Unconventional vortex dynamics in mesoscopic superconducting Corbino disks.

The discrete shell structure of vortex matter strongly influences the flux dynamics in mesoscopic superconducting Corbino disks. While the dynamical behavior is well understood in large and in very small disks, in the intermediate-size regime it occurs to be much more complex and unusual, due to (in)commensurability between the vortex shells. We demonstrate unconventional vortex dynamics (inversion of shell velocities with respect to the gradient driving force) and angular melting (propagating from the boundary where the shear stress is minimum, towards the center) in mesoscopic Corbino disks.

Complete nucleotide sequence of Dendrocalamus latiflorus and Bambusa oldhamii chloroplast genomes.

Although bamboo is one of the most important woody crops in Asia, information on its genome is still very limited. To investigate the relationship among Poaceae members and to understand the mechanism of albino mutant generation in vitro, the complete chloroplast genome of two economically important bamboo species, Dendrocalamus latiflorus Munro and Bambusa oldhamii Munro, was determined employing a strategy that involved polymerase chain reaction (PCR) amplification using 443 novel primers designed to amplify the chloroplast genome of these two species. The lengths of the B. oldhamii and D. latiflorus chloroplast genomes are 139,350 and 139,365 bp, respectively. The organization structure and the gene order of these two bamboos are identical to other members of Poaceae. Highly conserved chloroplast genomes of Poaceae facilitated sequencing by the PCR method. Phylogenetic analysis using both chloroplast genomes confirmed the results obtained from studies on chromosome number and reproductive organ morphology. There are 23 gaps, insertions/deletions > 100 bp, in the chloroplast genomes of 10 genera of Poaceae compared in this study. The phylogenetic distribution of these gaps corresponds to their taxonomic placement. The sequences of these two chloroplast genomes provide useful information for studying bamboo evolution, ecology and biotechnology.

Biological functions of the cytoplasmic TGBp1 inclusions of bamboo mosaic potexvirus.

The TGBp1 protein encoded by the first open reading frame of the triple-gene-block (TGB) of bamboo mosaic potexvirus (BaMV) plays important roles in virus movement; one of them is to shape the viral RNA before its being transported from cell to cell. However, TGBp1 mainly forms cytoplasmic inclusions which are devoid of the RNA-binding activity. With the purified TGBp1 inclusions of BaMV, we analyzed the possibility of dissociation of functional TGBp1 from the inclusions. Our data showed that the cytoplasmic inclusions were able to dissociate continuously functional TGBp1, which possessed ATP-binding, ATPase, and RNA-binding activities. Moreover, the dissociation of TGBp1 was significantly enhanced by the presence of viral RNA and capsid protein (CP), consistent with the finding that TGBp1 are able to form ribonucleoprotein complex with viral RNA and CP. Taken together, these results support the idea that the cytoplasmic TGBp1 inclusions are active pools of TGBp1, upon which viral RNA can be shaped into a transferable form.

Heterotrimeric G-protein and signal transduction in the nematode-trapping fungus Arthrobotrys dactyloides.

The fungus Arthrobotrys dactyloides produces specialized constricting rings to trap and then consume nematodes. The signal transduction pathway involved in the nematode-trapping process was examined. Mastoparan, an activator of G-protein, had a stimulatory effect on the inflation of ring cells, whereas a G-protein inhibitor, pertussis toxin, prevented ring-cell expansion. The 40-kDa G alpha of heterotrimeric G-proteins was specifically ADP-ribosylated by pertussis toxin. Using an antibody specific to the 35-kDa subunit G beta, we showed that immunogold-labeled G beta was more concentrated in ring cells than in the hyphae. In the absence of nematodes, the rings could be inflated by either pressurizing the culture in a syringe, raising intracellular Ca2+ concentrations, or adding warm water. We used these methods to reveal differences in responses to antagonists. The results support a model in which the pressure exerted by a nematode on the ring activates G-proteins in the ring cells. The activation leads to an increase in cytoplasmic Ca2+, activation of calmodulin, and finally the opening of water channels. The ring cells expand to constrict the ring and thus immobilize the nematode.

RNA degradosomes exist in vivo in Escherichia coli as multicomponent complexes associated with the cytoplasmic membrane via the N-terminal region of ribonuclease E.

RNase E isolated from Escherichia coli is contained in a multicomponent "degradosome" complex with other proteins implicated in RNA decay. Earlier work has shown that the C-terminal region of RNase E is a scaffold for the binding of degradosome components and has identified specific RNase E segments necessary for its interaction with polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and enolase. Here, we report electron microscopy studies that use immunogold labeling and freeze-fracture methods to show that degradosomes exist in vivo in E. coli as multicomponent structures that associate with the cytoplasmic membrane via the N-terminal region of RNase E. Whereas PNPase and enolase are present in E. coli in large excess relative to RNase E and therefore are detected in cells largely as molecules unlinked to the RNase E scaffold, immunogold labeling and biochemical analyses show that helicase is present in approximately equimolar amounts to RNase E at all cell growth stages. Our findings, which establish the existence and cellular location of RNase E-based degradosomes in vivo in E. coli, also suggest that RNA processing and decay may occur at specific sites within cells.

Functional analyses and identification of two arginine residues essential to the ATP-utilizing activity of the triple gene block protein 1 of bamboo mosaic potexvirus.

The TGBp1 of bamboo mosaic potexvirus (BaMV) is encoded by the first overlapping gene of the triple-gene-block (TGB), whose products are thought to play roles in virus movement between plant cells. This protein forms cytoplasmic inclusions associated with virus particles in the BaMV-infected tissues. It has been proposed that the inclusion is one of the active forms of TGBp1. To prove this idea, we purified the TGBp1 inclusions from both the BaMV-infected Chenopodium quinoa and Escherichia coli cells overexpressing this protein to test some of their biochemical activities. We found that the TGBp1 inclusions isolated from the infected plant leaves, but not from E. coli, possess the NTP-binding and NTPase activities. However, they lack the RNA-binding activity possessed by the soluble TGBp1. These results indicate that the TGBp1 proteins in the BaMV-infected tissues assume two different functional forms. Mutational analyses and competition experiments show that the two arginine residues, Arg-16 and Arg-21, essential to RNA binding, are also required for the ATP-utilizing activity of the soluble TGBp1. This indicates that a same-structure motif is required for the two functions of the soluble TGBp1. The location of the two arginine residues outside the seven conserved motifs of the NTP-utilizing superfamily I RNA helicases, to which TGBp1 belongs, suggests that an extra-structure motif, besides the seven conserved ones, is required for the NTP-utilizing activity of the TGBp1 protein of BaMV.

Generation of subgenomic RNA directed by a satellite RNA associated with bamboo mosaic potexvirus: analyses of potexvirus subgenomic RNA promoter.

Satellite RNA of bamboo mosaic potexvirus (satBaMV), a single-stranded positive-sense RNA encoding a nonstructural protein of 20 kDa (P20), depends on bamboo mosaic potexvirus (BaMV) for replication and encapsidation. A full-length cDNA clone of satBaMV was used to examine the sequences required for the synthesis of potexvirus subgenomic RNAs (sgRNAs). Subgenomic promoter-like sequences (SGPs), 107 nucleotides (nt) upstream from the capsid protein (CP) gene of BaMV-V, were inserted upstream of the start codon of the P20 gene of satBaMV. Insertion of SGPs gave rise to the synthesis of sgRNA of satBaMV in protoplasts of Nicotiana benthamiana and leaves of Chenopodium quinoa when coinoculated with BaMV-V genomic RNA. Moreover, both the satBaMV cassette and its sgRNA were encapsidated. From analysis of the SGPs by deletion mutation, we concluded that an SGP contains one core promoterlike sequence (nt -30 through +16), two upstream enhancers (nt -59 through -31 and -91 through -60), and one downstream enhancer (nt +17 through +52), when the transcription initiation site is taken as +1. Site-directed mutagenesis and compensatory mutation to disrupt and restore potential base pairing in the core promoter-like sequence suggest that the stem-loop structure is important for the function of SGP in vivo. Likewise, the insertion of a putative SGP of the BaMV open reading frame 2 gene or a heterologous SGP of potato virus X resulted in generation of an sgRNA. The satBaMV cassette should be a useful tool to gain insight into sequences required for the synthesis of potexvirus sgRNAs.

A defective RNA associated with bamboo mosaic virus and the possible common mechanisms for RNA recombination in potexviruses.

A naturally occurring 1.1 kb RNA was isolated from purified virions of bamboo mosaic potexvirus isolate S (BaMV-S). This RNA is a defective RNA (D RNA) derived from a single internal deletion of the BaMV genome. A cDNA clone representing the complete nucleotide sequence of the BaMV-S D RNA was generated and its nucleotide sequence was determined. The BaMV D cDNA is 1015 nts in length [excluding the poly(A) tail] and consists of two regions corresponding to 867 nts of the 5' terminus and 148 nts of the 3' terminus of the BaMV genomic RNA. BaMV D cDNA contains a single open reading frame (ORF) encoding a putative 29.7 kDa protein comprised of a fusion of the first 258 amino acids of BaMV ORF 1 and the last 2 amino acids of coat protein. The coding capacity of D RNA was verified by in vitro translation of native BaMV-S D RNA and of 1.1 kb RNA transcribed in vitro from the full-length D cDNA. BaMV D RNA can be reproducibly generated by serial passages of BaMV-S in Nicotiana benthamiana and is the first D RNA in the potexvirus group shown to be generated de novo. Alignments of sequences surrounding the 5' and 3' junction borders of reported potexvirus D RNAs reveal a 65.2-84.6% sequence identity, suggesting that common mechanisms for viral RNA recombination are involved in the generation of potexvirus D RNAs.

Identification of the RNA-binding sites of the triple gene block protein 1 of bamboo mosaic potexvirus.

The triple gene block protein 1 (TGBp1) encoded by open reading frame 2 of bamboo mosaic potexvirus (BaMV) was overexpressed in Escherichia coli and purified in order to test its RNA-binding activity. UV crosslinking assays revealed that the RNA-binding activity was present mainly in the soluble fraction of the refolded TGBp1. The binding activity was nonspecific and salt concentration-dependent: activity was present at 0-50 mM NaCl but was almost abolished at 200 mM. The RNA-binding domain was located by deletion mutagenesis to the N-terminal 3-24 amino acids of TGBp1. Sequence alignment analysis of the N-terminal 25 amino acids of the TGBp1 homologues of potexviruses identified three arginine residues. Arg-to-Ala substitution at any one of the three arginines eliminated most of the RNA-binding activity, indicating that they were all critical to the RNA-binding activity of the TGBp1 of BaMV.

Sufficient length of a poly(A) tail for the formation of a potential pseudoknot is required for efficient replication of bamboo mosaic potexvirus RNA.

RNAs transcribed from a full-length infectious cDNA clone of the bamboo mosaic potexvirus (strain O) genome, pBaMV-O, were infectious to Nicotiana benthamiana plants. Mutant genomes in which the poly(A) tail is absent or replaced by a 3' tRNA-like structure from turnip yellow mosaic virus RNA failed to amplify detectably in N. benthamiana protoplasts. No amplification was detected in protoplasts inoculated with transcripts containing 4, 7, or 10 adenylate residues at the 3' end, whereas transcript inocula with 15 adenylate residues resulted in coat protein accumulation to a level 26% of that resulting from inoculation with transcripts with 25 adenylate residues (designated as wild type). Coat protein accumulation levels of 69 and 98% relative to wild type were observed after inoculation of protoplasts with transcripts bearing poly(A) tails 18 and 22 nucleotides long, respectively. The presence of a putative 3' pseudoknot structure including at least 13 adenylate residues of the 3'-terminal poly(A) tail was supported by enzymatic and chemical structural analysis. The functional relevance of this putative pseudoknot was tested by mutations that affected basepairing within the pseudoknot. These results support the existence of functional 3' pseudoknot that includes part of the 3' poly(A) tail.

Bamboo mosaic potexvirus satellite RNA (satBaMV RNA)-encoded P20 protein preferentially binds to satBaMV RNA.

A satellite RNA of 836 nucleotides [excluding the poly(A) tail] depends on the bamboo mosaic potexvirus (BaMV) for its replication and encapsidation. The BaMV satellite RNA (satBaMV) contains a single open reading frame encoding a 20-kDa nonstructural protein (P20). The P20 protein with eight histidine residues at the C terminus was overexpressed in Escherichia coli. Experiments of gel retardation, UV cross-linking, and Northwestern hybridization demonstrated that purified P20 was a nucleic-acid-binding protein. The binding of P20 to nucleic acids was strong and highly cooperative. P20 preferred binding to satBaMV- or BaMV-related sequences rather than to nonrelated sequences. By deletion analysis, the P20 binding sites were mainly located at the 5' and 3' untranslated regions of satBaMV RNA, and the RNA-protein interactions could compete with the poly(G) and, less efficiently, with the poly(U) homopolymers. The N-terminal arginine-rich motif of P20 was the RNA binding domain, as shown by in-frame deletion analysis. This is the first report that a plant virus satellite RNA-encoded nonstructural protein preferentially binds with nucleic acids.

Subgenomic RNAs of bamboo mosaic potexvirus-V isolate are packaged into virions.

Purified virions of bamboo mosaic potexvirus-V isolate (BaMV-V) were found to contain three major RNA species, the 6.4 kb genomic RNA and two RNAs of 2.0 and 1.0 kb, in addition to associated satellite RNA (0.85 kb). Results of Northern blot hybridization, primer extension analysis and cDNA sequencing showed that the packaged 2.0 and 1.0 kb RNAs of BaMV-V were subgenomic RNAs. In contrast, in the BaMV-O isolate, only genomic RNA was packaged and encapsidated subgenomic RNAs were not detectable. The transcription initiation sites for the 2.0 and 1.0 kb subgenomic RNAs of BaMV-V were located 1 1 and 16 nt upstream of the initiation codon of open reading frames (ORFs) 2 and 5, respectively. The 2.0 and 1.0 kb subgenomic RNAs functioned as messengers for the ORF2 protein and capsid protein, respectively. Packaging of the 1.0 kb subgenomic RNAs resulted in the formation of rod-shaped particles about 70 nm in length. Our results indicate that BaMV isolates have evolved distinctly for packaging of subgenomic RNAs.

Subcellular localization of the 28 kDa protein of the triple-gene-block of bamboo mosaic potexvirus.

Open reading frame 2 of the bamboo mosaic potexvirus (BaMV) genome encodes a 28 kDa protein, the first of the "triple-gene-block' of BaMV which is believed to play a role in cell-to-cell movement of the virus in host plants. The 28 kDa protein was expressed in Escherichia coli and polyclonal antiserum was raised in a rabbit. Western blot analyses showed that the 28 kDa protein was associated mainly with components in the cell wall and 30000 g pellet fractions of a BaMV-infected leaf homogenate. Immunogold electron microscopy of infected leaf tissues revealed that the 28 kDa protein was associated with electron-dense crystal-line bodies (EDCBs) in the cytoplasm and nuclei. Nuclear EDCBs were found closely associated with nucleoli. Gold-labelled EDCB-like structures were also detected in the cytoplasm, but not within nuclei, in protoplasts up to 48 h post-inoculation. No specific labelling of the 28 kDa protein was found within any cytoplasmic structures or within cell walls.

Molecular evolution and phylogeny of satellite RNA associated with bamboo mosaic potexvirus.

Satellite RNA of bamboo mosaic potexvirus (satBaMV) is a linear RNA molecule which encodes a 20-kDa nonstructural protein. Sequences of seven different satBaMV isolates from bamboo hosts in three genera showed 0.7% to 7.5% base variation which spanned the whole RNA molecule. However, the putative 20-kDa open reading frame was all preserved in these isolates. The phylogenetic relationship based on the nucleotide sequence did not show particular grouping of satBaMV from the host in one genus; neither was the grouping of satBaMV evident by location of sampling. Putative secondary structures of the 3' untranslated regions showed a basic pattern with conserved hexanucleotides (ACCUAA) and polyadenylation signal (AAUAAA) located in the loop regions. Although the satBaMV-encoded 20-kDa protein is a nonstructural protein, its predicted secondary structure contains eight-stranded beta-sheets which may form "jelly-roll" structure similar to that found in capsid protein encoded by satellite virus of panicum mosaic virus.

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.

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.

The open reading frame of bamboo mosaic potexvirus satellite RNA is not essential for its replication and can be replaced with a bacterial gene.

A satellite RNA of 836 nt depends on the bamboo mosaic potexvirus (BaMV) for its replication and encapsulation. The BaMV satellite RNA (satBaMV) contains a single open reading frame encoding a 20-kDa nonstructural protein. A full-length infectious cDNA clone has been generated downstream of the T7 RNA polymerase promoter. To investigate the role of the 20-kDa protein encoded by satBaMV, satBaMV transcripts containing mutations in the open reading frame were tested for their ability to replicate in barley protoplasts and in Chenopodium quinoa using BaMV RNA as a helper genome. Unlike other large satellite RNAs, mutants in the open reading frame did not block their replication, suggesting that the 20-kDa protein is not essential for satBaMV replication. Precise replacement of the open reading frame with sequences encoding chloramphenicol acetyltransferase resulted in high level expression of chloramphenicol acetyltransferase in infected C. quinoa, indicating that satBaMV is potentially useful as a satellite-based expression vector.

Capsid protein of cucumber mosaic virus accumulates in the nuclei and at the periphery of the nucleoli in infected cells.

Tobacco leaves infected with two strains and their reciprocal RNA 3 pseudorecombinants of cucumber mosaic virus (CMV) were examined by immunoelectron microscopy. In addition to the regular detection of CMV in the cytoplasm and vacuoles, immunogold-labelled viral proteins occurred commonly in the nuclei and at the periphery of impacted nucleoli in all four samples examined. However, viral protein was present only in the euchromatin region and rare in the heterochromatin region.

Satellite RNA associated with bamboo mosaic potexvirus shares similarity with satellites associated with sobemoviruses.

A putative nonstructural protein encoded by a satellite RNA associated with bamboo mosaic potexvirus shares 46% identity with the capsid protein of satellite virus of panicum mosaic sobemovirus. The sequence similarity among satellite plant viruses which have no apparent relationship implies a common origin.

Nucleotide sequence of the genomic RNA of bamboo mosaic potexvirus.

The complete nucleotide sequence of the genomic RNA of bamboo mosaic virus (BaMV) was determined by sequencing a set of overlapping cDNA clones and by direct sequencing of the viral RNA. The RNA genome of BaMV is 6366 nucleotides long [excluding 3'poly(A) tail] and contains six open reading frames (ORFs 1 to 6) coding for polypeptides with M(r) values of 155K, 28K, 13K, 6K, 25K and 14K, respectively. The genome organization and sizes of the encoded proteins are very similar to those of other potexviruses which have been sequenced except that ORF 6 lies completely within ORF 1. The first five putative proteins of the BaMV genome show identities ranging between 44 to 59%, 26 to 49%, 30 to 53%, 15 to 35% and 20 to 30%, respectively, to the corresponding ORFs of other members of the potexvirus group. However the putative product ORF 6 shows no significant similarity to those of other potexvirus ORF products.

A satellite RNA associated with bamboo mosaic potexvirus.

A small RNA molecule with properties of a satellite RNA was found in an isolate of bamboo mosaic potexvirus (BaMV-V) from Bambusa vulgaris McClure. This RNA (sBaMV RNA) and the genomic RNA of BaMV shared no significant sequence homology as assessed by hybridization with cDNA probes derived from the genomes of BaMV and sBaMV RNA. Replication of sBaMV RNA in barley protoplasts or Chenopodium quinoa was supported by BaMV, but not by potato virus X, the type member of the potexvirus group, or other unrelated viruses. The complete nucleotide sequence of sBaMV RNA is 836 nucleotides (excluding the poly(A) tail) and contains an open reading frame which starts after a 159-nucleotide 5'-untranslated region and encodes a 20,154-mol wt protein. In an in vitro rabbit reticulocyte lysate system, sBaMV RNA directed the synthesis of a protein estimated to be 25 kDa by SDS-polyacrylamide gel electrophoresis. The sBaMV RNA-encoded protein was not immunoprecipitated with antiserum against BaMV capsid protein. However, the sBaMV RNA was encapsidated with BaMV capsid protein to form rod-shaped particles with an average length of 60 nm as shown by immunoelectron microscopy. This is the first satellite RNA found in the potexvirus group.