gene, while the RNA-binding domains vary within a single protein in their levels of conservation. Different levels of conservation among domains of different functional roles may reflect differences in editase substrate specificity and/or substrate sequence conservation.     

Mutation detection in Machado-Joseph disease using repeat expansion detection.

BACKGROUND: Several neurological disorders have recently been explained through the discovery of expanded DNA repeat sequences. Among these is Machado-Joseph disease, one of the most common spinocerebellar ataxias (MJD/SCA3), caused by a CAG repeat expansion on chromosome 14. A useful way of detecting repeat sequence mutations is offered by the repeat expansion detection method (RED), in which a thermostable ligase is used to detect repeat expansions directly from genomic DNA. We have used RED to detect CAG expansions in families with either MJD/SCA3 or with previously uncharacterized spinocerebellar ataxia (SCA). MATERIALS AND METHODS: Five MJD/SCA3 families and one SCA family where linkage to SCA1-5 had been excluded were analyzed by RED and polymerase chain reaction (PCR). RESULTS: An expansion represented by RED products of 180-270 bp segregated with MJD/SCA3 (p < 0.00001) in five families (n = 60) and PCR products corresponding to 66-80 repeat copies were observed in all affected individuals. We also detected a 210-bp RED product segregating with disease (p < 0.01) in a non-SCA1-5 family (n = 16), suggesting involvement of a CAG expansion in the pathophysiology. PCR analysis subsequently revealed an elongated MJD/SCA3 allele in all affected family members. CONCLUSIONS: RED products detected in Machado-Joseph disease families correlated with elongated PCR products at the MJD/SCA3 locus. We demonstrate the added usefulness of RED in detecting repeat expansions in disorders where linkage is complicated by phenotyping problems in gradually developing adult-onset disorders, as in the non-SCA1-5 family examined. The RED method is informative without any knowledge of flanking sequences. This is particularly useful when studying diseases where the mutated gene is unknown. We conclude that RED is a reliable method for analyzing expanded repeat sequences in the genome.     

Structural domains of the extracellular domain of human nerve growth factor receptor detected by partial proteolysis.

Using partial proteolytic cleavage, the nerve growth factor (NGF) binding site and the epitopes for two anti-NGF receptor (NGFR) monoclonal antibodies were localized on the recombinant extracellular domain (RED) of the NGFR. The RED was prepared in the baculovirus-insect cell system and was purified by immunoaffinity and ion-exchange chromatography. The four cysteine-rich repeat domains and some additional C-terminal sequences were resistant to proteolysis with papain or proteinase K. The Mr 32,000 papain-resistant fragment (P32) and the Mr 30,000 proteinase K-resistant fragment (K30) share the same N terminus as the intact RED and have C termini in the vicinity of residue 170. Even though P32 and K30 have the same N terminus and probably differ by only a small number of amino acids at the C terminus, P32, but not K30, binds 125I-NGF. As judged by Western blot analysis, two anti-NGFR antibodies (ME20.4 and NGFR5) bind to P32 but have a lesser affinity for K30. Since antibody ME20.4 inhibits NGF binding but antibody NGFR5 does not, these antibodies bind to distinct epitopes. However, these epitopes apparently are closely spaced since these antibodies compete with each other for binding to biotinylated RED. NGF, but not the control protein cytochrome c, protects RED from papain digestion. Therefore, the P32 C terminus is important for the expression of the NGF binding site and the antibody-defined epitopes, even though the NGF binding site and antibody-defined epitopes probably are not encoded by the P32 C terminus. These data suggest that complex interactions occur between different regions of the RED, and that optimum NGF binding requires the integrity of multiple RED domains, including a short sequence to the C terminus of residue 170.     

Effects of supplementing a CP-reduced diet with rumen-protected methionine on Fleckvieh bull fattening

The objective of this study was to evaluate the effect of supplementing a CP-reduced diet with rumen-protected methionine on growth performance of Fleckvieh bulls. A total of 69 bulls (367 ± 25 kg BW) were assigned to three feeding groups (n = 23 per group). The control (CON) diet contained 13.7% CP and 2.11 g methionine/kg diet (both DM basis) and was set as positive control. The diet reduced in CP (nitrogen) (RED) diet as negative control and the experimental RED + rumen-protected methionine (MET) diet were characterised by deficient CP concentrations (both 9.04% CP). The RED + MET diet differed from the RED diet in methionine concentration (2.54 g/kg DM vs. 1.56 g/kg DM, respectively) due to supplementation of rumen-protected methionine. Rumen-protected lysine was added to both RED and RED + MET at 2.7 g/kg DM to ensure a sufficient lysine supply relative to total and metabolisable protein intake. Metabolisable energy (ME) and nutrient composition were similar for CON, RED, and RED + MET. Bulls were fed for 105 days (d) on average. Individual feed intake was recorded daily; individual BW was recorded at the beginning of the experiment, once per month, and directly before slaughter. At slaughter, blood samples were collected and carcass traits were assessed. Reduction in dietary CP concentration reduced feed intake, and in combination with lower dietary CP concentration, daily intake of CP for RED and RED + MET was lower compared with CON (P < 0.01). Daily ME intake was reduced in RED and RED + MET compared with CON (P < 0.01). Consequently growth performance and carcass weights were reduced (both P < 0.01) in both RED and RED + MET compared with CON. Supplemental rumen-protected methionine was reflected in increased serum methionine concentration in RED + MET (P < 0.05) as compared to RED but it did not affect growth performance, carcass traits and serum amino acid (AA) concentrations, except for lysine which was reduced (P < 0.01) compared to CON and RED. In conclusion, bulls fed RED or RED + MET diets were exposed to a ruminal CP deficit and subsequently a deficit of prececal digestible protein, but methionine did not appear to be the first-limiting essential AA for growth under the respective experimental conditions.     

A Conditional Allele of the Saccharomyces Cerevisiae Hop1 Gene Is Suppressed by Overexpression of Two Other Meiosis-Specific Genes: Red1 and Rec104

The HOP1 gene of Saccharomyces cerevisiae is believed to encode a protein component of the synaptonemal complex, the structure formed when homologous chromosomes synapse during meiotic prophase. Five new mutant alleles (three conditional, two nonconditional) of HOP1 were identified by screening EMS-mutagenized cells for a failure to complement the spore viability defect of a hop1 null allele. Two high copy plasmids were found that partially suppress the temperature-sensitive spore inviability phenotype of one of these alleles, hop1-628. The suppression is allele-specific; no effect of the plasmids is observed in hop1 null diploids. Mutation of either of the two suppressor genes results in recessive spore lethality, indicating that these genes play important roles during meiosis. The DNA sequence of one high copy suppressor gene matched that of RED1, a previously identified meiosis-specific gene. Our data strongly support the idea that RED1 protein is also a component of the synaptonemal complex and further suggest that the RED1 and HOP1 gene products may interact. The second suppressor maps to the right arm of chromosome VIII distal to CDC12 and is REC104, a meiosis-specific gene believed to act early in meiosis.     

Actinorhodin biosynthesis: structural requirements for post-PKS tailoring intermediates revealed by functional analysis of ActVI-ORF1 reductase

Actinorhodin (ACT) produced by Streptomyces coelicolor A3(2) is an aromatic polyketide antibiotic, whose basic carbon skeleton is derived from type II polyketide synthase (PKS). Although an acyl carrier protein (ACP) serves as an anchor of nascent intermediates during chain elongation in the type II PKS complex, it generally remains unknown when an ACP-free intermediate is released from the complex to post-PKS modification ("tailoring") steps. In ACT biosynthesis, a stereospecific ketoreductase (RED1) encoded by actVI-ORF1 reduces the 3beta-keto group of a proposed bicyclic intermediate to an (S) secondary alcohol. The bicyclic intermediate is formed from the steps of PKS and its closely associated enzymes and lies at the interface toward ACT-tailoring steps. To clarify whether RED1 recognizes the ACP-bound bicyclic intermediate or the ACP-free bicyclic intermediate, recombinant RED1 was purified for enzymatic characterization. RED1 was heterologously expressed in Escherichia coli and purified using Ni-chelate and gel filtration column chromatographies to homogeneity in soluble form. Enzymatic studies in vitro on RED1 with synthetic analogues, in place of an unstable bicyclic intermediate, showed that RED1 recognizes 3-oxo-4-naphthylbutyric acid (ONBA) as a preferred substrate and not its N-acetylcysteamine thioester. This strongly suggests that RED1 recognizes ACP-free bicyclic beta-keto acid as the first committed intermediate of tailoring steps. Kinetic studies of RED1 showed high affinity with ONBA, consistent with the requirement for an efficient reduction of a labile beta-keto carboxylic acid. Interestingly, the methyl ester of ONBA acted as a competitive inhibitor of RED1, indicating the presence of strict substrate recognition toward the terminal acid functionality.     

Purification and characterization of the recombinant extracellular domain of human nerve growth factor receptor expressed in a baculovirus system

To obtain the large quantities of the extracellular domain of the nerve growth factor receptor (NGF-R) necessary for structural analyses, we produced this protein in the baculovirus expression system. A cDNA coding for the extracellular domain of the human NGF-R was first introduced into transfer vector pVL941. Recombinant baculovirus was produced by cotransfecting Spodoptera frugiperda cells with the transfer vector and DNA of Autographa californica nuclear polyhedrosis virus. Recombinant viral plaques were selected by morphology and dot hybridization. The expression of recombinant extracellular domain (RED) was analyzed by Western blot analysis using anti-NGF-R monoclonal antibody. Insect cells infected with recombinant virus synthesized RED and secreted it into the culture supernatant. RED was isolated by ammonium sulfate precipitation, immunoaffinity chromatography, and anion exchange chromatography yielding 4 mg of RED/liter of suspension culture. Since there was no apparent effect of endoglycosidase H or F on RED electrophoretic mobility, and since RED did not bind concanavalin A or soybean lectin, there appears to be little or no glycosylation of RED. Purified RED bound 125I-NGF as well as anti-NGF-R monoclonal antibodies. Sedimentation analysis and gel exclusion chromatography revealed that RED is an asymmetric molecule and may be a dimer.     

Generating carbon finance through avoided deforestation and its potential to create climatic, conservation and human development benefits

Recent proposals to compensate developing countries for reducing emissions from deforestation (RED) under forthcoming climate change mitigation regimes are receiving increasing attention. Here we demonstrate that if RED credits were traded on international carbon markets, even moderate decreases in deforestation rates could generate billions of Euros annually for tropical forest conservation. We also discuss the main challenges for a RED mechanism that delivers real climatic benefits. These include providing sufficient incentives while only rewarding deforestation reductions beyond business-as-usual scenarios, addressing risks arising from forest degradation and international leakage, and ensuring permanence of emission reductions. Governance may become a formidable challenge for RED because some countries with the highest RED potentials score poorly on governance indices. In addition to climate mitigation, RED funds could help achieve substantial co-benefits for biodiversity conservation and human development. However, this will probably require targeted additional support because the highest biodiversity threats and human development needs may exist in countries that have limited income potentials from RED. In conclusion, how successfully a market-based RED mechanism can contribute to climate change mitigation, conservation and development will strongly depend on accompanying measures and carefully designed incentive structures involving governments, business, as well as the conservation and development communities.     

Enhancing the examiner's resisting force improves the validity of manual muscle strength measurements: application to knee extensors and flexors

The purposes of this study were to test whether an examiner's strength may affect the validity of the knee muscle strength measurements using a hand-held dynamometer (HHD) and whether enhancing the forces applied by an examiner using a resistance-enhanced dynamometer (RED) would improve measurement validity. Twenty-five young male volunteers (mean [±SD] age: 22.5 ± 1.7 years) without a history of injury to the test limb and 6 male and 6 female experienced examiners participated in this study. Maximum resisting forces of the knee flexors and extensors were measured using RED, HHD, and a dynamometer (Kin-Com). For all testing conditions, poor to moderate associations were found between the HHD and Kin-Com, whereas there was a good to excellent relationship between RED and Kin-Com. The systematic variations between RED and Kin-Com were also smaller than those between HHD and Kin-Com. The force values measured by RED were very close to those measured by Kin-Com. An examiner's strength affects the validity of the measurements using HHD. Enhancing the forces applied by the examiner to the tested segment using RED appeared to improve the validity of muscle strength measurements.     

Circular dichroism and crosslinking studies of the interaction between four neurotrophins and the extracellular domain of the low-affinity neurotrophin receptor.

Interactions between the purified recombinant receptor extracellular domain (RED) of the human low-affinity neurotrophin receptor (LANR) and recombinant human brain-derived neurotrophic factor, neurotrophin-3 (NT-3) and neuotrophin-4/5 have been studied by chemical crosslinking and circular dichroism. Conformational changes subsequent to binding have been shown by these procedures. First, relative affinities of the neurotrophins for RED were determined by binding competition assays in which radioiodinated nerve growth factor (NGF) from mouse submaxillary gland was crosslinked to RED in the presence of varying amounts of unlabeled neurotrophin competitors. RED bound each of the 3 recombinant human neurotrophins with affinities that were indistinguishable from authentic mouse NGF. These results are the first measurement of binding of the neurotrophin family to their common receptor using purified components. In order to study the effect of binding on the conformation of the proteins, CD measurements were made before and after mixing neurotrophins and RED, as had previously been done with NGF and RED (Timm DE, Vissavajjhala P, Ross AH, Neet KE, 1992, Protein Sci 1:1023-1031). Similar changes in CD spectra occurred upon combination of each of the neurotrophins and RED, with negative changes near 220-225 nm and positive changes near 190-200 nm; however, significant differences existed among the various neurotrophin-RED difference spectra. The NT-3/RED complex showed the largest spectral change and NGF the smallest. Thus, specific conformational changes in secondary structure of neurotrophin, RED, or both accompany the binding of each neurotrophin to the extracellular domain of the LANR.     

Remarkably different structures and reaction mechanisms of ketoreductases for the opposite stereochemical control in the biosynthesis of BIQ antibiotics

Two ketoreductases, RED1 and RED2, are involved in the biosynthesis of actinorhodin in Streptomyces coelicolor A3(2) and dihydrogranaticin in S. violaceoruber Tu22, respectively. They are responsible for the stereospecific reductions of the bicyclic intermediate to give (S)- or (R)-DNPA, although there is no similarity between their amino acid sequences. Biotransformation using synthetic analogous substrates revealed that the substrate specificities are quite different. Homology modelling studies and site directed mutagenesis showed remarkable differences in three-dimensional structures and catalytic mechanisms between RED1 and RED2.     

Analysis of the motA flagellar motor gene from Rhodobacter sphaeroides a bacterium with a unidirectional, stop-start flagellum

Rhodobacter sphaeroides swims by unidirectional rotation of a single medial flagellum, re-orienting randomly by Brownian motion when flagellar rotation tops and restarts. Previously we identified a mutant with a paralysed flagellum, which was complemented by a Rhodobacter gene that had homology to motB of Escherichia coli , a bacterium with bidirectional flagella. In the current work, interposon mutagenesis upstream of the Rhodobacter motB gene gave rise to another paralysed mutant, RED5. DNA sequence analysis of this upstream region showed one open reading frame, the predicted polypeptide sequence of which shows homology to the MotA protein of E. coli . MotA is thought to be a proton 'pore' involved in converting proton-motive force into flagellar rotation. Several potential proton-binding amino acids were conserved between putative membrane-spanning regions of R. sphaeroides and E. coli MotA sequences, along with a highly charged cytoplasmic linker region. Complementation studies with mutant RED5 showed the presence of an active promoter upstream from motA which was found to be necessary for expression of both motA and motB , Examination of the upstream DNA sequence showed only one putative promoter-like sequence which resembled a σ⁵⁴- type promoter, including a potential enhancer binding site. The overall similarities between the R. sphaeroides MotA protein and those from other bacteria suggest that, despite the novel unidirectional rotation of he R. sphaeroides flagellum, the function of the MotA protein is similar to that in bacteria with bidirectional flagella.     

Fusarium culmorum is a single phylogenetic species based on multilocus sequence analysis

Fusarium culmorum is a major pathogen of wheat and barley causing head blight and crown rot in cooler temperate climates of Australia, Europe, West Asia and North Africa. To better understand its evolutionary history we partially sequenced single copy nuclear genes encoding translation elongation factor 1-α (TEF), reductase (RED) and phosphate permease (PHO) in 100 F. culmorum isolates with 11 isolates of Fusarium crookwellense, Fusarium graminearum and Fusarium pseudograminearum. Phylogenetic analysis of multilocus sequence (MLS) data using Bayesian inference and maximum parsimony analysis showed that F. culmorum from wheat is a single phylogenetic species with no significant linkage disequilibrium and little or no lineage development along geographic origin. Both MLS and TEF and RED gene sequence analysis separated the four Fusarium species used and delineated three to four groups within the F. culmorum clade. But the PHO gene could not completely resolve isolates into their respective species. Fixation index and gene flow suggest significant genetic exchange between the isolates from distant geographic regions. A lack of strong lineage structure despite the geographic separation of the three collections indicates a frequently recombining species and/or widespread distribution of genotypes due to international trade, tourism and long-range dispersal of macroconidia. Moreover, the two mating type genes were present in equal proportion among the F. culmorum collection used in this study, leaving open the possibility of sexual reproduction.     

Spectroscopic and chemical studies of the interaction between nerve growth factor (NGF) and the extracellular domain of the low affinity NGF receptor.

Nerve growth factor (NGF) interacts with a cell surface receptor on responsive neurons to initiate a series of cellular events leading to neuronal survival and/or differentiation. The first step in this process is the binding of NGF to a low affinity and/or a high affinity receptor. In the present report, we have studied the conformation and stability of recombinant receptor extracellular domain (RED) from the human low affinity receptor and the structural basis of its interaction with NGF. Circular dichroism (CD) studies indicate that the RED is primarily random coil in nature with little regular secondary structure. Thermal stability studies have shown that this irregular conformation is a specific structure that can undergo a reversible two-state thermal denaturation with a concomitant fluorescent and CD change. During heating at 100 degrees C for 15 min, the structure of RED is sufficiently unfolded for a reducing agent, dithiothreitol, to inactivate the receptor toward NGF binding and cross-linking. The complex formation between the RED and NGF has been examined by differential CD measurements, and we have shown that a small, reproducible change in conformation occurs in RED or NGF upon interaction. These results are interpreted in terms of the initiation of NGF cell surface binding and possible modes of signal transduction.     

Genetic characterization of two S-adenosylmethionine-induced ABC transporters reveals their roles in modulations of secondary metabolism and sporulation in Streptomyces coelicolor M145

S-Adenosylmethionine (SAM) was previously documented to activate secondary metabolism in a variety of Streptomyces spp. and to promote actinorhodin (ACT) and undecylprodigiosin (RED) in Streptomyces coelicolor. The SAM-induced proteins in S. coelicolor include several ABC transporter components (SCO5260 and SCO5477) including BldKB, the component of a well-known regulatory factor for differentiations. In order to assess the role of these ABC transporter complexes in differentiation of Streptomyces, SCO5260 and SCO5476, the first genes from the cognate complex clusters, were individually inactivated by gene replacement. Inactivation of either SCO5260 or SCO5476 led to impaired sporulation on agar medium, with the more drastic defect in the SCO5260 null mutant (ASCO5260). ASCO5260 displayed growth retardation and reduced yields of ACT and RED in liquid cultures. In addition, SAM supplementation failed in promoting the production of ACT and RED in ASCO5260. Inactivation of SCO5476 gave no significant change in growth and production of ACT and RED, but impaired the promoting effect of SAM on ACT production without interfering with the effect on RED production. The present study suggests that SAM induces several ABC transporters to modulate secondary metabolism and morphological development in S. coelicolor.     

Escherichia coli MTC, a NADPH cytochrome P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450: comparison of three types of expression systems

Currently three different methods have been taken to develop new mutagenicity tester strains containing human cytochrome P450s (CYPs). Each of these use a single expression vector. In this paper we describe a fourth approach, i.e., the coexpression of a CYP and its electron-transfer flavoprotein, NADPH CYP reductase (RED), encoded by two different expression vectors. The Escherichia coli mutagenicity tester strain BMX100 has been expanded to a strain, MTC which stably expresses human RED. This new tester strain permits the biplasmid coexpression of human CYP1A2 and RED (MTC1A2). This novel strain can be used for the determination of the mutagenicity of chemicals known to be procarcinogens and metabolized by CYP1A2. The mutagenicity tester strain MTC1A2 was compared with: (i) BMX100 using the post-mitochondrial rat liver fraction (S9); (ii) BMX100 with expressing CYP1A2 alone (iii) or with expressing CYP1A2 fused to rat RED or (iv) with expressing CYP1A2, bicistronically coexpressed with rat RED. The biplasmid RED/CYP coexpression system generated a strain with the highest methoxy- and ethoxy-resorufin dealkylase activities and the highest mutagenic activities for the procarcinogens 2-aminoanthracene (2AA), aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). Furthermore, the metabolism of 2AA and IQ was detected more efficiently using the MTC1A2 strain than with the BMX100 strain plus the standard rodent liver S9 metabolic system.