SEARCH · Search PubMed

Results for “Assembly process”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 199 records · Page 11Linked to original sources

PubMed1984

[Synthesis of 10S particles in cells infected with aphthovirus].

In the present study, evidence is presented for the existence of a morphogenetic intermediary that may be a precursor of the procapsids in the assembling process. BHK21 clone 13S cells were infected with Aphthovirus A24 (Cruzeiro strain), and pulse-chase experiments were carried out using 3H-leucine. Cytoplasmic extracts were then prepared at appropriate times, and analyzed by sucrose-gradient ultracentrifugation. After preliminary assays (Fig. 1), working conditions were standardized so as to obtain maximal recovery of the morphogenetic intermediary, as well as consistency of results. Only in the presence of DOC-Brij58 and Mg++ could a 10S sedimentation coefficient peak be seen (Fig. 1 a). A heterogeneous zone, with 4,5-5S as sedimentation coefficient, was also observed. The degree of labeling in the region 4,5-5S compared with that in the 10S portion, depends on the time within the infectious cycle when cells were pulse-labeled. Maximal levels for the ratio 10S/4,5-5S are reached when pulse-labeling takes place at the time when the amount of RNA viral synthesis reaches 80% of its total value (Fig. 2). Similar experiments, performed with third passage bovine fetal kidney cells, were confirmatory of the presence of a structure sedimenting at 10S, as well as of a heterogeneous zone of 4,5-5S (Fig. 3). It would appear that assembling of Aphthoviruses is accomplished through an intermediary unit which differs from that found for other Picornaviruses, the latter being the result of the union of 12 pentamers. The capsid of Aphthoviruses, also composed of 60 identical sub-units, would instead derive from the joining of 20 trimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

The formation of a defective small subunit of the mitochondrial ribosomes in petite mutants of Saccharomyces cerevisiae.

The involvement of mitochondrial protein synthesis in the assembly of the mitochondrial ribosomes was investigated by studying the extent to which the assembly process can proceed in petite mutants of Saccharomyces cerevisiae which lack mitochondrial protein synthetic activity due to the deletion of some tRNA genes and/or one of the rRNA genes on the mtDNA. Petite strains which retain the 15-S rRNA gene can synthesize this rRNA species, but do not contain any detectable amounts of the small mitochondrial ribosomal subunit. Instead, a ribonucleoparticle with a sedimentation coefficient of 30 S (instead of 37 S) was observed. This ribonucleoparticle contained all the small ribosomal subunit proteins with the exception of the var1 and three to five other proteins, which indicates that the 30-S ribonucleoparticle is related to the small mitochondrial ribosomal subunit (37 S). Reconstitution experiments using the 30-S particle and the large mitochondrial ribosomal subunit from a wild-type yeast strain indicate that the 30-S particle is not active in translating the artificial message poly(U). The large mitochondrial ribosomal subunit was present in petite strains retaining the 21-S rRNA gene. The petite 54-S subunit is biologically active in the translation of poly(U) when reconstituted with the small subunit (37 S) from a wild-type strain. The above results indicate that mitochondrial protein synthetic activity is essential for the assembly of the mature small ribosomal subunit, but not for the large subunit. Since the var1 protein is the only mitochondrial translation product known to date to be associated with the mitochondrial ribosomes, the results suggest that this protein is essential for the assembly of the mature small subunit.

DNA, Mitochondrial↗

PubMed1983

Flexibility in tobacco mosaic virus.

Tobacco mosaic virus (TMV) particles are rod-like, 300 nm long and 18 nm in diameter. TMV consists of 2140 protein subunits, each with a relative molecular mass of 17420 (158 residues), arranged on a helix of pitch 2.3 nm with 16 1/3 subunits per turn. Winding through this helix is a single strand of RNA 6400 nucleotides long. Three bases are bound to each protein subunit. TMV has a central hole of diameter 4.0 nm. Assembly of TMV occurs by the threading of the RNA through the central hole of the growing rodlet of viral coat protein and involves a preassembled double disk as intermediate. Given the structure of the subunit, such a mechanism requires that the segment of polypeptide chain which separates the nucleic acid binding site from the lumen of the cylinder should be able to move out of the way during the assembly process. Evidence from diffraction studies and from proton nuclear magnetic resonance spectroscopy points to a segment of about 20 amino acid residues being very flexible in the disk. In the helical virus these residues take on a well-defined conformation which completely shields the nucleic acid from the central channel.

Magnetic Resonance Spectroscopy↗

PubMed1994

A conserved region in the tail domain of vimentin is involved in its assembly into intermediate filaments.

Although the head and rod domains of intermediate filament (IF) proteins are known to play significant roles in filament assembly, the role of the tail domain in this function is unclear and the available information supports contradictory conclusions. We examined this question by comparing transfection of the same cDNA constructs, encoding vimentins with modified tail domains, into cell lines that do and do not contain endogenous IF proteins. By this approach, we were able to distinguish between the ability of a mutant IF protein to initiate assembly de novo, from that of incorporating into existing filament networks. Vimentins with modifications at or near a highly conserved tripeptide, arg-asp-gly (RDG), of the tail domain incorporated into existing IF networks in vimentin-expressing (vim+) cells, but were assembly-incompetent in cells that did not express IF proteins (vim-). The failure of the RDG mutant vimentins to assemble into filament arrays in vim- cells was reversible by re-introducing a wild-type vimentin cDNA, whereupon both wild-type and mutant vimentins coassembled into one and the same IF network. We conclude that the function of the tail domain of type III IF proteins, and possibly of keratins K8 and K18, in IF assembly is distinct from those of other domains; a region encompassing the RDG tripeptide appears to be important in the assembly process.

Adrenal Gland Neoplasms↗

PubMed1989

Surface expression of the T cell receptor complex requires charged residues within the alpha chain transmembrane region.

The T cell receptor (TcR) complex is a multi-subunit glycoprotein comprising at least five transmembrane polypeptides. An unusual characteristic of each of the transbilayer domains is the presence of charged amino acids. To examine the importance of these residues for the association and consequent surface expression of the components of the complex, a TcR alpha chain containing either charged or neutral residues within its transbilayer segment was introduced into the human T cell line MOLT-4, and the appearance of the TcR complex at the cell surface was assayed. Surface expression was observed only in MOLT-4 cells transfected with the alpha chain containing charged transbilayer residues. Thus, these residues most probably play a crucial role in the assembly process.

Amino Acids↗

PubMed1984

The mechanism of self-assembly of the multi-enzyme complex tryptophan synthase from Escherichia coli.

The alpha subunit is bound with negative cooperativity to the holo beta 2 subunit of tryptophan synthase in phosphate buffer. Thus it is feasible to measure separately the rates of formation both of the stable alpha beta 2 subcomplex from beta 2, and of the mature alpha 2 beta 2 complex from alpha beta 2, using stopped-flow techniques. Addition of each alpha subunit proceeds in two steps; an initial alpha beta protomer is formed rapidly, which subsequently isomerizes slowly to the equilibrium state. The rates of dissociation of both the alpha beta 2 and alpha 2 beta 2 complexes were measured by trapping released alpha subunit with enzymically inactive reduced beta 2 subunit. The reversal of the slow isomerization both determines the rate of dissociation, and accounts for the high overall affinity of the beta protomer for the alpha subunit. The data fit to a sequential assembly mechanism consisting of seven protein species and yields values for most of the rate constants and all of the microscopic equilibrium constants. Negative cooperativity arises from a weaker initial binding of the second alpha subunit, as expressed by its larger off-constant, possibly due to steric hindrance. The kinetics of binding of L-serine and indolepropanol phosphate during the assembly process shows that the beta protomer is already partially activated in the initial alpha beta complex. Full activation is achieved in the slow isomerization reaction. In contrast, the alpha subunit gains high affinity for indolepropanol phosphate only in the isomerization reaction. These observations indicate that the isomerization involves synchronous conformation changes of both alpha and beta protomers.

Bacterial Proteins↗

PubMed1984

Assembly of transfected DNA into chromatin: structural changes in the origin-promoter-enhancer region upon replication.

Chimeric SV40 DNA containing only the early region, or plasmid DNA harboring the origin-promoter-enhancer region of SV40, when introduced into CV-1 or Cos-1 monkey cells by DEAE-dextran mediated transfer are rapidly assembled in a typical chromatin structure revealed by the generation of a regular 190 bp repeat ladder after micrococcal nuclease digestion. DNA replication is not required for this assembly process. Chromatin-specific DNase I hypersensitive sites are observed in the enhancer region of these minichromosomes. The pattern of the sites differs between non-replicating and post-replicated chromatin. The latter is identical to that observed in the lytic cycle. The presence of large T antigen is not sufficient for the shift in the structure of the chromatin. These experiments suggest that replication can modulate protein-DNA interactions during viral infection or upon cell differentiation.

Animals↗

PubMed1986

Both hydrophobic domains of M13 procoat are required to initiate membrane insertion.

M13 procoat protein has two hydrophobic domains, one in the leader peptide and one which anchors the mature coat protein in the membrane. Disruption of the membrane anchor region by insertion of arginyl residues does not yield periplasmic coat protein. Instead, the rate of membrane assembly is slowed greater than 100-fold (t1/2 less than 5 s for wild-type, t1/2 greater than 10 min for mutant). The hydrophobic region of mature coat protein not only functions as a membrane anchor, but has an important role in the membrane assembly process per se.

Amino Acid Sequence↗

PubMed1989

Isolation of scid pre-B cells that rearrange kappa light chain genes: formation of normal signal and abnormal coding joins.

Consistent with an ordered immunoglobulin (Ig) gene assembly process during precursor (pre-) B cell differentiation, we find that most Abelson murine leukemia virus (A-MuLV)-transformed pre-B cells derived from scid (severe combined immune deficient) mice actively form aberrant rearrangements of their Ig heavy chain locus but do not rearrange endogenous kappa light chain variable region gene segments. However, we have identified several scid A-MuLV transformants that transcribe the germline Ig kappa light chain constant region and actively rearrange the kappa variable region gene locus. In one case progression to the stage of kappa light chain gene rearrangement did not require expression of Ig mu heavy chains; furthermore, this progression could not be efficiently induced following expression of mu heavy chains from an introduced vector. As observed in pre-B cell lines from normal mice, attempted V kappa-to-J kappa rearrangements in scid transformants occur by inversion at least as frequently as by deletion. The inverted rearrangements result in retention of both products of the recombination event in the chromosome, thus allowing their examination. scid kappa coding sequence joins are aberrant and analogous in structure to previously described scid heavy chain coding joins. In contrast, the recognition signals that flank involved coding segments frequently are joined precisely back-to-back in normal fashion. The scid VDJ recombinase defect therefore does not significantly impair recognition of, site-specific cutting at, or juxtaposition and appropriate ligation of signal sequences. Our finding that the scid defect prevents formation of correct coding but not signal joins distinguishes these events mechanistically.

Abelson murine leukemia virus↗

PubMed1994

The influenza virus hemagglutinin cytoplasmic tail is not essential for virus assembly or infectivity.

The influenza A virus hemagglutinin (HA) glycoprotein contains a cytoplasmic tail which consists of 10-11 amino acids, of which five residues re conserved in all subtypes of influenza A virus. As the cytoplasmic tail is not needed for intracellular transport to the plasma membrane, it has become virtually dogma that the role of the cytoplasmic tail is in forming protein-protein interactions necessary for creating an infectious budding virus. To investigate the role of the HA cytoplasmic tail in virus replication, reverse genetics was used to obtain an influenza virus that lacked an HA cytoplasmic tail. The rescued virus contained the HA of subtype A/Udorn/72 in a helper virus (subtype A/WSN/33) background. Biochemical analysis indicated that only the introduced tail- HA was incorporated into virions and these particles lacked a detectable fragment of the helper virus HA. The tail- HA rescued virus assembled and replicated almost as efficiently as virions containing wild-type HA, suggesting that the cytoplasmic tail is not essential for the virus assembly process. Nonetheless, a revertant virus was isolated, suggesting that possession of a cytoplasmic tail does confer an advantage.

Amino Acid Sequence↗

PubMed1995

In vivo iodination of a misfolded proinsulin reveals co-localized signals for Bip binding and for degradation in the ER.

The signal for degradation of proteins in the endoplasmic reticulum (ER) is thought to be the exposure of internal domains which are buried when the protein has adopted its correct conformation and which are also exposed in assembly intermediates. This raises the question of why the intermediates are not degraded. We developed a system based on the peroxidase-catalyzed iodination of tyrosine residues which continuously monitors the exposure of internal domains of proinsulin. In CHO cells this system discriminated between assembly intermediates of wild type (wt) proinsulin and misfolded proinsulin, as shown by the exclusive iodination of a misfolded mutant which was finally degraded in the ER. Iodination in vitro showed that the assembly intermediates of wt proinsulin also exposed internal domains. This iodination was inhibited by the addition of the molecular chaperone Bip which was co-immunoprecipitated with proinsulin in CHO cells. The results obtained with the mutant proinsulin support the assumption that exposed internal domains represent the signal for degradation in the ER. Observations of wt proinsulin show that Bip masks internal domains of normal assembly intermediates during the entire assembly process, thereby suppressing their degradation. We propose that internal domains contain co-localized signals for Bip binding and for degradation.

Adenosine Triphosphate↗

PubMed1995

A domain sharing model for active site assembly within the Mu A tetramer during transposition: the enhancer may specify domain contributions.

The functional configuration of Mu transposase (A protein) is its tetrameric form. We present here a model for the organization of a functional Mu A tetramer. Within the tetramer, assembly of each of the two active sites for Mu end cleavage requires amino acid contributions from the central and C-terminal domains (domains II and III respectively) of at least two Mu A monomers in a trans configuration. The Mu enhancer is likely to function in this assembly process by specifying the two monomers that provide their C-terminal domains for strand cleavage. The Mu B protein is not required in this step. Each of the two active sites for the strand transfer reaction is also organized by domain sharing (but in the reverse mode) between Mu A monomers; i.e. a donor of domain II (also the recipient of domain III) during cleavage is a recipient of domain II (and the donor of domain III) during strand transfer. The function of the Mu B protein (which is required at the strand transfer step) and that of the enhancer element may be analogous in that their interactions with Mu A (domain III and domain I alpha respectively) promote conformations of Mu A conducive to strand cleavage or strand transfer.

Bacteriophage mu↗

PubMed1976

The oral apparatus of Tetrahymena pyriformis, strain WH-6. II. Cytochalasin B inhibition of oral apparatus morphogenesis.

The effects of cytochalasin B on oral apparatus morphogenesis and cell division were studied in synchronized Tetrahymena pyriformis, strain WH-6 syngen 1. Cytochalasin B brought about the rapid arrest of oral apparatus primordium development when added prior to the completion of oral apparatus membranelle differentiation. Cells arrested in development did not divide. When cytochalasin B was added after this transition point, oral apparatus morphogenesis and cell division were completed. The effects of cytochalasin B could be reversed by washing it from the medium. Even though cytochalasin B (at 400 mug/ml) reduced protein synthesis by 30%, the data are consistent with the interpretation that cytochalasin B prevents an assembly process during the membranelle differentiation phase of oral apparatus development.

Animals↗

PubMed1995

Reassembly of the 66 kD neurofilament protein in vitro following isolation and purification from bovine spinal cord.

NF-66, also known as alpha-internexin, has been characterized as a 66 kD mammalian neurofilament (NF) protein whose expression in developing rat brain precedes that of the low molecular weight NF protein (NF-L). NF-66 is thought to assemble into 10 nm diameter intermediate filaments in vitro, although the precise nature of the assembly process remains obscure. Likewise, the ability of NF-66 to polymerize with the low (NF-L), middle (NF-M), and high (NF-H) M(r)NF proteins has not been defined. This investigation describes the reassembly of bovine NF-66 regarding its formation into 10 nm diameter filaments as well as its potential for polymerization with other type IV intermediate filaments. NF-66 and the NF triplet proteins were isolated from bovine spinal cord using established biochemical extraction and isolation procedures (Balin et al., Brain Res 556:181-195, 1991), and purified by a combination of high performance liquid chromatography (HPLC) (DEAE anion exchange and hydroxylapatite column chromatography) and gel elution strategies. In vitro reassembly experiments revealed that NF-66 formed approximately 10 nm diameter filaments of varying length; immunoelectron microscopy demonstrated labeling of these filaments by a monoclonal antibody to intermediate filament antigen (IFA), a polyclonal antibody against rat NF-66 and by a monoclonal antibody generated against the core region of NF-M but cross-reactive with NF-66. This report is the first investigation to look at the in vitro interaction between NF-66 and other type IV intermediate filament proteins (NF-H, -M, and -L) and establishes that NF-66 forms heteropolymeric filaments with these other neurofilament proteins, as confirmed by double immunolabeling. These studies suggest that NF-66 could provide a nucleation site for the polymerization of later-expressed proteins during neuronal development.

Animals↗

PubMed1988

Comparison of the dynamics of the membrane-bound form of fd coat protein in micelles and in bilayers by solution and solid-state nitrogen-15 nuclear magnetic resonance spectroscopy.

Solid-state and solution 15N nuclear magnetic resonance experiments on uniformly and specifically 15N labeled coat protein in phospholipid bilayers and in detergent micelles are used to describe the dynamics of the membrane-bound form of the protein. The residues in the N- and C-terminal portions of the coat protein in both phospholipid bilayers and in detergent micelles are mobile, while those in the hydrophobic midsection are immobile. There is evidence for a gradient of mobility in the C-terminal region of the coat protein in micelles; at 25 degrees C only the last two residues are mobile on the 10(9)-Hz timescale, while the last six to eight residues appear to be mobile on slower timescales and highly mobile at higher temperatures. Since all of the C-terminal residues are immobile in the virus particles, the mobility of these residues in the membrane-bound form of the protein may be important for the formation of protein-DNA interactions in the assembly process.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1995

Redistribution of nuclear envelope associated antigen during the mitotic cycle.

Murine hybridomas were generated to DNA/tight binding proteins complex isolated from the residual nuclear structure following a procedure analogous to that yielding "empty" shells of nuclear envelope. A monoclonal antibody designated 2A8 was selected because of its differential immunostaining of mitotic cells of a synchronized mouse fibroblast cell culture L-929. The target antigen was rendered insoluble by a sequence of extractions of isolated nuclei of diverse cell types with detergents, urea, DNase I and alkali thus reproducing some solubility properties of proteins constituting an operationally defined residual nuclear matrix. The cognate polypeptide was localized on a subset of proteins of M(r) 58-65 kDa, 70 kDa in isolated fibroblast nuclear matrices. The functional implication of the antigen in mitosis-related disassembly-assembly process of the nuclear matrix/envelope was detected. At prophase the antibody decorated the nuclear periphery and nuclear envelope fixed inward filaments. A fibrous network of cytoplasmic localization was stained in metaphase. At anaphase the antigen was dispositioned into peripheral fibrogranular clusters of polar orientation predominantly on one side of the nucleus. Proceeding to telophase a spreading fluorescence was manifested over the entire contour of the nuclear periphery to delineate the reforming nucleus. By immunogold electron microscopy of interphase cells the antigen was identified as evenly distributed in chromatin and interchromatin regions. At initiation of chromosome condensation in mitosis the label was detected predominantly in the chromosomal area.

Animals↗

PubMed1995

The ribosomal DNA loci in Plasmodium falciparum accumulate mutations independently.

Homogeneity of rDNA sequence within a cell is maintained by mechanisms working at the DNA level. The imperative to maintain homogeneity is thought to result from pressure to maintain the sequence of the rRNA transcript. We have investigated the extent of sequence variation within and between members of a species that is unable to utilize some standard mechanisms of rDNA sequence correction. We have compared the sequence of the internal transcribed spacer (ITS1) located between the 18 S rRNA and 5.8 S rRNA genes of five different loci of a single Plasmodium falciparum genotype. The ITS1 sequences are identical at 80 to 91% of the positions among the three asexually expressed genes (A-types) and 75% between the two genes expressed during sporogony (S-types), with only 42 to 57% identity between the types. This is rather startling in that the differences described here for a single genome are greater than those normally seen when comparing rDNA units from distantly related organisms. We observe an apparent conservation of secondary structure within ITS1 sequences from the different transcription units, which would reflect a level of selection at the rRNA but the organism seems to be quite tolerant of primary sequence variation. Investigation of the mature coding region within the 18 S rRNA genes did not reveal sequence variation within A- and S-types from a single genotype. However, comparison of the 18 S rRNA coding region from 17 geographically distinct strains reveals up to 10% sequence variation within a 400 nucleotide region. Hence homogeneity of rRNA units within a species does not seem to be an imperative driven totally by selection at the RNA level. The extraordinary maintenance of homogeneity within rDNA units normally seen within a species appears to have significance beyond those that can be ascribed to the events involved in processing, assembly and function of the ribosome.

Animals↗

PubMed1994

Characterization of hepatitis B virus core mutants that inhibit viral replication.

We have generated and functionally characterized dominant negative core protein variants of the hepadnaviruses to determine their effects on "wild type" viral replication. Plasmids expressing these constructs were introduced into hepatoma cell lines by transient transfection and effects on wild type woodchuck hepatitis virus (WHV) and hepatitis B virus (HBV) replication were evaluated by Southern blot analysis of purified viral core particles. WHV and HBV constructs expressing a truncated core protein fused in frame with the C-terminus of the small surface protein were found to inhibit viral replication by 90-95% due to disruption of the viral nucleocapsid assembly process and preventing encapsidation of pregenomic RNA. The antiviral effects were found to be specific for the targeted virus. These results demonstrate that mutants of hepadnaviral core protein may represent a novel class of antiviral agents.

DNA Replication↗