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At least 1,279 records · Page 71Linked to original sources
PubMed2006

EGassembler: online bioinformatics service for large-scale processing, clustering and assembling ESTs and genomic DNA fragments.

Expressed sequence tag (EST) sequencing has proven to be an economically feasible alternative for gene discovery in species lacking a draft genome sequence. Ongoing large-scale EST sequencing projects feel the need for bioinformatics tools to facilitate uniform EST handling. This brings about a renewed importance for a universal tool for processing and functional annotation of large sets of ESTs. EGassembler (http://egassembler.hgc.jp/) is a web server, which provides an automated as well as a user-customized analysis tool for cleaning, repeat masking, vector trimming, organelle masking, clustering and assembling of ESTs and genomic fragments. The web server is publicly available and provides the community a unique all-in-one online application web service for large-scale ESTs and genomic DNA clustering and assembling. Running on a Sun Fire 15K supercomputer, a significantly large volume of data can be processed in a short period of time. The results can be used to functionally annotate genes, to facilitate splice alignment analysis, to link the transcripts to genetic and physical maps, design microarray chips, to perform transcriptome analysis and to map to KEGG metabolic pathways. The service provides an excellent bioinformatics tool to research groups in wet-lab as well as an all-in-one-tool for sequence handling to bioinformatics researchers.

Computational Biology↗
PubMed1997

Expression, assembly, and proteolytic processing of Helminthosporium victoriae 190S totivirus capsid protein in insect cells.

The dsRNA genome (5.2 kbp) of Helminthosporium victoriae 190S totivirus (Hv190SV) consists of two large overlapping open reading frames (ORFs). The 5' proximal ORF codes for the capsid protein (CP) and the 3' ORF codes for an RNA-dependent RNA polymerase. Although the capsid of Hv190SV is encoded by a single gene, it is composed of two major closely related polypeptides, either p88 and p83 or p88 and p78. Whereas p88 and p83 are phosphoproteins, p78 is nonphosphorylated. Expression of the CP ORF in insect cells generated both p78 and p88 which assembled into virus-like particles. The finding that p78, p83, and p88 share a common N-terminal amino acid sequence is consistent with the determination that N-terminal, but not C-terminal, CP deletions were incompetent for assembly. Evidence was obtained that p78 is derived from p88 via proteolytic cleavage at the C-terminus. Proteolytic processing may play a regulatory role in the virus life cycle since it leads to dephosphorylation of CP and a subsequent decrease in virion transcriptional activity.

Animals↗
PubMed2001

Retinyl ester secretion by intestinal cells: a specific and regulated process dependent on assembly and secretion of chylomicrons.

Retinyl esters (RE) have been used extensively as markers to study chylomicron (CM) catabolism because they are secreted in the postprandial state with CM and do not exchange with other lipoproteins in the plasma. To understand the mechanism of secretion of RE by the intestine under the fasting and postprandial states, differentiated Caco-2 cells were supplemented with radiolabeled retinol under conditions that support or do not support CM secretion. We observed that these cells assimilate vitamin A by a rapid uptake mechanism. After uptake, cells store retinol in both esterified and unesterified forms. Under fasting conditions, cells do not secrete RE but secrete free retinol unassociated with lipoproteins. Under postprandial conditions, cells secrete significant amounts of RE only with CM. The secretion of RE with CM was independent of the rate of uptake of retinol and intracellular free and esterified retinol levels, and was absolutely dependent on the assembly and secretion of CM. The secretion of RE was correlated with the secretion of CM and not with the secretion of total apolipoprotein B. Inhibition of CM secretion by Pluronic L81 decreased the secretion of RE and did not result in their increased secretion with smaller lipoproteins. These data strongly suggest that RE secretion by the intestinal cells is a specific and regulated process that occurs in the postprandial state and is dependent on the assembly and secretion of CM. We propose that RE are added to CM during final stages of lipoprotein assembly and may serve as signposts for these steps.

Caco-2 Cells↗
PubMed1997

An algorithm to assemble pathways from processes.

To understand or to modify a biological pathway, the first step is to determine the patterns of coupling among its processes that are compatible with its input-output relation. Algorithms for this purpose have been devised for metabolic pathways, in which the reactions typically leave the enzymes unmodified. As shown here, one of these algorithms can also assemble molecular networks in which reactions modify proteins, if the proteins are included among the inputs to the reactions. Thus one procedure suffices to assemble pathways for metabolism, cytoplasmic signal transduction, and gene regulation.

Algorithms↗
PubMed1994

The brain Kv1.1 potassium channel: in vitro and in vivo studies on subunit assembly and posttranslational processing.

While combined cloning, mutagenesis, and electrophysiological techniques have provided great insight into K+ channel structure/function relationships, little is known about K+ channel biosynthesis. To examine K+ channel biosynthesis, immune purifications were conducted on Triton X-100 extracts of 35S-met-labeled channels from in vitro translations and transfected mouse L-cells. When Kv1.1 and Kv1.4 were cotranslated in vitro, isoform-specific antisera copurified both proteins even at early time points, suggesting rapid subunit assembly. The non-Shaker Kv2.1 channel did not assemble with Kv1.1 or Kv1.4. Mouse L-cells transfected with Kv1.1 cDNA yielded 1000-4000 functional surface channels, and immune purification from Kv1.1 cells with Kv1.1 antisera produced a 57-59 kDa doublet on SDS-PAGE but not in sham-transfected cells. Immune purification of surface channels isolated both the 57 and 59 kDa proteins, suggesting cell surface channels are represented by two species. Pulse-chase metabolic labeling studies were consistent with a precursor-product relationship with the 57 kDa species giving rise to the 59 kDa protein within several minutes of synthesis. At longer chase times, the 57 kDa species reappeared, indicating both an early precursor and a mature protein ran with identical electrophoretic mobility. Mutation of the extracellular glycosylation site (N207) yielded two proteins at steady state, a 55 kDa core peptide and a 57 kDa species. Lack of glycosylation at N207 had little effect on channel synthesis, turnover, or function. Together these results suggest (1) heteromeric assembly of Shaker-like channels is cotranslational, and (2) N207 glycosylation of Kv1.1 occurs but is not required for subunit assembly, transport, or function.

Amino Acid Sequence↗
PubMed1990

Quantum mechanics and cellular information processing: the self-assembly paradigm.

Biological cells have greater information processing efficiency than the programmable computers used to model them. In part this is due to the larger number of interactions that can contribute to function. General arguments suggest that systems in which quantum features play a prominent role are more powerful than classical physical-dynamical analogs. A hypothetical model, involving macromolecular self-assembly, is used to illustrate how the parallelism inherent in the quantum mechanical wave function could play a role in cellular pattern processing. Signals impinging on the external membrane of the cell trigger the release of specifically shaped macromolecules. These aggregate into a mosaic shape features that reflect different groupings of the signal input patterns. The shape features are in turn read out and connected to effector actions by adaptor molecules. The self-assembly model fits into a more general hierarchical scheme of biological information processing in which macroscopic signals are transduced to mesoscopic and then microphysical representations, processed largely at the microphysical level, and then amplified for macroscopic action. The physical dynamics are controlled by proteins and other macromolecules that are molded through the evolutionary process of variation and selection. The organizational requirements for evolutionary moldability and for efficient information processing function are completely consistent. They include high dimensionality, multiplicity of weak interactions, and hierarchical-compartmental structure.

Biological Evolution↗
PubMed1995

Characterization of revertants of a Sindbis virus 6K gene mutant that affects proteolytic processing and virus assembly.

Alphaviruses of the Togaviridae encode a small hydrophobic polypeptide of 55 amino acids, noted as the 6K protein, that is synthesized as part of a polyprotein containing the sequences of the two major transmembranal viral structural glycoproteins. Mutations, insertions and deletions in the 6K appear to selectively interfere with the final stages of virus assembly and budding, producing aberrant, multi-cored infectious viruses. In addition, some of these mutations were pleiotropic and much more inhibitory to virus formation. One of the latter, a substitution of alanine in the wild-type Sindbis virus 6K gene by arginine, has been studied further and shown to interfere with normal proteolytic processing of the polyprotein. Cells infected with this mutant but not the wild-type virus also displayed viral antigens in nuclear membranes and released fragments of membranes into the cell culture media. A revertant, obtained by enrichment for a faster growing strain, 'suppressed' these defects and genetic mapping showed that the arginine codon had been modified to encode a methionine. However, the sequence of the 6K protein in this revertant was not wild-type and the revertant was still defective in assembly as demonstrated by formation of aberrant particles. A complete restoration of wild-type particle formation for this revertant could be effected by modifying the E2 glycoprotein sequence.

Animals↗
PubMed1996

Eggshell assembly in Drosophila: processing and localization of vitelline membrane and chorion proteins.

The Drosophila eggshell consists of three major proteinaceous layers: the vitelline membrane, the inner chorionic layer, and the outer endochorion. During the latter stages of oogenesis, the proteins that comprise these layers are synthesized and secreted by epithelial follicle cells which surround the maturing oocyte. While there is considerable knowledge of the structural units which comprise the eggshell layers, there is little knowledge of how individual proteins function or interact with one another to form the structure. Immunoelectron microscopy was used to follow the distribution of four different eggshell proteins in the assembling and mature eggshell. sV23 and sV17, follicle cell proteins synthesized during the early stages of eggshell formation (stages 8-10), were distributed within the vitelline membrane layer at all stages. Despite marked temporal differences in their accumulation profiles, s36 and s18, putative chorion proteins, were similarly distributed throughout the floor, pillars, and roof of the endochorion. Although the vitelline membrane appears to be morphologically complete by stage 11, developmental Western blots and immunolocalization data indicate that molecular dynamism persists within the layer throughout the subsequent choriogenic stages. During early chorion formation the vitelline membrane appears to act as a reservoir for chorion proteins since s36 was found predominantly in the vitelline membrane layer of stage 12 egg chambers. During the late choriogenic stages (13-14), both sV17 and sV23 are processed to smaller derivatives. Interactions between the eggshell layers were suggested by ultrastructural analysis of a sV23 protein null mutant which showed that the structural integrity of the outer chorion is dependent upon the presence of a vitelline membrane component.

Animals↗
PubMed2004

Coupling between snoRNP assembly and 3' processing controls box C/D snoRNA biosynthesis in yeast.

RNA polymerase II transcribes genes encoding proteins and a large number of small stable RNAs. While pre-mRNA 3'-end formation requires a machinery ensuring tight coupling between cleavage and polyadenylation, small RNAs utilize polyadenylation-independent pathways. In yeast, specific factors required for snRNA and snoRNA 3'-end formation were characterized as components of the APT complex that is associated with the core complex of the cleavage/polyadenylation machinery (core-CPF). Other essential factors were identified as independent components: Nrd1p, Nab3p and Sen1p. Here we report that mutations in the conserved box D of snoRNAs and in the snoRNP-specific factor Nop1p interfere with transcription and 3'-end formation of box C/D snoRNAs. We demonstrate that Nop1p is associated with box C/D snoRNA genes and that it interacts with APT components. These data suggest a mechanism of quality control in which efficient transcription and 3'-end formation occur only when nascent snoRNAs are successfully assembled into functional particles.

Base Sequence↗
PubMed2002

Room temperature microspectrofluorimetry as a useful tool for studying the assembly of the PSII chlorophyll-protein complexes in single living cells of etiolated Euglena gracilis Klebs during the greening process.

The assembly kinetics of the PSII chlorophyll-protein complexes was followed during the greening of Euglena gracilis by microspectrofluorimetry in vivo, at room temperature, on single living cells. The study was correlated to micro- and submicroscopic events accompanying the proplastid to chloroplast transformation and with the immunolocalization of the LHCPII. Etiolated cells of Euglena gracilis were grown in darkness in Mego's heterotrophic liquid medium under shaking at 25+/-1 degrees C. At the stationary phase of growth, they were exposed to continuous light (330 micromol m(-2) s(-1)) for 72 h. The analyses were carried out on samples collected at different times of illumination. Microspectrofluorimetric data were recorded in the 620-780 nm range (excitation at 436 nm) and were resolved into Gaussian components corresponding to the reaction centres (RCII) and the inner antennae (CP(43-47)) of the PSII and LHCPII. From the RCII/CP(43-47) and LHCPII/PSII ratios, it was inferred that (1) a disconnection between RCII and CP(43-47) syntheses occurs during the lag phase of chloroplast differentiation, RCII being synthesized before the inner antennae. This results in the accumulation of uncoupled PSII Chl-protein complexes; (2) after lag phase, the RCII and CP(43-47) syntheses are connected one to another; (3) the freshly synthesized LHCPII complexes are immediately assembled with the PSII, suggesting that the outer antennae always maintain the form bound to PSII. Micro- and submicroscopical observations and LHCPII immunolocalization were in agreement. These data suggest that microspectrofluorimetry may constitute a useful non-destructive tool for studying the assembly kinetics of PSII, under fully physiological life conditions.

Animals↗
PubMed1997

Synthesis and assembly of the D1 protein into photosystem II: processing of the C-terminus and identification of the initial assembly partners and complexes during photosystem II repair.

In previous studies [van Wijk, K. J., Bingsmark, S., Aro, E.-M., & Andersson, B. (1995) J. Biol. Chem. 270, 25685-25695; van Wijk, K. J., Andersson, B., & Aro, E.-M. (1996) J. Biol. Chem 271, 9627-9636], we have demonstrated that D1 protein synthesized in isolated chloroplasts and thylakoids is incorporated into the photosystem II (PSII) core complex. By pulse-chase experiments in these in vitro systems, followed by sucrose gradient fractionation of solubilized thylakoid membranes, it was shown that this assembly proceeded stepwise; first the D1 protein was incorporated to form a PSII reaction center complex (PSII rc), and through additional assembly steps the PSII core complex was formed. In this study, we have analyzed this assembly process in more detail, with special emphasis on the initial events, through further purification and analysis of the assembly intermediates by nondenaturing Deriphat-PAGE and by flatbed isoelectric focusing. The D2 protein was found to be the dominant PSII reaction center protein initially associating with the new D1 protein. This strongly suggests that the D2 protein is the primary "receptor" or stabilizing component during or directly after synthesis of the D1 protein. After formation of the D1-D2 heterodimer, cyt b559 became attached, whereas the psbI gene product was assembled as a subsequent step, thereby forming a PSII reaction center complex. Subsequent formation of the PSII core occurred by binding of CP47 and then CP43 to the PSII rc. The rapid radiolabeling of a minor population of a PSII core subcomplex without CP43 indicated that an association of newly synthesized D1 protein with a preexisting complex consisting of D2/cyt b55q/psbI gene product/CP47 was possibly occurring, in parallel to the predominant sequential assembly pathway. The kinetics of synthesis and processing of the precursor D1 protein were followed in isolated chloroplasts and were compared with its incorporation into PSII assembly intermediates. No precursor D1 protein was found in PSII core complexes, indicating either that incorporation into the PSII core complex facilitates the cleavage of the C-terminus or, more likely, that processing is more rapid than the assembly into the PSII core.

Centrifugation, Density Gradient↗
PubMed2006

On the mechanism of nucleosome assembly by histone chaperone NAP1.

The process of mononucleosome assembly mediated by histone chaperone NAP1 was investigated using DNA fragments 146 and 207 bp in length containing the Lytechinus variegatus 5 S rDNA nucleosome positioning sequence. A quantitative description was derived using gel electrophoresis and fluorescent anisotropy data. First, NAP1-bound H3.H4 was released forming a DNA-histone tetramer complex with a time constant of k(1) = (2.5 +/- 0.7) . 10(4) m(-1) s(-1). The tetrasome was converted quickly (k(2) = (4.1 +/- 3.5) . 10(5) m(-1) s(-1)), by the addition of a single H2A.H2B dimer, into a "hexasome," i.e. a nucleosome lacking one H2A.H2B dimer. From this intermediate a nucleosome was formed by the addition of a second H2A.H2B dimer with an average rate constant k(3) = (6.6 +/- 1.4) . 10(3) m(-1) s(-1). For the back-reaction, significant differences were observed between the 146- and 207-bp DNA upon substitution of the canonical H2A histone with H2A.Z. The distinct nucleosome/hexasome ratios were reflected in the corresponding equilibrium dissociation constants and revealed some differences in nucleosome stability. In a fourth reaction, NAP1 mediated the binding of linker histone H1 to the nucleosome, completing the chromatosome structure with k(4) = (7.7 +/- 3.7) . 10(3) m(-1) s(-1). The activity of the chromatin remodeling complex ACF did not increase the kinetics of the mononucleosome assembly process.

Anisotropy↗
PubMed1993

Assembly of a processive messenger RNA polyadenylation complex.

Polyadenylation of mRNA precursors by poly(A) polymerase depends on two specificity factors and their recognition sequences. These are cleavage and polyadenylation specificity factor (CPSF), recognizing the polyadenylation signal AAUAAA, and poly(A) binding protein II (PAB II), interacting with the growing poly(A) tail. Their effects are independent of ATP and an RNA 5'-cap. Analysis of RNA-protein interactions by non-denaturing gel electrophoresis shows that CPSF, PAB II and poly(A) polymerase form a quaternary complex with the substrate RNA that transiently stabilizes the binding of poly(A) polymerase to the RNA 3'-end. Only the complex formed from all three proteins is competent for the processive synthesis of a full-length poly(A) tail.

Adenosine Triphosphate↗
PubMed2005

Structural relationship between a host included chain of spirocyclic water hexamers and bulk water--the role of water clusters in self assembly and crystallization processes.

Infinite chains of spirocyclic water hexamers are included in the crystal lattice of a tin complex with a curved, hydrophobic surface and only weak intermolecular bonding interactions between the host molecules, so that the enclosed water clusters might be reminiscent of the solvation sphere in solution.

Journal Article↗
PubMed1997

Human recombinant alpha1(V) collagen chain. Homotrimeric assembly and subsequent processing.

Human embryonic kidney cells (293-EBNA) have been transfected with the full-length human alpha1 chain of collagen V using an episomal vector. High yields (15 microgram/ml) of recombinant collagen were secreted in the culture medium. In presence of ascorbate, the alpha1(V) collagen is correctly folded into a stable triple helix as shown by electron microscopy and pepsin resistance. Circular dichroism data confirm the triple-helix conformation and indicate a melting temperature of 37.5 degrees C for the recombinant homotrimer. The major secreted form is a 250-kDa polypeptide (alpha1FL). N-terminal sequencing and collagenase digestion indicate that alpha1FL retains the complete N-propeptide but lacks the C-propeptide. However, alpha1FL might undergo a further N-terminal trimming into a form (alpha1TH) corresponding to the main triple-helix domain plus the major part of the NC2 domain. This processing is different from the one of the heterotrimeric (alpha1(V))2alpha2(V) and could have some physiological relevance. Analysis of cell homogenates indicates the presence of a 280-kDa polypeptide that is disulfide-linked through its C-terminal globular domain. This C-propeptide is rapidly cleaved after secretion in the medium, giving the first evidence of a C-terminal processing of recombinant fibrillar collagens. Rotary shadowing observations not only confirm the presence of a globular domain at the N-terminal end of the molecule but reveal the presence of a kink within the triple helix in a region poor in iminoacids. This region could represent a target for proteases. Together with the thermal stability data, these results might explain the low amount of (alpha1(V))3 recovered from tissues.

Ascorbic Acid↗