Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Assembly”

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.

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

At least 1,207 records · Page 67Linked to original sources
PubMed1983

Pathways of assembly of nucleohistone complexes formed in vitro under physiological conditions. Implications for the structure of the nucleosome.

The composition and structure of three discrete nucleohistone particles produced by micrococcal nuclease digestion of the complex formed when the four core histones are mixed directly with DNA under conditions that are close to physiological have been reported in accompanying articles (Ellison, M. J., and Pulleyblank, D. E. (1983) J. Biol. Chem. 258, 13307-13313; 13314-13320). These include a peak containing a compact hexameric complex composed of one pair of (H3,H4) and two pairs of (H2A,H2B) (P2) and a peak containing complexes with the composition and sedimentation properties of nucleosome cores (P3). We have examined the pathways of assembly of P2 and P3 by determining the effect of order of histone addition on the yields of these species. The assembly of P2 is initiated by the binding of an (H2A,H2B) pair to the DNA which then directs the placement of an (H3,H4) pair. The incorporation of the final (H2A,H2B) pair to complete the particle is probably cooperative. The assembly of P3 is less dependent than the assembly of P2 upon the order in which the histone pairs are added to the DNA. More than one pathway has been implicated in the formation of these particles. P3 contains a component which has many but not all of the properties of nucleosome cores assembled at elevated ionic strength. Alternative pathways of assembly of histone pairs onto DNA are discussed which could lead to the formation of the discrete histone DNA structures that have been isolated here. A model is proposed in which the nucleosome unfolds and refolds into an alternative configuration.

Animals↗
PubMed1995

Oct-1 protein promotes functional transcription complex assembly on the mouse mammary tumor virus promoter.

The ubiquitous transcription factor Oct-1 stimulates basal transcription from the mouse mammary tumor virus (MMTV) promoter by binding to octamer-related sequences present in the proviral long terminal repeat. The mechanism of transcriptional activation by Oct-1 was investigated using in vitro transcription assays with a HeLa cell nuclear extract depleted of endogenous Oct-1. Oct-1-mediated transcriptional activation could be reconstituted by addition of bacterially expressed recombinant Oct-1 protein. The stimulatory effect of Oct-1 was observed only when the protein was present during formation of transcription preinitiation complexes and not when added to fully assembled complexes. Furthermore, assembled MMTV preinitiation complexes were resistant to inhibition by a competitor oligonucleotide containing MMTV octamer-related elements that could eliminate Oct-1-mediated stimulation when present during the assembly process. The time course of transcription complex assembly revealed that Oct-1 increases the number of templates on which functional transcription complexes form. Finally, experiments designed to exploit the sensitivity of discrete steps in transcription complex assembly to the anionic detergent Sarkosyl demonstrated that Oct-1 must be present during formation of an early intermediate in the assembly process.

Animals↗
PubMed1995

[Studies on the microtubules assembly of multidrug-resistant human leukemic cells].

The development of drug resistance in cancer cells is a significant clinical problem for the successful cancer chemotherapy. Since the cytoskeleton, including microtubules, may be involved in modulating cellular signal transduction, morphological and structural changes, the microtubules assembly of multidrug resistant cells was examined using Confocal Laser Microscope MRC500 system (Bio Rad). In this study, multidrug resistant cells were established by the continuous exposure to ADR(adriamycin) starting with 20 nM up to 1 microM. The expression of MDR-1 (multidrug resistance) gene was detected in K562 leukemia cells and to more extent in the multidrug resistant K562/ADR cells, but not in HL-60 leukemia cells and multidrug resistant HL-60/ADR cells by RT-PCR method. The chronological features of microtubules assembly in the parent cell lines were lost on day 3, after incubation with 20nM of ADR. In accordance with development of drug resistance, the microtubules assembly appeared to be more dense and stronger than that of parent cells. During the development of drug resistant cells, the ADR-accumulation in the nucleus was decreased according to the increase of microtubules assembly. In the case of incubation with 0.5 microM colcemid, an inhibitor of microtubules polymerization, for 3 hours, the stainings of microtubules were lost their fine network and appeared to be diffuse and dot-like pattern. At the same time, both untreated HL-60/ADR and K562/ADR showed the decrease of ADR-accumulation, but the accumulations both colcemid treated resistant cells were increased the same level of their parent cells at the point of 120 min. These results suggested that the resistance to ADR in human leukemia cells correlated with microtubules assembly, and the microtubules assembly played an important role of drug resistance with or without MDR-1 gene overexpression.

Demecolcine↗
PubMed1994

26 amino acids of an extracellular domain of the Na,K-ATPase alpha-subunit are sufficient for assembly with the Na,K-ATPase beta-subunit.

Chimeric cDNAs encoding a sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA1) and regions of the Na,K-ATPase alpha-subunit were constructed to seek the minimal region of the alpha-subunit sufficient for assembly with the Na,K-ATPase beta-subunit. cDNAs encoding a chimera and the chicken beta-subunit were coexpressed in mammalian cells and assembly was assayed by immune precipitation of the chimeric subunit with a monoclonal antibody to the chicken beta-subunit. A chimera containing 26 amino acyl residues of the Na,K-ATPase alpha 1-subunit (NDVEDSYGQQWTFEQRKIVEFTCHTA) (Asn894 to Ala919) that replaced the corresponding avian SERCA1 Ca-ATPase amino acyl residues (Thr871 to Thr898) was able to assemble with the chicken beta-subunit. This alpha-subunit region is predicted to be extracellular, located between membrane-spanning domains 7 and 8 (H7-H8). Chimeras that assembled with full-length beta-subunit also assembled with a beta-subunit chimera that retained only the ectodomain of the chicken beta 1-subunit. These results suggest that the Na,K-ATPase alpha-subunit has the same topology in the membrane as the sarcoplasmic reticulum Ca-ATPase, probably with 10 membrane-spanning domains, and that the aminoacyl residues between membrane domains H7 and H8 are involved in assembly with the beta-subunit in the extracellular/lumenal space.

Amino Acid Sequence↗
PubMed1994

Studies on the assembly of apolipoprotein B-100- and B-48-containing very low density lipoproteins in McA-RH7777 cells.

The mechanisms by which apolipoprotein B-100 (apoB-100) and apoB-48 assemble lipoproteins have been studied in the McA-RH7777 cell line. After incubation for 2 h with 360 microM oleic acid, the McA-RH7777 cells secreted apoB-100 mainly on very low density lipoprotein (VLDL) particles, as judged from sucrose gradient and sequential ultracentrifugation. ApoB-48 was secreted on both VLDL and on denser, high density lipoprotein (HDL)-like lipoproteins. Both apoB-48 and apoB-100 occurred on VLDL particles in the luminal content of the total microsomal fraction. In addition, both proteins were present on denser particles, in particular, those that banded in the HDL region. The denser particles containing apoB-48 were secreted from the cells, whereas those containing apoB-100 were retained in the cell and degraded. Pulse-chase experiments showed that apoB-100 on VLDL was present in the secretory pathway already after a labeling period as short as 3 min. Thus, this particle was the first apoB-100-containing lipoprotein that could be detected in the microsomal fraction of the cell. The assembly of labeled apoB-100 VLDL was acutely (within min) and completely blocked by cycloheximide, if the cycloheximide was added after the pulse labeling period. If, on the other hand, a 15-min chase was introduced after the labeling period, there was no effect of cycloheximide on the assembly of apoB-100 VLDL. A 15-min chase is enough to allow the pulse-labeled apoB nascent polypeptides to be completed to apoB-100. These results indicated that the assembly of apoB-100-containing VLDL is dependent upon ongoing protein biosynthesis during the time when the nascent polypeptide elongate. After this period, the assembly and secretion of the particles are independent of ongoing protein biosynthesis. The first apoB-48-containing particle seen in the luminal content of the microsomal fraction was the HDL-like particle. Pulse-chase experiments as well as experiments with different lengths of the radioactive pulse indicated that the formation of apoB-48-containing VLDL was delayed compared with the formation of the apoB-48-containing HDL-like particle and also in relation to the assembly of the different apoB-100-containing particles, including VLDL. After a 30-min pulse with [35S]methionine followed by a 120-min chase the secretory pathway of the cell was depleted of almost all lipoproteins except the HDL-like apoB-48-containing particle.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Charge interactions in the rod domain drive formation of tetramers during intermediate filament assembly.

The purpose of this study was to test a long standing hypothesis regarding the forces that drive the assembly of intermediate filaments (IFs). The initial step of IF assembly is the formation of dimeric, alpha-helical coiled coils. On the outside of the coiled coils, charged amino acids are distributed periodically such that positively and negatively charged residues are arranged in alternating zones, 9.5/2 residues wide (Parry et al., 1977; McLachlan and Stewart, 1982). This structural feature has given rise to the hypothesis that, if neighboring coiled coils were staggered axially by an odd multiple of a charged zone, electrostatic interactions between them could provide the driving force for the assembly of higher order oligomers or filaments (Fraser et al., 1986; Parry and Steinert, 1992). Using the IF protein vimentin as a model system, we carried out deletion mutagenesis experiments to test this hypothesis. We generated mutant vimentin proteins lacking 14, 21, and 28 residues in Helix 1B of the rod domain, and analyzed their assembly properties by DNA transfection into IF null cells, in vitro assembly, and chemical cross-linking. Results from these experiments are consistent with, and support, the hypothesis that charge complementation plays a key role in the assembly and stabilization of intermediate filaments.

Animals↗
PubMed1994

Minichromosome replication in vitro: inhibition of re-replication by replicatively assembled nucleosomes.

Single-stranded circular DNA, containing the SV40 origin sequence, was used as a template for complementary DNA strand synthesis in cytosolic extracts from HeLa cells. In the presence of the replication-dependent chromatin assembly factor CAF-1, defined numbers of nucleosomes were assembled during complementary DNA strand synthesis. These minichromosomes were then induced to semiconservatively replicate by the addition of the SV40 initiator protein T antigen (re-replication). The results indicate that re-replication of minichromosomes appears to be inhibited by two independent mechanisms. One acts at the initiation of minichromosome re-replication, and the other affects replicative chain elongation. To directly demonstrate the inhibitory effect of replicatively assembled nucleosomes, two types of minichromosomes were prepared: (i) post-replicative minichromosomes were assembled in a reaction coupled to replication as above; (ii) pre-replicative minichromosomes were assembled independently of replication on double-stranded DNA. Both types of minichromosomes were used as templates for DNA replication under identical conditions. Replicative fork movement was found to be impeded only on post-replicative minichromosome templates. In contrast, pre-replicative minichromosomes allowed one unconstrained replication cycle, but re-replication was inhibited due to a block in fork movement. Thus, replicatively assembled chromatin may have a profound influence on the re-replication of DNA.

Centrifugation↗
PubMed1994

Self-assembly into fibrils of collagen II by enzymic cleavage of recombinant procollagen II. Lag period, critical concentration, and morphology of fibrils differ from collagen I.

A recently developed recombinant system for synthesis of human procollagen II by stably transfected host cells was used to prepare adequate amounts of protein to study the self-assembly of collagen II into fibrils. The procollagen II was cleaved to pCcollagen II by procollagen N-proteinase (EC 3.4.24.14), the pCcollagen II was chromatographically purified, and the pCcollagen II was then used as a substrate to generate collagen II fibrils by cleavage with procollagen C-proteinase. The kinetics for assembly of collagen II fibrils were similar to those observed previously for the self-assembly of collagen I in that a distinct lag phase was observed followed by a sigmoidal propagation phase. However, under the same experimental conditions, the lag time for assembly of collagen II fibrils was 5-6-fold longer, and the propagation rate for collagen II fibrils was about 30-fold lower than for collagen I fibrils. The relatively long lag time for the assembly of collagen II into fibrils made it possible to demonstrate that most of the conversion of pCcollagen II to collagen II occurred in the solution phase. The critical concentration at 37 degrees C for collagen II was about 50-fold greater than the critical concentration for collagen I. The Gibbs free energy change for the assembly of collagen II into fibrils was -40 kJ/mol, a value that was about 14 kJ/mol less than the free energy change for collagen I and about the same as the free energy change for the homotrimer of collagen I. Dark-field light microscopy and negative-staining electron microscopy demonstrated that the collagen II fibrils were thin and formed network-like structures. The results demonstrated, therefore, that the structural information of the monomer is sufficient to explain the characteristically small diameters and arcade-like geometry of collagen II fibrils found in cartilage and other tissues.

Cell Line↗
PubMed1994

Self-assembly of collagen I from a proband homozygous for a mutation that substituted serine for glycine at position 661 in the alpha 2(I) chain. Possible relationship between the effects of mutations on critical concentration and the severity of the phenotype.

Procollagen I was isolated from cultured skin fibroblasts from a proband who was homozygous for a mutation in the COL1A2 gene that substituted a serine codon for a glycine codon at position 661 of the alpha 2(I) chain. The procollagen I was cleaved to pCcollagen I by procollagen N-proteinase and the pCcollagen I was used as a substrate for assay of self-assembly of collagen I into fibrils. The mutated pCcollagen I was cleaved to collagen I by procollagen C-proteinase at the same rate as control pCcollagen I. However, self-assembly of the mutated collagen I had a lag period that was 15-fold greater than the lag period observed with normal collagen I under the same conditions. Also, self-assembly of the mutated collagen I had a propagation rate of about one-fourth of the propagation rate of normal collagen I. In addition, the critical concentration for fibril assembly was slightly increased. Rotary shadowing electron microscopy of the mutated procollagen I did not reveal any increased flexibility of the triple helix as was seen previously with two mutated procollagens I in which there were substitutions of cysteine for glycine residues in the alpha 1(I) chain (Vogel, B. E., Doelz, R., Kadler, K. E., Hojima, Y., Engel, J., and Prockop, D. J. (1988) J. Biol. Chem. 263, 19249-19255; Lightfoot, S. J., Holmes, D. F., Brass, A., Grant, M. E., Byers, P. H., and Kadler, K. E. (1992) J. Biol. Chem. 267, 25521-25528). However, morphometric analysis by dark-field light microscopy and electron microscopy showed that the fibrils formed from the mutated collagen I appeared thicker in diameter than the fibrils formed from the normal collagen I. Comparison of the results with similar data on four mutated procollagens previously studied raised the possibility that mutations which markedly increase the critical concentration of fibril assembly produce more severe phenotypes than mutations which change other parameters of fibril assembly.

Amino Acid Sequence↗
PubMed1994

Role of interchain disulfide bonds on the assembly and secretion of human fibrinogen.

The cysteines involved in joining the 2 half-molecules of fibrinogen and also those located on either side of the alpha-helical coiled-coil region, were substituted, by site-directed mutagenesis, with serine. Fibrinogen assembly and secretion were determined in transiently transfected COS cells. Our studies indicate that in order to assemble the 2 half-molecules into a dimer, it is not sufficient to only have the disulfide linkages which keep the 2 half-molecules intact. The disulfide rings which flank the coil-coiled region also play important roles in dimer assembly. Intact interchain disulfide linkages at the NH2-terminal end of the coiled-coil region are essential for assembly of the 2 half-molecules. Disruption of these disulfide rings leads to the formation and secretion of half-molecules. Disruption of the interchain disulfide rings at the COOH-terminal end of the coiled-coil region allows dimer formation, but the 6-chain molecule which is assembled is not secreted. Disruption of both disulfide rings at either end of the coiled-coil region disallows assembly of half-molecules and of dimeric fibrinogen.

Animals↗
PubMed1993

Specific contribution of different phospholipid surfaces to the activation of prothrombin by the fully assembled prothrombinase.

This paper addresses, in thermodynamic and kinetic terms, the reasons for the acidic lipid specificity of the human prothrombinase complex. We obtained, from the measured lipid titrations of the initial rates of prothrombin activation, the empirical binding constants for prothrombinase assembly on different membranes. These favored assembly on phosphatidylserine (PS)- as opposed to phosphatidylglycerol (PG)-containing membranes. In addition, we have used full time courses of prothrombin activation, in conjunction with a calculation of the equilibrium distribution of factor Xa between four enzymatic forms, to obtain the intrinsic kinetic constants of the prothrombinase assembled on PS- or PG-containing membranes. The resulting values of kcat, Km, and kcat/Km increased as acidic lipid content increased, and kcat/Km reached a plateau at 12 mol % PS and 50 mol % PG. Using the measured assembly and kinetic constants, the observed shapes of the phospholipid titration curves of human prothrombin activation were interpreted. We conclude that the difference in activity of prothrombinase assembled on PS- versus PG-containing membranes results both from the different binding properties of factors Xa and Va to these surfaces and from the different intrinsic activities of the prothrombinase when assembled on different membranes.

Arginine↗
PubMed1996

Improving the quality of automatic DNA sequence assembly using fluorescent trace-data classifications.

Virtually all large-scale sequencing projects use automatic sequence-assembly programs to aid in the determination of DNA sequences. The computer-generated assemblies required substantial hand-editing to transform them into submissions for GenBank. As the size of sequencing projects increases, it becomes essential to improve the quality of the automated assemblies so that this time consuming hand-editing may be reduced. Current ABI sequencing technology uses base calls made from fluorescently-labeled DNA fragments run on gels. We present a new representation for the fluorescent trace data associated with individual base calls. This representation can be used before, during, and after fragment assembly to improve the quality of assemblies. We demonstrate one such use-end-trimming of sub-optimal data-that results in a significant improvement in the quality of subsequent assemblies.

Algorithms↗
PubMed1996

In vivo membrane assembly of the E.coli polytopic protein, melibiose permease, occurs via a Sec-independent process which requires the protonmotive force.

To investigate the mechanism of polytopic membrane protein insertion in Escherichia coli, we have examined the protein and energy requirements for in vivo membrane assembly of the prototypic 12 transmembrane domain sugar co-transporter, melibiose permease (MelB). MelB membrane assembly was analyzed both kinetically, by pulse labeling experiments, and functionally by measuring the activity of the inserted permease. Strikingly, the rate of MelB membrane assembly is decreased approximately 4-fold upon dissipation of the transmembrane electrochemical proton gradient, delta(mu)H+, indicative of a strong requirement for delta(mu)H+. Interestingly, selective dissipation of either the electrical (delta(psi)) or the chemical (delta(pH)) component of delta(mu)H+ demonstrates that either form of energy is required for MelB membrane assembly. In contrast, MelB membrane assembly does not require SecA, SecY or SecE, all three proteins which are strictly required for protein translocation. Neither the rate of MelB membrane assembly nor the amount of functional permease is affected by inactivation or depletion of these Sec proteins. These results strongly suggest that polytopic membrane proteins such as MelB insert into the cytoplasmic membrane by a mechanism fundamentally different from protein translocation.

Adenosine Triphosphatases↗
PubMed1997

Roles of integrins in fibronectin matrix assembly.

Fibronectin (Fn) matrix assembly is a dynamic cellular process in which the soluble dimeric Fn molecules are assembled into insoluble, disulfide bond stabilized fibrillar polymeric matrix. Fn matrix assembly requires specific Fn binding integrins. Several Fn binding integrins that are capable of mediating Fn matrix assembly have been identified. They include alpha5beta1, alphaIIb(beta3) and alpha(v)beta3 integrins. Cells regulate the matrix assembly process not only by controlling cell surface expression level of the Fn binding integrins but also by modulating Fn binding and cytoskeleton binding activities of the integrins. A major challenge of future studies is to delineate the signal transduction pathway that regulates Fn matrix assembly.

Amino Acid Sequence↗
PubMed1999

Chromatin assembly: biochemical identities and genetic redundancy.

Investigations on chromatin assembly in vitro implicate chromatin assembly factor 1 (CAF1) as a chaperone for histones H3/H4 and nucleosome assembly protein 1 (NAP1) as a chaperone for histones H2A/H2B. Deletion analysis of CAF1 in vivo suggests multiple redundant pathways for deposition of the histones. Histone deposition requires acetylation of the amino-terminal tails and analysis of mutants suggests a specific but redundant role for acetylation of the tails in assembly. Furthermore, studies on the HAT1 acetyltransferase raise the possibility that acetylation of histones occurs following their transport into the nucleus but prior to their deposition onto DNA. Identification of the factors involved in the redundant pathways of assembly is awaited.

Acetylation↗
PubMed1999

Chromatin assembly during DNA replication in somatic cells.

Newly replicated DNA is assembled into chromatin through two principle pathways. Firstly, parental nucleosomes segregate to replicated DNA, and are transferred directly to one of the two daughter strands during replication fork passage. Secondly, chromatin assembly factors mediate de-novo assembly of nucleosomes on replicating DNA using newly synthesized and acetylated histone proteins. In somatic cells, chromatin assembly factor 1 (CAF-1) appears to be a key player in assembling new nucleosomes during DNA replication. It provides a molecular connection between newly synthesized histones and components of the DNA replication machinery during the S phase of the cell division cycle.

Cell Cycle Proteins↗
PubMed1998

Nucleosome assembly activity and intracellular localization of human CAF-1 changes during the cell division cycle.

We characterized changes of nucleosome assembly activity, intracellular localization, and reversible phosphorylation of the human chromatin assembly factor CAF-1 during the somatic cell division cycle. HeLa cells were synchronized in the G1, S, G2, and M phases of the cell cycle. All three subunits of human CAF-1 (p150, p60, and p48) are present during the entire cell cycle. In interphase, p150 and p60 are bound to the nucleus, but they predominantly dissociate from chromatin during mitosis. During S phase, p150 and p60 are concentrated at sites of intranuclear DNA replication. Only a fraction of total p48 is associated with p150 and p60, and the majority is present in other high molecular weight complexes. The other nucleosome assembly protein, NAP-1, is predominantly cytosolic throughout the cell cycle. Human CAF-1 efficiently mediates nucleosome assembly during complementary DNA strand synthesis in G1, S, and G2 phase cytosolic extracts. Active CAF-1 can be isolated as a 6.5 S complex from G1, S, and G2 phase nuclei. In contrast, CAF-1 isolated from mitotic cytosol does not support nucleosome assembly during DNA synthesis. In mitosis, the p60 subunit of inactive CAF-1 is hyperphosphorylated, whereas active CAF-1 in interphase contains hypophosphorylated and/or phosphorylated forms of p60.

Cell Cycle↗
PubMed2005

Micelle formation and crystallization as paradigms for virus assembly.

Self-assembly processes of crystallization, micelle formation and virus assembly, by their creation of geometric order from disordered components, represent first-order phase transitions that arise through the formation of partially ordered intermediates. The self-assembly of protein subunits into the geometric shells of polyhedral viruses may proceed through formation of reverse micelles, and be driven by condensation of encapsidated nucleic acid complexed with the amino terminal polypeptides of the coat proteins. Restructuring of subunits on the fluid, micellar surface, analogous to processes on the surfaces of growing crystals, then leads to symmetrical, icosahedral capsids. Such a pathway for viral assembly is attractive because it utilizes only physical properties inherent to the system, and it shares many characteristics that we know to be associated with those two other preeminent examples of self-assembly, micelles and crystals.

Amino Acid Sequence↗