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1.
The polymerase-like core of brome mosaic virus 2a protein, lacking a region interacting with viral 1a protein in vitro, maintains activity and 1a selectivity in RNA replication. 总被引:2,自引:2,他引:0
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Brome mosaic virus (BMV), a member of the alphavirus-like super-family of positive-strand RNA viruses, encodes two proteins required for viral RNA replication: 1a and 2a. 1a contains m7G methyltransferase- and helicase-like domains, while 2a contains a polymerase (pol)-like core flanked by N- and C-terminal extensions. Genetic studies show that BMV RNA replication requires 1a-2a compatibility implying direct or indirect 1a-2a interaction in vivo. In vitro, la interacts with the N-terminal 125-amino-acid segment of 2a preceding the pol-like core, and prior deletion studies suggested that this 2a segment was essential for RNA replication. We have now used protein fusions and deletions to explore possible parallels between noncovalent 1a-2a interaction and covalent fusion of similar protein domains in tobacco mosaic virus and to see whether the N-terminal 2a-1a interaction was the primary basis for 1a-2a compatibility in vivo. We found that 2a can function as part of a tobacco mosaic virus-like 1a-2a fusion and that a 2a segment (amino acids 162 to 697) comprising the pol-like core was sufficient to provide 2a functions in such a fusion. Unexpectedly, the unfused 2a core segment also supported RNA replication when it and wild-type la were expressed as separate proteins. Moreover, in gene reassortant experiments with the related cowpea chlorotic mottle virus, the unfused 2a core segment showed the same 1a compatibility requirements as did wild-type BMV 2a. Thus, the pol-like core of 2a must interact with la in a way that is selective and essential for RNA synthesis, and 1a-2a interactions are more complex than the single, previously mapped interaction of the N-terminal 2a segment with 1a. 相似文献
2.
Rearrangement of VHa1-encoding Ig gene segment to the a2 chromosome in an a1/a2 heterozygous rabbit. Evidence for trans recombination 总被引:1,自引:0,他引:1
VDJ genes were cloned from leukemic B cells of an a1/a2 heterozygous Emu-cmyc transgenic rabbit. Restriction mapping and nucleotide sequence analysis indicated that one clone, 5C3, had a VHa1-encoding gene segment functionally rearranged to a JH gene segment from the a2 chromosome. This VDJ gene may be the result of a trans recombination between a VH gene on the a1 chromosome and a JH gene segment on the a2 chromosome or, it may be the result of a cis recombination if the a2 chromosome contains VHa1-encoding gene segments. 相似文献
3.
Increasing evidence suggests that the assembly of lipoprotein[a] (Lp[a]) proceeds in two steps. In the first step, non-covalent interactions between apolipoprotein[a] (apo[a]) and apolipoprotein B (apoB) of low density lipoprotein (LDL) form a dissociable apo[a]:LDL complex. In the second step, a covalent disulfide linkage forms the stable Lp[a] particle. Several methods are currently used to study the assembly of Lp[a], however, these methods are laborious, time-consuming, and not suitable for a high throughput screening. We report here the development of a rapid and simple assay based on the binding of labeled LDL to a Lp[a]/apo[a] substrate which is immobilized on the surface of a microtiter plate. Quantification of bound LDL provides a measure of the extent of complex formation. Labeled LDL bound to both Lp[a] and apo[a] substrates with similar affinity. Plasma lipoproteins containing apoB as well as free apo[a] were capable of competing with LDL binding. The binding of LDL to Lp[a]/apo[a] was inhibited by L-proline and lysine analogs, which are known to inhibit the non-covalent association between apo[a] and apoB. Using this method we have found that nicotinic acid and captopril are able to inhibit the association of apo[a] with apoB. This method is compatible with automation and can be applied to a high throughput screening of inhibitors of Lp[a] formation. 相似文献
4.
5.
The motion of a metal plasma flow of a vacuum-arc discharge in a transportation plasma-optical system with a curvilinear magnetic
field is studied experimentally and numerically. The flow position at the output of the system is shown to depend on the cathode
material, which determines the mass-to-charge ratio of plasma ions. As a result, the flow with a greater ion mass-to-charge
ratio moves along a trajectory with a larger radius. A similar effect is observed in the case of a multicomponent plasma flow
generated by a composite cathode. The results of two-fluid MHD simulations of a plasma flow propagating in a curvilinear magnetic
field agree qualitatively with the experimental data. 相似文献
6.
The assembly of lipoprotein(a) (Lp(a)) particles occurs via a two-step mechanism in which noncovalent interactions between apolipoprotein(a) (apo(a)) and the apolipoproteinB-100 component of low density lipoprotein precede the formation of a single disulfide bond. Although we have previously demonstrated that the rate constant for the covalent step of Lp(a) assembly can be enhanced by altering the conformational status of apo(a), the resultant rates of covalent Lp(a) particle formation measured in vitro are relatively slow. The large excess of Lp(a) (over apo(a)) observed in vivo can be accounted for by a preferential clearance of apo(a) over Lp(a) and/or a sufficiently high rate of covalent Lp(a) assembly. In the present study, we report that cultured human hepatoma cells secrete an oxidase activity that dramatically enhances the rate of covalent Lp(a) assembly. This activity is likely possessed by a protein because it is heat-sensitive and is retained in the concentrate following ultrafiltration through a 5 kDa cutoff filter. However, a small molecule cofactor for the activity is suggested by the observation that the activity is lost upon dialysis. Plots of Lp(a) assembly rate versus input apo(a) concentration gave rectangular hyperbolae; the reaction displayed an unusual dependence on the concentration of apoB-100, with increasing concentrations of apoB-100 resulting in slower rates of Lp(a) assembly at low concentrations of apo(a), an effect that was alleviated by higher apo(a) concentrations. Interestingly, V(max(app))/K(m(app)) ratios were insensitive to apoB-100 concentration, which is diagnostic of a ping-pong reaction mechanism. In this way, the putative Lp(a) oxidase may be functionally analogous to protein disulfide isomerase, which exhibits a similar mechanism during the catalysis of disulfide bond formation during protein folding, although we have ruled out a role for this enzyme in Lp(a) assembly. 相似文献
7.
The interconversion of chlorophyll a and chlorophyll b, referred to as the chlorophyll cycle, plays a crucial role in the processes of greening, acclimation to light intensity, and senescence. The chlorophyll cycle consists of three reactions: the conversions of chlorophyll a to chlorophyll b by chlorophyllide a oxygenase, chlorophyll b to 7-hydroxymethyl chlorophyll a by chlorophyll b reductase, and 7-hydroxymethyl chlorophyll a to chlorophyll a by 7-hydroxymethyl chlorophyll a reductase. We identified 7-hydroxymethyl chlorophyll a reductase, which is the last remaining unidentified enzyme of the chlorophyll cycle, from Arabidopsis thaliana by genetic and biochemical methods. Recombinant 7-hydroxymethyl chlorophyll a reductase converted 7-hydroxymethyl chlorophyll a to chlorophyll a using ferredoxin. Both sequence and biochemical analyses showed that 7-hydroxymethyl chlorophyll a reductase contains flavin adenine dinucleotide and an iron-sulfur center. In addition, a phylogenetic analysis elucidated the evolution of 7-hydroxymethyl chlorophyll a reductase from divinyl chlorophyllide vinyl reductase. A mutant lacking 7-hydroxymethyl chlorophyll a reductase was found to accumulate 7-hydroxymethyl chlorophyll a and pheophorbide a. Furthermore, this accumulation of pheophorbide a in the mutant was rescued by the inactivation of the chlorophyll b reductase gene. The downregulation of pheophorbide a oxygenase activity is discussed in relation to 7-hydroxymethyl chlorophyll a accumulation. 相似文献
8.
Increased virulence and competitive advantage of a/alpha over a/a or alpha/alpha offspring conserves the mating system of Candida albicans 总被引:1,自引:0,他引:1
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The majority of Candida albicans strains in nature are a/alpha and must undergo homozygosis to a/a or alpha/alpha to mate. Here we have used a mouse model for systemic infection to test the hypothesis that a/alpha strains predominate in nature because they have a competitive advantage over a/a and alpha/alpha offspring in colonizing hosts. Single-strain injection experiments revealed that a/alpha strains were far more virulent than either their a/a or alpha/alpha offspring. When equal numbers of parent a/alpha and offspring a/a or alpha/alpha cells were co-injected, a/alpha always exhibited a competitive advantage at the time of extreme host morbidity or death. When equal numbers of an engineered a/a/alpha2 strain and its isogenic a/a parent strain were co-injected, the a/a/alpha2 strain exhibited a competitive advantage at the time of host morbidity or death, suggesting that the genotype of the mating-type (MTL) locus, not associated genes on chromosome 5, provides a competitive advantage. We therefore propose that heterozygosity at the MTL locus not only represses white-opaque switching and genes involved in the mating process, but also affects virulence, providing a competitive advantage to the a/alpha genotype that conserves the mating system of C. albicans in nature. 相似文献
9.
Purification of proteins is commonly a multiple-step process involving size exclusion, ion exchange, affinity, hydrophobic, and other modes of chromatography. In an effort to circumvent the laborious process of collecting the solutes from each column and reintroducing them onto a second column, a valving system is described that directs the samples eluted from a high-performance liquid chromatographic column through a detector with a high-pressure cell into either a second column or into storage loops of a multiloop value. This multiloop value is referred to as a high-pressure fraction collector. After development of the first column is complete, a second solvent can be directed to the second column or high-pressure fraction collector to elute the solutes back through the detector and onto any other column in the system. The process of eluting a sample from a column through a single detector and directing it to the high-pressure fraction collector or any other column in the system may be repeated a number of times. Such valving systems make it possible to chromatograph a single protein component on two or three columns in a short time. 相似文献
10.
Identification of the domains required for direct interaction of the helicase-like and polymerase-like RNA replication proteins of brome mosaic virus. 总被引:6,自引:0,他引:6
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Brome mosaic virus is a positive-strand RNA virus whose RNA replication requires viral protein 1a, which has putative helicase and capping functions, and 2a, which has putative polymerase function. Since domains of related sequence are conserved in a wide range of plus-strand RNA viruses, analysis of 1a and 2a function should have applicability to many other viruses. We have recently demonstrated that 1a and 2a form a complex in vivo and in vitro. Using immune coprecipitation and mutant polypeptides made in reticulocyte lysates, we have now mapped both the 1a and 2a domains necessary for complex formation. The sequences needed to bind 2a map to the carboxy-terminal helicase-like domain of 1a. Truncated polypeptides containing this domain were able to bind to 2a, while several small insertions in the helicase-like domain disrupted binding. The sequence required for binding 1a lies within a 115-residue subset of the 2a N-terminal segment preceding the polymerase-like domain. Truncations or fusion polypeptides containing this segment can bind 1a. We also determined that highly purified 2a protein made in insect cells can form a complex with highly purified 1a helicase-like domain made in Escherichia coli, suggesting that no other factor is required to mediate 1a-2a interaction. Previous genetic analyses of 1a and 2a are consistent with this mapping and show that the newly defined 1a and 2a binding regions are required for RNA synthesis. The locations of these interacting regions are discussed with regard to models of viral replication and the evolution of positive-strand RNA virus genomes. 相似文献
11.
Results are reported of experimental investigations that have revealed the presence of a plasma in the interaction region of a model wideband relativistic microwave amplifier—a dielectric Cherenkov maser. The electrodynamic properties of a hybrid system—a waveguide with an annular dielectric liner and a plasma layer adjacent to its inner wall—are analyzed. Experiments with a high-current accelerator have revealed that the power of the emitted microwaves at the output of the system increases strongly when an external microwave source at different frequencies in the X-band is switched on. However, this effect was found to be hard to reproduce. Indirect evidence is obtained of the fact that, during the transport of an electron beam and under the action of the signal from a high-power pulsed magnetron, the plasma in the system is created at the surface of the dielectric. In the model of a cold magnetized plasma, a dispersion relation is derived for axisymmetric waves in a system with a wall plasma layer. The spectra of the waveguide and plasma modes in the system and the transverse structure of their electromagnetic fields are investigated thoroughly as functions of the plasma density and layer thickness. It is shown that even a very thin layer of a high-density plasma results in a large frequency shift of the dispersion curve of the waveguide mode, in which case the coupling impedance at a fixed frequency decreases sharply. On the other hand, a layer of a moderately dense plasma increases the coupling impedance for the waveguide mode. It is established that, in a configuration with a wall plasma layer, the longitudinal component of the electric field of a plasma mode whose power flux in the dielectric is of a volumetric nature reverses direction across the layer. 相似文献
12.
13.
Two neuregulin-1 isoforms highly expressed in the nervous system are the type III neuregulin III-beta1a and the type I neuregulin I-beta1a. The sequence of these two isoforms differs only in the region that is N-terminal of the bioactive epidermal growth factor-like domain. While the biosynthetic processing of the I-beta1a isoform has been well characterized, the processing of III-beta1a has not been reported. In this study, we compared III-beta1a and I-beta1a processing. Both III-beta1a and I-beta1a were synthesized as transmembrane proproteins that were proteolytically cleaved to produce an N-terminal fragment containing the bioactive epidermal growth factor-like domain. For I-beta1a, this product was released into the medium. However, for III-beta1a, this product was a transmembrane protein. In cultures of cells expressing III-beta1a, the amount of neuregulin at the cell surface was much greater, and the amount in the medium was much less than in cultures expressing I-beta1a. Phorbol ester treatment and truncation of the cytoplasmic tail had markedly different effects on III-beta1a and I-beta1a processing. These results demonstrate an important role for the N-terminal region in determining neuregulin biosynthetic processing and show that a major product of III-beta1a processing is a tethered ligand that may act as a cell surface signaling molecule. 相似文献
14.
Selecting pedigrees for linkage analysis of a quantitative trait: the expected number of informative meioses. 总被引:1,自引:1,他引:0
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With evidence of segregation at a major locus for a quantitative trait having been found, a logical next step is to select a subset of the pedigrees to include in a linkage study to map the major locus. Ideally this subset should include much of the linkage information in the sample but include only a fraction of the pedigrees. We previously described a strategy for selecting pedigrees for linkage analysis of a quantitative trait on the basis of a pedigree likelihood-ratio statistic. For quantitative traits controlled by a major locus with a rare dominant allele, the likelihood-ratio strategy extracted nearly all the information for linkage while typically requiring marker data on only about one-third of the pedigrees. Here, we describe a new strategy to select pedigrees for linkage analysis on the basis of the expected number of potentially informative meioses in each pedigree. We demonstrate that this informative-meioses strategy provides an efficient and more general means to select pedigrees for a linkage study of a quantitative trait. 相似文献
15.
A. Borghi E. Foa R. Balossino F. Migliavacca 《Computer methods in biomechanics and biomedical engineering》2013,16(4):367-377
Today the most popular approach for the prevention of the restenosis consists in the use of the drug eluting stents. The stent acts as a source of drug, from a coating or from a reservoir, which is transported into and through the artery wall. In this study, the behaviour of a model of a hydrophilic drug (heparin) released from a coronary stent into the arterial wall is investigated. The presence of the specific binding site action is modelled using a reversible chemical reaction that explains the prolonged presence of drug in the vascular tissue. An axi-symmetric model of a single stent strut is considered. First an advection–diffusion problem is solved using the finite element method. Then a simplified model with diffusion only in the arterial wall is compared with: (i) a model including the presence of reversible binding sites in the vascular wall and (ii) a model featuring a drug reservoir made of a degradable polymeric matrix. The results show that the inclusion of a reversible binding for the drug leads to delayed release curves and that the polymer erosion affects the drug release showing a quicker elution of the drug from the stent. 相似文献
16.
Genetic linkage between lipoprotein(a) phenotype and a DNA polymorphism in the plasminogen gene 总被引:12,自引:0,他引:12
D T Drayna R A Hegele P E Hass M Emi L L Wu D L Eaton R M Lawn R R Williams R L White J M Lalouel 《Genomics》1988,3(3):230-236
Coronary heart disease risk correlates directly with plasma concentrations of lipoprotein(a) (Lp(a)), a low-density lipoprotein-like particle distinguished by the presence of the glycoprotein apolipoprotein(a) (apo(a)), which is bound to apolipoprotein B-100 (apoB-100) by disulfide bridges. Size isoforms of apo(a) are inherited as Mendelian codominant traits and are associated with variations in the plasma concentration of lipoprotein(a). Plasminogen and apo(a) show striking protein sequence homology, and their genes both map to chromosome 6q26-27. In a large family with early coronary heart disease and high plasma concentrations of Lp(a), we found tight linkage between apo(a) size isoforms and a DNA polymorphism in the plasminogen gene; plasma concentrations of Lp(a) also appeared to be related to genetic variation at the apo(a) locus. We found free recombination between the same phenotype and alleles of the apoB DNA polymorphism. This suggests that apo(a) size isoforms and plasma lipoprotein(a) concentrations are each determined by genetic variation at the apo(a) locus. 相似文献
17.
The Col2a1 gene is expressed in notochord, otic vesicle, cartilaginous tissue and the anlage of endochondral bone during development in higher vertebrates. Type II collagen, a homotrimeric product of the Col2a1 gene, functions as a key regulatory protein for cartilage development and endochondral ossification. In medaka and zebrafish, a single homolog of the col2a1 gene has been identified. However, it is necessary to note that many genes are duplicated in teleost fishes. To clarify function of col2a1 genes in teleost fishes and to further understand the process of cartilage development and endochondral ossification, we cloned and mapped the gene loci of two col2a1 orthologs in medaka. The proteins encoded by both medaka col2a1a and col2a1b genes were highly conserved (85.3% and 82.6%) relative to human COL2A1, but synteny was not observed. We also examined the expression patterns of col2a1a and col2a1b during embryonic development. Whole-mount insitu hybridization data suggests that expression patterns of both medaka co2a1a and col2a1b genes are similar to that of zebrafish co2a1 in the early embryonic stages. In medaka, the two col2a1 genes show a closely correlated pattern of spatial and temporal expression. In late embryonic stages, however, there were differences in both expression patterns in the pectoral fin. This study is the first report of two homologs of col2a1 in teleosts and also the first examination of col2a1a and col2a1b expression patterns in this group. 相似文献
18.
The Action of Homothallism Genes in Saccharomyces Diploids during Vegetative Growth and the Equivalence of hma and HMalpha Loci Functions 总被引:3,自引:3,他引:0
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The action of homothallism genes in vegetatively growing diploid cells was examined. The results demonstrate that homothallism genes function during regular vegetative growth cycles as well as during the first few divisions after spore germination. A procedure based on ultraviolet-induced reciprocal mitotic recombination monitored by homozygosity for cryptopleurine resistance (a recessive marker closely linked to the mating-type locus) allowed us to identify and recover Saccharomyces cerevisiae colonies sectored for the mating-type locus i.e., a/a and alpha/alpha. Homothallism genes can switch a/a or alpha/alpha vegetative diploid cells, generated from a strain with genotype a/alpha HO/ho HMalpha/HMalpha HMa/HMa, to a/alpha diploids or a/a/alpha/alpha tetraploids during a given mitotic division cycle. We found that both a/a and alpha/alpha sectors generated from a strain with genotype a/alpha HO/HO hmalpha/hmalpha hma/HMa switch to a/alpha diploids or a/a/alpha/alpha tetraploids. This finding supports Naumov and Tolstorukov's suggestion (1973) that the hm a allele provides for the same functions as the HMalpha allele, namely, a switch at the mating-type locus from alpha to a. The HO allele is dominant to ho but hma and HMa alleles are codominant. A loose linkage between the mating-type and the HMalpha loci ( approximately 55cM), confirming Harashima, Nogi and Oshima (1974) data, was observed. 相似文献
19.
A missense mutation in the 20S proteasome β2 subunit of Great Danes having harlequin coat patterning
Harlequin is a pigmentary trait of the domestic dog that is controlled by two autosomal loci: the melanosomal gene, SILV, and a modifier gene, harlequin (H), previously localized to chromosome 9. Heterozygosity for a retrotransposon insertion in SILV and a mutation in H causes a pattern of black patches on a white background. Homozygosity for H is embryonic lethal. Fine mapping of the harlequin locus revealed a 25 kb interval wherein all harlequin Great Danes are heterozygous for a common haplotype. This region contains one gene, PSMB7, which encodes the β2 catalytic subunit of the proteasome. Sequence analysis identified a coding variant in exon 2 that segregates with harlequin patterning. The substitution predicts the replacement of a highly conserved valine with a glycine. Described herein is the identification of a naturally-occurring mutation of the ubiquitin proteasome system that is associated with a discernable phenotype of dogs. 相似文献
20.
Wnt proteins and Wnt signalings have been implicated in a variety of development and cell processes, while aberrant activation of Wnt signaling is linked to a range of cancers in many tissues. In this study, we used the HEK293 cell line to investigate the effects of Wnt3a and Wnt5a on proliferation and apoptosis in a serum starvation culture. After Wnt3a and Wnt5a proteins were expressed, they both promoted the proliferation of HEK293 cells under serum starvation. After 48h of serum starvation, both Wnt3a and Wnt5a inhibited serum starvation-induced apoptosis of HEK293 cells and continued up to 96h. We demonstrated that Wnt3a and Wnt5a can promote proliferation of HEK293 cells and inhibit serum starvation-induced apoptosis, which implies that Wnt3a and Wnt5a can maintain the survival of HEK293 cells under stress, and also provide a novel insight into the role of Wnt3a and Wnt5a and their related signalings in carcinogenesis. 相似文献