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PubMed1989

Assembly and properties of chromatin containing histone H1.

The Xenopus oocyte supernatant (oocyte S-150) forms chromatin in a reaction that is affected by temperature and by the concentration of ATP and Mg. Under optimal conditions at 27 degrees C, relaxed DNA plasmids are efficiently assembled into supercoiled minichromosomes with the endogenous histones H3, H4, H2A and H2B. This assembly reaction is a gradual process that takes four to six hours for completion. Micrococcal nuclease digestions of the chromatin assembled under these conditions generate an extended series of DNA fragments that are, on average, multiples of 180 base-pairs. We have examined the effect of histone H1 in this system. Exogenous histone H1, when added at a molar ratio of H1 to nucleosome of 1:1 to 5:1, causes an increase in the micrococcal nuclease resistance of the chromatin without causing chromatin aggregation under these experimental conditions. Furthermore, the periodically arranged nucleosomes display longer internucleosome distances, and the average length of the nucleosome repeat is a function of the amount of histone H1 added, when this histone is present at the onset of the assembly process. In contrast, no major change in the length of the nucleosome repeat is observed when histone H1 is added at the end of the chromatin assembly process. Protein analyses of the purified minichromosomes show that histone H1 is incorporated in the chromatin that is assembled in the S-150 supplemented with histone H1. The amount of histone H1 bound to chromatin is a function of the total amount of histone H1 added. We define here the parameters that generate histone H1-containing chromatin with native nucleosome repeats from 160 to 220 base-pairs, and we discuss the implications of these studies.

Adenosine Triphosphate↗
PubMed1994

The amino acid residues 1-128 in the alpha subunit of the nicotinic acetylcholine receptor contain assembly signals.

Expression of nicotinic acetylcholine receptor (AChR) involves complex processes including assembly of different receptor subunits into hetero-oligomers. To identify the minimal N-terminal region involved in AChR subunit association, we used a dominant negative assay. Co-expression of fragments of the alpha subunit, containing the N-terminal extracellular domain and transmembrane domain 1 (TM 1), with the parental AChR subunits in Xenopus oocytes blocked functional expression of the receptor. In contrast, co-expression of N-terminal extracellular fragments without TM1 failed to inhibit functional expression of AChRs, but altered the functional properties of co-expressed parental AChRs. Furthermore, when these alpha subunit fragments were co-expressed with the beta, gamma, and delta subunits, they were co-immunoprecipitated with a mixture of beta, gamma, and delta subunit specific antibodies. These results suggest that 'assembly signals' are confined to a local structure in the N-terminal extracellular domain. Our findings also indicate that an assembly step may be a target for genetic intervention not only to block the expression of functional receptors, but also to alter the function of the receptor.

Amino Acids↗
PubMed1980

Membrane attack complex of complement: a structural analysis of its assembly.

This study was conducted to gain insight into the process of assembly of the membrane attack complex (MAC) of complement through structural analysis. Four intermediate complexes and the MAC were examined by electron microscopy and by sucrose density-gradient ultracentrifugation. The C5b-6 complex has a sedimentation rate of 11S, an elongated, slightly curved shape and dimensions of 160 x 60 x 60 A. At protein concentrattions greater than 1 mg/ml, and physiologic ionic strength and pH, the complex forms paracrystals that have the appearance of parallel strands. Equimolar quantities of C5b-6 and C7 mixed in the absence of lipids or detergents give rise to C5b-7 protein micelles which are soluble in aqueous media and have a sedimentation rate of 36S, suggesting a tetrameric composition. Ultrastructurally, C5b-7 protein micelles consist of four half-rings, each measuring 200 x 50 A, which are connected to one another by short stalks extending from the convex side of the half-rings. C5b-7 bound to dioleoyl lecithin (DOL) vesicles has a similar ultrastructural appearance. After extraction with deoxycholate (DOC), C5b-7 has a sedimentation velocity of 36S which further suggests the occurrence of C5b-7 in the form of tetrameric protein micelles. Attachment of C8 to vesicle-bound C5b-7 results in dissociation of the protein micelles. An individual C5b-8 complex appears as a half-ring attached to the DOL-vesicle via a 100-A-long and 30-A-wide stalk. After extraction from the DOL-vesicles with DOC, C5b-8 has a sedimentation velocity of approximately 18S. Binding of C9 to DOL-vesicle bound C5b-8 induces the formation of the typical ultrastructural complement lesions. C5b-9 extracted from the vesicles with DOC has a sedimentation rate of 33S, which is characteristic of the C5b-9 dimer. It is concluded that dimerization is a function of C9. C5b-9 monomers are visualized when a single C5b-9 complex or an odd number of complexes were bound per DOL-vesicle. The C5b-9 monomer has an ultrastructural appearance that is theoretically expected of a half-dimer: a 200- x 50-A half-ring which is attached to the DOL-vesicle by a 100- x 80-A appendage. Extracted with DOC, the C5b-9 monomer has a sedimentation rate of 23S. At a higher multiplicity of MAC per DOL-vesicle, large structural defects in the lipid bilayer are seen which are attributed to direct physical destruction of membranes by the known lipid-binding capacity of the MAC. It is proposed that protein micelle formation at the C5b-7 stage of MAC assembly and dissociation of these micelles upon binding of C8 are events that facilitate dimerization of C5b-9 and thus MAC formation.

Binding Sites↗
PubMed1995

Intracellular transport of invariant chain-MHC class II complexes to the peptide-loading compartment.

Th cells recognize peptide fragments of foreign Ags bound to MHC class II molecules. Upon synthesis in the endoplasmic reticulum, the alpha- and beta-chains of the class II molecules rapidly associate with invariant chains (li). The dissociation of li from class II molecules precedes binding of processed Ag and the formation of SDS-stable alpha beta dimers. We previously showed that functional, processed Ag-class II complexes are assembled in a dense lysosome-like compartment that contains stable class II molecules, but no li, referred to in this work as the peptide-loading compartment. We also identified a separate compartment that contains predominantly SDS-unstable li-class II complexes. Because we were unable to identify known organelle markers associated with this compartment, we refer to it as the X compartment. In this work, we provide results that indicate that the X compartment is composed of transport vesicles that move li-class II complexes to the peptide-loading compartment, where all events in the assembly of processed Ag-class II complexes occur.

Antigens, Differentiation, B-Lymphocyte↗
PubMed2000

Ebp2p, yeast homologue of a human protein that interacts with Epstein-Barr virus nuclear antigen 1, is required for pre-rRNA processing and ribosomal subunit assembly.

BACKGROUND: A defect in the secretory pathway causes the transcriptional repression of both rRNA and ribosomal protein genes in Saccharomyces cerevisiae, suggesting a coupling of ribosome synthesis and plasma membrane synthesis. Rrs1p, an essential nuclear protein, is required for the secretory response. RESULTS: EBP2, encoding the yeast homologue of a human protein that interacts with Epstein-Barr virus Nuclear Antigen 1, was cloned in a two-hybrid screen using RRS1 as a bait. The rrs1-1 mutation, which produces Rrs1p without the C-terminal half and causes a defect in the secretory response, almost abolished the interaction with Ebp2p. Ebp2p is essential for growth and is mainly localized in the nucleolus. The effects of Ebp2p depletion on ribosome biogenesis is quite similar to that of Rrs1p depletion; in the Ebp2p-depleted cells, the rate of pre-rRNA processing is slower, and significantly less mature 25S rRNA is produced compared to those in wild-type cells. The polysome pattern indicates that Ebp2p-depletion causes a decrease of 80S monosomes and polysomes, an accumulation of 40S subunits, and the appearance of half-mer polysomes. CONCLUSIONS: Ebp2p is required for the maturation of 25S rRNA and 60S subunit assembly. Ebp2p may be one of the target proteins of Rrs1p for executing the signal to regulate ribosome biogenesis.

Blotting, Northern↗
PubMedDate not supplied

Non-homogenous neural networks with chaotic recursive nodes: connectivity and multi-assemblies structures in recursive processing elements architectures.

This paper addresses recurrent neural architectures based on bifurcating nodes that exhibit chaotic dynamics, with local dynamics defined by first order parametric recursions. In the studied architectures, logistic recursive nodes interact through parametric coupling, they self organize, and the network evolves to global spatio-temporal period-2 attractors that encode stored patterns. The performance of associative memories arrangements is measured through the average error in pattern recovery, under several levels of prompting noise. The impact of the synaptic connections magnitude on architecture performance is analyzed in detail, through pattern recovery performance measures and basin of attraction characterization. The importance of a planned choice of the synaptic connections scale in RPEs architectures is shown. A strategy for minimizing pattern recovery degradation when the number of stored patterns increases is developed. Experimental results show the success of such strategy. Mechanisms for allowing the studied associative networks to deal with asynchronous changes in input patterns, and tools for the interconnection between different associative assemblies are developed. Finally, coupling in heterogeneous assemblies with diverse recursive maps is analyzed, and the associated synaptic connections are equated.

Algorithms↗
PubMedDate not supplied

[Modification of fibrin assembly as a method for studying the mechanism of this process].

A study of monomeric fibrin assembly--the process at which the ordered fibres are formed and at the definite moment there arises an elastic three-dimension net (coagulation occurs) made it possible to establish the following. Fibrinogen and high-molecular fragment D formed in fibrinogen enzymatic hydrolysis inhibit the assembly and prevent from creating the regular morphological structure of the fibres. This effect is determined by the presence in fibrinogen and fragment D. of a part of those specific binding centres by means of which the fibrin molecules recognize each other and realize the assemply. The early but not late stages of the assembly are sensitive to fibrinogen and fragment D. This fact evidences for difference in the mechanisms acting at these stages of the process. A moderate increase in the ionic strength prolongs sharply the clotting time of fibrin monomer, that is connected with both retardation in the assembly and prolongation of the pathway in the transformation itself. Transition from the high ionic strength to the low one produces the opposite effect--acceleration in the assembly and shortening of the pathway. For the subsequently appearing intermediate products of the assembly there exists a threshold at which a slow stage of growth and ordering is replaced by the fast terminal reaction of fibre formation. The threshold height is not constant, it depends on the conditions under which the assembly proceeds. The hypothetic explanations of the obtained results are advanced.

Binding Sites↗
PubMed1977

Membrane assembly: synthesis and intracellular processing of the vesicular stomatitis viral glycoprotein.

The glycoprotein (G) of vesicular stomatitis virus (VSV) is synthesized on membrane-bound polyribosomes. Approximately 30 min after its synthesis, it reaches the surface plasma membrane where it is incorporated into budding virus. The first part of this paper focuses on the 2 intracellular, membrane-bound, glycosylated forms of the glycoprotein which are intermediates in its biogenesis. All glycosylation and processing is completed in the smooth microsome fraction before the protein reaches the surface. Next, we turn to the mechanism by which G is synthesized on membrane-bound polyribosomes. All of the G mRNA is bound to membranes, and studies with puromycin suggest that this attachment of G mRNA is mediated by the nascent glycoprotein chain. After its synthesis G is a transmembrane protein with about 30 amino acids at the carboxyl terminus remaining on the cytoplasmic side of the endoplasmic reticulum. Since 95% of the glycoprotein, containing the carbohydrate residues, is resistant to attack by external proteases, it appears to be within the lumen of the endoplasmic reticulum or embedded within the lipid bilayer. Finally, we show that synthesis, glycosylation, and proper asymmetric insertion of G into the ER can be achieved in cell-free extracts. Both glycosylation of G and proper insertion into the ER membrane in this cell-free system require concomitant protein synthesis.

Cell Line↗
PubMed2002

The mRNA assembly line: transcription and processing machines in the same factory.

Processing of RNA precursors to their mature form often occurs co-transcriptionally. Consequently, the ternary complex of DNA template, RNA polymerase and nascent RNA chain is the physiological substrate for factors that modify the nascent RNA by capping, splicing and cleavage/polyadenylation. mRNA production is thought to occur within a "factory" that contains the RNA polymerase II transcription machine and the processing machines. Newly discovered protein-protein contacts between RNA polymerase and factors that process mRNA precursors are beginning to illuminate how the "mRNA factory" works.

3' Untranslated Regions↗
PubMed2006

Heterogeneous electron transfer processes in self-assembled monolayers of amine terminated conjugated molecular wires.

A versatile synthesis of triarylamine and phenothiazine end-capped oligo(phenyleneacetylene) molecular wires which are terminated by thiol functions is described. The repetitive synthesis allows the preparation of molecular wires with different chain length and different substituents attached to the wire backbone. These molecular wires were used to form dense self-assembled monolayers (SAM) on gold substrates as proved by cyclic voltammetry and quartz crystal microbalance measurements. The heterogeneous electron transfer rate constant of these SAMs was measured by impedance spectroscopy between 1 MHz and 0.1 Hz. The rate constants are somewhat larger for the triarylamine terminated systems than for the phenothiazine compound, due to the higher reorganization energy in the latter. While the molecular wires with electron withdrawing substituents display an electron transfer which is slow enough to be measurable with our impedance setup, we were unable to determine the rate of molecular wires with electron donating substituents.

Journal Article↗
PubMed2000

The HIV-1 vpr protein induces anoikis-resistance by modulating cell adhesion process and microfilament system assembly.

We have previously shown that CD4+ T Jurkat cells constitutively expressing low levels of the human immunodeficiency virus 1 (HIV-1) vpr protein were less susceptible to undergo apoptosis than control cells.1 In this study we have investigated the role of vpr in affecting mechanisms of importance in the control of apoptosis. Vpr-expressing clones consistently aggregated in clusters with time in culture, whereas mock-transfected cells grew as dispersed cultures. The analysis of adhesion molecules involved in cell-to-cell as well as in cell-substrate interactions showed a higher expression of cadherin and integrins alpha5 and alpha6 in vpr-transfected clones with respect to mock-transfected cells. This up-modulation was specifically blocked by cell exposure to antisense oligonucleotides targeted at the vpr. In addition, F-actin microfilament cytoskeletal organization, known to be involved in cell-cell interaction pathways and in the modulation of cell surface molecule expression, was significantly improved in vpr-expressing clones, in which filament polymerization was increased. We thus envisage that vpr viral protein can maintain cell survival via a specific activity on cytoskeleton-dependent cell adhesion pathways, i.e. by inducing anoikis-resistance. These particular effects of vpr might enhance the homing, spreading and survival of the infected lymphocytes, thus contributing to virus persistence in the course of acute HIV-1 infection.

Actin Cytoskeleton↗
PubMed2002

The association of ASAP1, an ADP ribosylation factor-GTPase activating protein, with focal adhesion kinase contributes to the process of focal adhesion assembly.

ASAP1 (ADP ribosylation factor [ARF]- GTPase-activating protein [GAP] containing SH3, ANK repeats, and PH domain) is a phospholipid-dependent ARF-GAP that binds to and is phosphorylated by pp60(Src). Using affinity chromatography and yeast two-hybrid interaction screens, we identified ASAP1 as a major binding partner of protein tyrosine kinase focal adhesion kinase (FAK). Glutathione S-transferase pull-down and coimmunoprecipitation assays showed the binding of ASAP1 to FAK is mediated by an interaction between the C-terminal SH3 domain of ASAP1 with the second proline-rich motif in the C-terminal region of FAK. Transient overexpression of wild-type ASAP1 significantly retarded the spreading of REF52 cells plated on fibronectin. In contrast, overexpression of a truncated variant of ASAP1 that failed to bind FAK or a catalytically inactive variant of ASAP1 lacking GAP activity resulted in a less pronounced inhibition of cell spreading. Transient overexpression of wild-type ASAP1 prevented the efficient organization of paxillin and FAK in focal adhesions during cell spreading, while failing to significantly alter vinculin localization and organization. We conclude from these studies that modulation of ARF activity by ASAP1 is important for the regulation of focal adhesion assembly and/or organization by influencing the mechanisms responsible for the recruitment and organization of selected focal adhesion proteins such as paxillin and FAK.

Animals↗
PubMed2000

Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a major role in the processing, subcellular localization, and assembly of these envelope proteins.

For most membrane proteins, the transmembrane domain (TMD) is more than just an anchor to the membrane. The TMDs of hepatitis C virus (HCV) envelope proteins E1 and E2 are extreme examples of the multifunctionality of such membrane-spanning sequences. Indeed, they possess a signal sequence function in their C-terminal half, play a major role in endoplasmic reticulum localization of E1 and E2, and are potentially involved in the assembly of these envelope proteins. These multiple functions are supposed to be essential for the formation of the viral envelope. As for the other viruses of the family Flaviviridae, these anchor domains are composed of two stretches of hydrophobic residues separated by a short segment containing at least one fully conserved charged residue. Replacement of these charged residues by an alanine in HCV envelope proteins led to an alteration of all of the functions performed by their TMDs, indicating that these functions are tightly linked together. These data suggest that the charged residues of the TMDs of HCV glycoproteins play a key role in the formation of the viral envelope.

Amino Acid Sequence↗